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https://openalex.org/W2076535906	https://europepmc.org/articles/pmc2834645?pdf=render	English	null	Endothelial progenitor cells: what use for the cardiologist?	Journal of angiogenesis research	2,010	cc-by	12,248	* Correspondence: g.y.h.lip@bham.ac.uk † Contributed equally 1Haemostasis, Thrombosis and Vascular Biology Unit, University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, UK © 2010 Siddique 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 Endothelial Progenitor Cells (EPC) were first described in 1997 and have since been the subject of numerous inves- tigative studies exploring the potential of these cells in the process of cardiovascular damage and repair. Whilst their exact definition and mechanism of action remains unclear, they are directly influenced by different cardiovas- cular risk factors and have a definite role to play in defining cardiovascular risk. Furthermore, EPCs may have impor- tant therapeutic implications and further understanding of their pathophysiology has enabled us to explore new possibilities in the management of cardiovascular disease. This review article aims to provide an overview of the vast literature on EPCs in relation to clinical cardiology. Endothelial progenitor cells: what use for the cardiologist? Aurangzeb Siddique1†, Eduard Shantsila1†, Gregory YH Lip1, Chetan Varma2 Introduction As an example, CD34 antibody coated stents designed to attract EPCs to the healing endothelium are under intensive clinical trial investigation. However, the precise role of EPCs in vascular pathology still needs to be further clarified as results of some currently available studies in the literature are controversial. Maintenance of endothelial integrity and functioning is vital to the preservation of a healthy vasculature [1]. Thus, the impairment as well as insufficient recovery potential of the endothelial cell monolayer is believed to be a critical factor during the initiaton and progression of atherosclerosis [2]. Indeed, endothelial damage/dys- function has been proved to be involved in the patho- genesis of atherothrombotic vascular disease, with important prognostic and therapeutic implications. This article aims to provide an overview of the vast literature on EPCs in relation to clinical cardiology. JOURNAL OF ANGIOGENESIS RESEARCH JOURNAL OF ANGIOGENESIS RESEARCH Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 EPCs and cardiovascular risk factors Hypercholesterolemia negatively affects both EPC num- ber and function. Indeed, EPC count has an inverse rela- tionship with total cholesterol and LDL-cholesterol levels [9]. Enhanced oxidative stress associated with dys- lipidaemia may at least be partly involved in the dysre- gulation of EPC mobilisation, maturation and survival. Of note, circulating EPCs are very sensitive to oxidized LDL, resulting in premature apoptosis [21,22]. Admittedly, some controversy still exists on the rela- tionship of age to EPC levels. For example, Pelliccia et al failed to find any difference in absolute numbers of CD34+, CD133+, CD105+, and CD14+ cells in older patients with CAD indicating a strong impact of factors other than age for the presence in atherosclerosis and re-emphasising the necessity of having a precise defini- tion of EPC populations studied [13]. The decrease in EPC recruitment in the elderly may be associated with downregulation of tissue hypoxia-inducible factor 1 or insufficient local expression of VEGF, one of the key attractors of EPCs [14]. Furthermore, over an indivi- dual’s lifetime, long-lived cells such as bone marrow endothelial precursors incur repeated exposures to oxi- dative stress; initially, EPCs may compensate by increas- ing their antioxidant responses to prevent oxidant injury. Over time, oxidant damage is likely to accumu- late, thus diminishing the functional properties of EPCs. Enhanced endothelial dysfunction and damage may result in higher tissue demand for EPCs and their increased turnover. Hypercholesterolaemia may also directly affect the bone marrow, resulting in depletion or exhaustion of the bone marrow pool of endothelial progenitors, with a consequent limited supply of EPCs released into circulation [23]. Also, LDL cholesterol levels have an inverse relation with EPC migratory capa- city [23]. Elevated LDL cholesterol and oxidized LDL levels impair EPC migration, via a VEGF-mediated path- way, and oxidized LDL blocks VEGF-induced EPC migration through the inhibition of NO production [24,25]. Physical activity effectively promotes EPC health in terms of quantity, functional capacity and the prevention of apoptosis [15]. This effect is rapid and sustained, at least for 4 weeks [15]. Importantly, physical activity positively affects both bone marrow and peripheral EPC levels [15]. Furthermore, studies in animals have shown that physical activity enhances replacement of the dys- functional endothelium by bone marrow-derived cells [16]. Reduced levels of EPCs have been described in both type 1 and type 2 diabetes mellitus [26]. EPCs ageing and physical activity Although the possibility that adult endothelial precur- sors may exist was suggested more than four decades ago, Asahara et al. first reported isolation of putative adult endothelial precursors which we now recognize as endothelial progenitor cells (EPCs), only in 1997 [3,4]. Endothelial progenitors were able to differentiate into mature endothelial cells and promote repair of damaged endothelium [5-8]. Progressively increasing number of studies has been devoted to these enigmatic cells in the recent years and their close association with multiple markers of cardiovascular health is now well-recognised. Physiological factors and conventional risk factors for atherosclerosis are associated with variations of the number and activity of endothelial progenitors and may be the bridge linking EPCs to common cardiovascular disorders such as coronary artery disease (CAD), myo- cardial infarction and heart failure. EPC characteristics are closely associated with the pre- sence of various cardiovascular risk factors (Table 1). EPC numbers or function - or both - may be affected. For example, smoking contributes towards reducing the number of circulating EPCs, whilst hypertension reduces migratory capacity [9]. Serum LDL cholesterol levels, a positive family history of CAD, and age have all been shown to influence the number and migration of circu- lating CD34+ cells and EPCs [9]. Apart from a diagnostic and prognostic role, EPCs may be attractive target for treatment and, at the same time, be used themselves in an attempt to stimulate angiogenesis, vasculogenesis and cardiac performance. There is an age-related quantitative decline in bone marrow cells expressing endothelial progenitor markers [3]. Jie et al analysed the number of circulating CD34 There is an age-related quantitative decline in bone marrow cells expressing endothelial progenitor markers [3]. Jie et al analysed the number of circulating CD34 +/KDR+ EPCs in healthy subjects aged from 1 to 81 years old, and an inverse relationship with age was Correspondence: g.y.h.lip@bham.ac.uk † Contributed equally 1Haemostasis, Thrombosis and Vascular Biology Unit, University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, UK y g +/KDR+ EPCs in healthy subjects aged from 1 to 81 years old, and an inverse relationship with age was Page 2 of 13 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. EPCs ageing and physical activity Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Table 1 Effects of cardiovascular risk factors on EPC number and function Cardiovascular risk factors Effect on EPC number and function EPC category References Hypercholesterolemia Reduced EPC number, impaired EPC migratory capacity Circulating EPCs (CD133, 34, 45) CFU-ECs [9,23] Diabetes Mellitus Reduced EPC number, impaired EPC migratory capacity Circulating EPCs (CD31, 34, KDR) CFU-ECs [26,27,29,30] Hypertension Inverse relationship of EPC number with systolic blood pressure Circulating EPCs (CD133, 34, KDR) [9,34] Smoking Affects EPC number in a dose-dependent manner Circulating EPCs (CD133, 34, KDR) CFU-ECs [35,36] Ageing Reduced migration and proliferation Circulating EPCs (CD133, 34, 45) CFU-ECs [10-14,113] Exercise Increased EPC number and function Circulating EPCs (CD133, 34, 45) CFU-ECs [15,17-20] EPC - endothelial progenitor cells Table 1 Effects of cardiovascular risk factors on EPC number and function Cardiovascular risk factors Effect on EPC number and function EPC - endothelial progenitor cells [17,18]. Again, NO-dependent mechanisms may by involved, given that physical exercise increases NO bioavailability [19]. The effect of physical activity on EPCs may be reduced by inhibition of endogenous NO synthase [20]. observed [10]. The progressive reduction in different progenitor cell populations with age was also confirmed by Shaffer et al, both in healthy donors and in patients with peripheral arterial disease [11]. Impairment of the functional activity of endothelial progenitors also paral- lels downregulation of their numbers [12]. This decline in EPC clonogenic capacity appears to occur at an ear- lier age, followed by a decline in migratory activity. EPCs and cardiovascular pathology Atherosclerotic disease progression Reduced levels of circulating EPCs have been shown to be independent predictors of atherosclerotic disease pro- gression [37]. Indeed, endothelial integrity is a fine bal- ance between endothelial damage and repair [37]. Since atherosclerotic risk factors are associated with reduced numbers and function of circulating EPCs, it is possible that progression of atherosclerosis is ‘driven’ by an impairment of EPC repairing capacity [9]. Systolic blood pressure has a negative correlation with the number of circulating EPCs, but the clonogenic potential (number of CFU-ECs) is not impaired by arter- ial hypertension [9]. Angiotensin II accelerates the onset of EPC senescence, leading to impaired proliferation of EPCs; this seems to be inhibited by treatment with the angiotensin II type 1 receptor blocker, valsartan [29]. Ramipril also improves the proliferation and migration of EPCs, as well as in vitro vasculogenesis in patients with CAD [30]. In addition, disease processes that ‘damage’ the endothelium per se lead to endothelial cell detachment, resulting in increased levels of circulating endothelial cells (CECs) in the blood [38]. CECs are thought to be mature cells that have detached from the intimal mono- layer in response to endothelial injury and are a differ- ent cell population to EPCs [38]. There is increasing evidence to support a relation between endothelial damage/dysfunction and CECs [39,40]. An inverse rela- tionship has been found between EPCs and CECs, as elevated numbers of CECs have been seen in patients with CAD whereas a reduction in the number of circu- lating EPCs has been associated with CAD [39-41]. These observations were confirmed in the Endothelial Progenitor Cells in Coronary Artery Disease (EPCAD) study, demonstrating that angiotensin-converting enzyme inhibitor treatment was associated with increased num- bers and improved clonogenic potential of circulating EPCs, when compared with patients who were not taking angiotensin-converting enzyme inhibitors [31]. Whilst smoking leads to a reduction in EPC counts, nicotine itself may have a positive effect on EPC num- bers and functional activity at low concentrations [32,33]. This is supported by the finding that the use of nicotine patches slightly increases the magnitude of the rise in EPC levels after smoking cessation [32]. However, higher nicotine levels will have cytotoxic effects on EPCs, indicating complex effects of nicotine on EPCs [33]. Indeed, the number of EPCs is reduced in chronic smokers, whilst cessation of smoking leads to rapid restoration of EPC levels [32]. Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Page 3 of 13 Hyperglycemia significantly reduces eNOS production by EPCs with a corresponding decline in nitric oxide (NO) bioavailability [31]. The effects of high glucose could be ameliorated by co-incubation of EPCs with the NO donor sodium nitroprusside or p38 mitogen-acti- vated protein kinase inhibitor, and deteriorated by eNOS inhibitor [28]. In contrast, antioxidants including vitamin C, N-acetylcysteine and polyethylene glycol-con- jugated superoxide dismutase, and polyethylene glycol- catalase have no significant effects on EPCs [28]. This suggests that the inhibitory effects of high glucose on EPC could be reversed by NO donors, but not by var- ious antioxidants. Although mechanisms linking cardiovascular risk fac- tors and the impairment of EPC mobilization and func- tion are not sufficiently well understood, the number of studies strongly indicates that chronic inflammation and oxidative stress may play a critical role, despite substan- tial resistance of endothelial progenitors to oxidative burden [34-36]. EPCs and cardiovascular pathology Atherosclerotic disease progression In contrast, smoking ces- sation leads to rapid recovery of the circulating EPC population, especially amongst light smokers [32]. EPCs and cardiovascular risk factors EPC recruit- ment for re-endothelialization after vascular injury is impaired in patients with diabetes, and as a consequence of such EPC dysfunction, the vascular regenerative potential of this disease group may be reduced - thus, contributing to the development of vascular complica- tions [27]. Indeed, decreased numbers and functional activity of early EPCs are significantly associated with the pathogenesis of vascular complications in either type 1 or type 2 diabetes [26,27]. A rapid increase of EPC numbers following physical exertion may be attributable to acute mobilisation of the bone marrow EPC pool or a shear stress-induced release into circulation of vessel-wall resident EPCs Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 g Stable coronary artery disease An inverse relationship has been found between circu- lating EPCs and CAD severity, independently of tradi- tional risk factors [42] (Table 2). Indeed, patients with multivessel CAD, had significantly lower EPC counts as compared to those without - for every increase in 10 clusters of CFU-ECs, a patient’s likelihood for multives- sel CAD declined by 20% [42]. In contrast, Guven et al [43] showed that the number of EPCs was increased among patients with significant CAD especially in those requiring coronary intervention, and EPC numbers cor- related with the maximum angiographic stenosis sever- ity. The discrepancy between these two studies might be attributable to the degree of ischemia experienced by patients included as more severe ischemia in patients required intervention could be responcible for EPC mobilization. Recent studies have established a clear link between levels of circulating EPCs and the cumulative cardiovas- cular risk profile [8]. For example, Hill et al[8] hypothe- sized that EPCs derived from the bone marrow have a role in ongoing endothelial repair and thus, the deple- tion of these cells contributes to cardiovascular disease progression. Also, the number of CFU-EC colonies from peripheral blood correlates closely with endothelial func- tion. Thus, EPC levels could be used as a ‘biological marker’ (or biomarker) for vascular function and the relationship to cumulative cardiovascular risk. Measurement of EPCs is of predictive value for cardi- ovascular outcomes in stable CAD patients. For exam- ple, Werner et al measured EPCs positive for the CD34 and KDR in 519 patients with CAD confirmed on cor- onary angiography, and evaluated the association between baseline levels of EPCs and major adverse Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. g Stable coronary artery disease Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Page 4 of 13 Table 2 Effect of cardiovascular diseases on EPC number and function Cardiovascular disorders Effect on EPC number and function EPC category References Acute coronary syndrome Increased EPC number and function Circulating EPC (CD34, 45,133) CFU-ECs [45-48] Atherosclerotic disease progression Reduced EPC number Circulating EPC (CD14) CFU-ECs [37] Severity of coronary artery disease Reduced EPC number/Increased EPC number Circulating EPC (CD34, 133, KDR) CFU-ECs [34,42,43] Heart failure Increased EPC number at early mild heart failure, decreased EPC number in severe heart failure CFU-ECs [52,53] EPC - endothelial progenitor cells Table 2 Effect of cardiovascular diseases on EPC number and function Cardiovascular disorders Effect on EPC number and function Of note, the type of revascularization (ie, thrombolytic therapy or primary angioplasty) does not seem to affect EPC mobilization [50]. However, ischemia per se may be a primary factor for EPC mobilization, given a signifi- cant rise of EPC numbers in patients with unstable angina [50]. An inflammatory state, as shown by high C-reactive protein levels, is also involved in the modula- tion of adhesive properties of EPCs in ACS [51]. cardiac event rates at 12 months [34]. After adjustment for age, sex, vascular risk factors and other relevant vari- ables, increased levels of EPCs were associated with a reduced risk of death from cardiovascular causes, a first major cardiovascular event, revascularization and hospi- talization. This association was independent of severity of CAD, cardiovascular risk factors and drug therapies known to influence cardiovascular outcomes. Of interest, EPC number and function is closely asso- ciated with coronary endothelial function. In 90 patients with CAD coronary endothelial function was assessed using an intracoronary acetylcholine with quantitative coronary angiography [44]. The number of circulating CD133+ or CD34+/KDR+ EPCs and EPC function esti- mated by proliferation of CFU-ECs inversely correspon- dent with the degree of endothelial function impairment [44]. Multivariate analysis showed that the number of EPCs predicted severe endothelial dysfunction indepen- dently of classical cardiovascular risk factors. Congestive heart failure Biphasic changes of the number of CD34+ cells and EPCs have been observed in patients with heart failure. EPCs are significantly up-regulated in mild heart failure (NYHA class I-II) but their mobilization is severely depressed in patients with advanced heart failure (NYHA III-IV) irrespectively of the origin of the disease [52,53]. Depletion in EPCs has been correlated with high levels of TNF-a indicating that endothelial precur- sors arem additional ‘victim’ of excessive inflammation seen in heart failure [52]. These data are in accordance with observations in animal model that statin-induced prevention of left ventricular dysfunction was strongly associated the ability of statins to mobilize EPCs [54]. Acute coronary syndromes Acute coronary syndromes (ACS) are associated with increased levels of inflammatory and haematopoietic cytokines, which in turn can mobilise progenitor cells from the bone marrow [45-47]. Of the many studies in this patient group, Shintani et al were the first to describe a rapid and significant increase in the CD34+ EPC numbers, which reached a maximum at 7 days after the onset of ischaemia in acute myocardial infarc- tion (AMI) [48]. Similarly, Massa et al described sponta- neous mobilisation and a 5.8-fold increase of CD34+ progenitor cells, which peaked about 3 hours after the onset of symptoms, significantly decreased after 7 days, and reached levels comparable with those of healthy subjects within 2 months [46]. Other cell subpopula- tions, such as CD34+/CD117+, CD34+/CXCR4+, CD34 +/CD38+ and CD34+/CD45+ broadly follow the same pattern as that of EPCs [45,46]. Sufficient EPC numbers, as well as the capacity to differentiate into mature endothelial cells are considered to be essential for myo- cardial functional recovery and infarct size reduction after AMI [49]. The proportion of CD34+ cells in hospitalised patients with congestive heart failure increases with the improve- ment of clinical status and is correlated with BNP levels. Of note, Michowitz et al have recently demonstrated that the CFU-EC numbers, together with age and pre- sence of diabetes were independent predictor of all- cause mortality in 107 patients with congestive heart failure [55]. Coronary artery in-stent restenosis and cardiac surgery Coronary artery in-stent restenosis and cardiac surgery Coronary artery in stent restenosis and cardiac surgery Restenosis after coronary artery stenting remains a sig- nificant problem in the practice of interventional cardi- ology [56]. Prevention of restenosis may be promoted by endothelial regeneration through the administration of growth factors, endothelial cell seeding, vessel recon- struction with autologous endothelial cell/fibrin matrix, and the use of stents designed to attract and capture EPCs [56]. Traditionally, the regeneration of injured endothelium has been believed to be due to the migration and Page 5 of 13 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 paclitaxel) reduce neointimal formation by impeding smooth muscle cells proliferation and migration, as well as cause impairment of the normal process of healing of the injured arterial wall, leading to delayed re-endothe- lialization [67]. proliferation of neighboring endothelial cells. This hypothesis has been challenged by animal studies, which reveal that transfusion of EPCs led to reduced intimal thickening of the injured arterial wall, as a result of accelerated reendothelialization [57]. Stents coated with an integrin-binding cyclic Arg-Gly-Asp peptide appear to limit coronary neointima formation and accelerates endothelialization by attracting EPCs, at least in an ani- mal model [58]. Are EPCs really a negative factor in interventional procedures? In contrast to the data where increased numbers of EPCs lead to excessive neointima hyperpla- sia, Matsuo et al have demonstrated that circulating EPCs in patients with in-stent restenosis grow less colo- nies of CFU-ECs and these cells have signs of increased senescence compared to patients with successful arterial healing [68]. Increased number of senescent EPCs were an independent predictor of stent restenosis, but these data also suggest that functional characteristics of EPCs - rather than numbers per se - may be critical for effi- cient re-endothelisation. However, one has to keep in mind that circulating CD34+ cells are not exclusively made up of endothelial precursors but also of hemato- poietic stem cells and attraction of CD34+ cells may also involve homing of certain populations of inflamma- tory leukocytes to sites of vascular damage. What are the implications for therapy? The MAGIC cell trial demonstrated that G-CSF therapy with intra- coronary infusion of peripheral blood stem cells improved cardiac function, and promoted angiogenesis in patients with acute myocardial infarction(AMI) [59]. Coronary artery in-stent restenosis and cardiac surgery At the same time, the concern over aggravation of reste- nosis was raised. However, the MAGIC Cell-3-DES trial showed that G-CSF - based stem cell therapy was both feasible and safe with DES implantation, eliminating the risk of restenosis [60]. Nonetheless, G-CSF administration itself has been shown to be associated with enhanced neointimal hyper- plasia, possibly by the stimulation of excessive prolifera- tion and the migration of smooth muscle cells, thus promoting re-stenosis of stented arteries [61]. Moreover, the possibility remains that EPCs may be independently responsible for stent restenosis, as a strong correlation has been shown between circulating CD34+ cells and late luminal loss following coronary angiography [62]. In a multivariate regression model, a change in CD34+ cells independently predicts late lumen loss [63]. How- ever, CD34 cells may be the ones that aggravated in stent restenosis per se. CD34 cells are not authentic EPCs but myeloid stem cells/progenitors that may dif- ferentiate into smooth muscle cells depending on the situation or in the presence of platelet derived growth factor [63]. The numbers of circulating EPCs may be significantly affected by cardiac surgery, including coronary artery bypass grafting and valve surgery. For example, Smadja et al demonstrated selective mobilisation of endothelial progenitors after different cardiac operations [69], prob- ably in response to tissue damage and application of cardiovascular bypass. Indeed, the mobilization of EPCs from the bone marrow may be promptly stimulated by cardiopulmonary bypass but the extent of such mobilisa- tion is significantly affected by pre-existing risk factors and the European System for Cardiac Operative Risk Evaluation (EuroSCORE) score. Further research is needed to establish the dynamics of EPC during or fol- lowing cardiac surgery, and how this affects prognosis [70]. The role of EPCs in process of post-arterial injury recovery/impairment is pretty complex. The ability of EPCs to home to arterial neointima was initially shown in a experimental minipig model [64]. Inoue et al sub- ssequently confirmed that an increase in EPC numbers following bare-metal stent implantation in patients with CAD and the extent of EPC elevation correlated with the risk of stent restenosis [65]. In contrast, DESs (eg, sirolimus-eluting stents) are capable of preventing pro- cedure-related EPC mobilization [65]. Therapeutic modification of EPCs Statins Statin therapy is associated with an increase in the num- ber of circulating EPCs in patients with CAD [20] (Table 3). The increase in EPCs and improvement of their migratory capacity was significant as early as at 1 week after the initiation of treatment with atorvastatin, with a 3-fold increase at 3 to 4 weeks of therapy [20]. The mobilization of circulating EPCs, along with their enhanced functional activity may contribute to the bene- ficial effects of statins in patients with CAD [20]. The migration and incorporation of EPCs to the sites of re- endothelialization was found significantly increased after statin administration [20,71]. Rapamycin has been also shown to inhibit prolifera- tion, migration, and differentiation of human EPCs in vitro, suggesting that the targeting of EPCs may be an additional mechanism of their successful prevention of re-stenosis by the employment of DESs [66]. Indeed, a reduction of EPC numbers may be one mechanism through which drug-eluting stents actually inhibit reste- nosis. Of note, cell-cycle inhibitors (sirolimus and While short-term treatment with statins has been seen to increase both EPC number and function, more Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Page 6 of 13 Page 6 of 13 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Table 3 Effect of drug treatments on EPC number and function Medication Effect on EPC number and function Reference Atorvastatin Increased EPC number and migration [20] Simvastatin, Mevastatin, Atorvastatin Increased proliferation [114] Simvastatin Increased adhesion [69] Atorvastatin, ezetimibe Increased EPC number [115] Valsartan Reduced senescence [32] Ramipril Increased EPC number, proliferation, migration, adhesion, in vitro vasculogenesis capacity [33] Rosiglitazone Increase in EPC culture and migration [116] Table 3 Effect of drug treatments on EPC number and function Medication Effect on EPC number and function Enalapril also caused a 6-fold increase in the contribu- tion of bone marrow-derived EPCs to the ischemia- induced neovascularization. long-term treatment with statins may have effects to the contrary. Indeed, there is a biphasic effect of statins on EPC counts. In one study of 144 patients with angiogra- phically documented CAD, the administration of statins for more then 4 weeks resulted in a significant decrease of EPC numbers, as determined by flow cytometry and in vitro culture [72]. Therapeutic modification of EPCs Statins However, only CFU-ECs were eval- uated in this study, and as previously discussed, this spe- cial subpopulation of EPCs is probably monocytic in origin, with limited capacity to be incorporated into the microvasculature, but may still promote angiogenesis by the release of proangiogenic factors [73]. These observa- tions were further substantiated by Deschaseaux et al [74], who demonstrated that statin-induced depression of EPCs was only attributed to CFU-ECs, whereas the ECFC populations, were preserved or even enhanced in the circulation by long-term statin treatment. Administration of the PPAR-gamma agonists has been associated with elevation of EPC numbers and improve- ment of their function [77]. For example, rosiglitazone was shown to attenuate the negative effects of C-reactive protein on EPCs and enhance NO-production of EPCs [78]. Other available data indicate that positive effects of PPAR-gamma agonists on EPCs may be one of mechan- isms on the cardiovascular system [78,79]. In addition to the effects of PPAR-gamma agonists on EPCs, another PPAR subtype -the PPAR agonists- has recently attracted attention. For example, Han et al showed PPAR agonists modulate CFU-EC promoting vasculo- genesis [80]. There are also provasculogenic effects of PPAR agonists on ECFCs [81]. In summary, the effects of statins on EPCs are com- plex. They help to mobilize early EPCs, which is espe- cially important in acute ischaemic conditions such as AMI, but strong long-term positive effects of statins may be partly explained by augmentation of ECFC, which are believed to be ‘true EPCs’ responsible for vasculogenesis. Oestrogens augment production and survival of EPCs, thereby increasing the circulating levels of these cells [82]. Moreover, oestrogens not only increase EPC count, but also their ability to effectively home to the sites of vascular lesions [82]. Oestradiol may enhances EPC mobilization though NO-mediated pathways [83]. Implantation of CD34 antibody coated stents ACE inhibitors and Angiotensin II receptor blockers The first of these studies was the Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man (HEALING-FIM) Reg- istry which demonstrated feasibility and safety of EPC capture by CD34 covered stents in stable CAD [89]. Subsequently, the HEALING II and the HEALING III studies, and ultimately the real-world e-Healing multi- center registry of the Genous™pro-healing EPC captur- ing stent (OrbusNeich, Netherlands) have been performed. The majority of completed randomized trials have demonstrated some benefits of stem cells treatment, and studies uniformly report the safety of this approach with no specific adverse events observed (including proar- rhythmia, oncology or excessive inflammatory burden) [95,96]. High rate of in-stent restenosis in the MAGIC study where cell therapy was combined with G-CSF administration has been discussed above [59]. In the BOOST trial, intracoronary implantation of bone marrow cells did not provide long-term benefit on left ventricular systolic function after AMI compared to a randomized control group; however, stem cell therapy was associated with acceleration of left ventricular recovery [97]. The e-Healing registry established that the rates of cardiac death, MI and target lesion revascularization with the CD34 covered stents were low and comparable to the early Taxus registry studies [90]. These stents were also tested for use in primary PCI in patients with AMI. Implantation of these stents was associated with relatively low rate of major adverse cardiac events: 1.6% in-hospital, 4.2% at 30 days, 5.8% at 6 months, and 9.2% at 1 year; there was 1 patient each with acute and suba- cute stent thrombosis but no incidence of late stent thrombosis [91]. When used in ‘high risk’ patients (33% with diabetes, 73% with ACS, 8% with left ventricular dysfunction, 9% with multivessel intervention, 56% type B2/C lesions) 14 month follow-up revealed an event rate of 13% for noncardiac death and AMI, 13% for repeated percutaneous revascularization and 4% for de novo lesions [92]. No bypass surgery was preformed. The study showed that the CD34-covered stents were safe and effective, with satisfactory immediate results and mid-term outcome, without evidence of stent thrombosis y In the study by Janssens et al [98], transfer of bone marrow stem cells to the coronary artery in 67 patients with AMI did not contributed to improvement of the global left ventricular function, but did favorably affected infarct remodelling at 4 months follow-up, with a reduction in infarct size. ACE inhibitors and Angiotensin II receptor blockers Treatment options for CAD include the mechanical dilatation of one or more areas of narrowing within the coronary artery using percutaneous coronary interven- tion (PCI). The original ‘balloon-only’ technique was improved by the use of bare-metal stents (BMS). How- ever, BMSs are still prone to restenosis in up to 30% of patients [84]. To reduce stent restenosis, the use of DESs which can elute antimitotic medications has become widespread. However, DES use has been asso- ciated with late stent thrombosis [85,86]. Indeed, the risk of stent thrombosis with DES is greater than that seen with BMS due to impaired endothelial healing and delayed endothelialisation [87]. Angiotensin II subtype 1-receptor blockade increases the number of EPCs, an effect which seems to be common to all angiotensin II receptor antagonists [75]. VEGF appears to be involved in angiotensin receptor blocker- mediated EPC stimulation [32]. Min et al showed that treatment with ramipril 5 mg daily for 4 weeks in patients with CAD was associated with an approximately 1.5-fold increase in the number of circulating EPCs within 1 week of initiating treatment [33]. This trend persisted with increased levels to approximately 2.5-fold throughout the 4-week study period. In addition, increases in the functional activity of EPCs - as assessed by their proliferation, migration, adhesion and in vitro vasculogenesis capacity - was also seen. Broadly similar effects were also seen with enala- pril, as shown in a study by Wang et al, whereby patients on enalapril displayed a significant increase in circulating EPCs in response to ischemic stress [76]. In contrast, endothelialisation with a third type of stent, the CD34 antibody coated stents based on EPC capture technology, usually occurs within 7 days in ani- mal studies [88]. Rapid endothelialisation and restora- tion of endothelial function may have several Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Page 7 of 13 advantages, including reduced restenosis and reduced stent thrombosis rate. In addition, early cessation of dual antiplatelet therapy may be possible, reducing the inherent risk of this. improvement in cardiac vascularisation and performance while others failing to show any benefits. Results of ran- domized clinical studies are on cardiac transplantation of cells with endothelial progenitor potential are sum- marized in table 4. Effectiveness of CD34 covered stents has been tested in a series of studies performed under the HEALING (Healthy Endothelial Accelerated Lining Inhibits Neoin- timal Growth) program. ACE inhibitors and Angiotensin II receptor blockers In the double-blind, placebo- controlled multicentre REPAIR-AMI trial, 204 patients with MI were randomized to bone marrow-derived cells or placebo [99]. At 12 months, the pre-specified cumu- lative endpoint of death, myocardial infarction, or neces- sity for revascularization was significantly reduced in the stem cell-therapy group compared with placebo; of note, stem cells therapy was an independent predictor of a favorable clinical outcome in this study [99]. Alternative to intracoronary infusion routs of stem cell delivery such as intramyocardial implantation (either during cardiac surgery on NOGA system) have also been tested [100,101]. In the only randomised with per- cutaneous transendocardial injections of CD34+ cells to patient with severe intractable angina, this method was found to be feasible and safe and is being extended into a larger ongoing phase IIb study [102]. In a recent report from the ongoing multi-centre, ran- domized the Tri-stent adjudication (TRIAS) study CD34-covered stents were as effective as paclitaxel-elut- ing stents for the prevention of re-stenosis during 12- month follow-up; however, 4 cases of out-of-hospital thrombotic events were reported for paclitaxel-covered stents, whilst no such events were reported in patients with implanted CD34-covered stents [93]. Given the controversy of trial results, important issues have arisen about the factors affecting the efficacy of such therapy. In addition to route of administration, the time of stem cell delivery (ie. after AMI), origin and number of cells used may be critical. In fact, the major- ity of studies have used unselected mononuclear cells with unknown but evidently very low proportion of endothelial progenitors but with progenitors of other origins (eg. mesenchimal stem cells), this could be potentially favorable for cardiac recovery. Indeed, when either bone marrow or circulating mononuclear cells were delivered to infarct-related coronary arteries, treat- ment with bone morrow cells resulted in a better improvement of left ventricular contractility, when Transplantation of EPCs The available data that demonstrate angiogenic proper- ties of EPCs and favorable outcomes of animal studies have encouraged clinical trials in patients with ischemic heart disease, particularly in the AMI setting [94]. How- ever, results of human studies have proved to be contro- versial with some trials reporting significant Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Page 8 of 13 Table 4 Randomized clinical studies on transplantation of cells which include endothelial progenitors Study/year Disorder N Delivery route Cells delivered Follow-up (months) Effectiveness Strauer et al. (2002) [117] AMI 20 Intracoronary BM-MNCs 4 Effective Kang et al. MAGIC (2004) [92] AMI Old MI 27 Intracoronary PB-MNCs and G- CSF 6 Effective, but high rate of in-stent re-stenosis Schachinger et al. TOPCARE-AMI (2004) [118] AMI 59 Intracoronary PB-MNCs or BM- MNCs 12 Effective Ruan et al. (2005) [119] Acute MI 20 Intracoronary BM-MNCs 6 Effective Strauer et al. IACT (2005) [120] Chronic CAD 36 Intracoronary BM-MNCs 3 Effective Bartunek et al. (2005) [111] Recent MI 35 Intracoronary CD133+ 4 Effective Erbs et al. (2005) [112] Chronic CAD 26 Intracoronary Cultured PB-EPCs 3 Effective Assmus et al. (2006) [111] Chronic CAD 53 Intracoronary BM-MNCs 3 Effective Meyer et al. BOOST (2006) [104] Acute MI 60 Intracoronary BM-MNCs 18 Effective at 6 month, ineffective at 18 month Hendrikx et al. (2006) [121] Heart failure 20 Intramyocardial BM-MNCs 4 Ineffective Janssens et al. (2006) [105] MI 67 Intracoronary BM-MNCs 4 Ineffective Kang et al. MAGIC Cell-3-DES (2006) [60] Acute MI Old MI 82 Intracoronary PB-MNCs and G- CSF 6 Effective Schachinger et al. REPAIR-AMI (2006) [106,122] AMI 201 Intracoronary BM-MNCs 12 Effective Ge et al. TCT-STAMI (2006) [95] MI 20 Intracoronary BM-MNCs 6 Effective Meluzin et al. (2007) [122] Acute MI 60 Intracoronary BM-MNCs 12 Effective AMI - acute myocardial infarction, BM - bone marrow, CAD - coronary artery disease, G-CSF - granulocyte-colony stimulating factor, MI - myocardial infarction, MNC - mononuclear cell, PB - peripheral blood. 4 Randomized clinical studies on transplantation of cells which include endothelial progenitors dies on transplantation of cells which include endothelial progenitors compared to peripheral blood cells [103]. Several studies with selected CD34+, CD133+ cells or cultured periph- eral blood EPCs have been successfully performed but large controlled studies are required to evaluate their clinical utility [102,104,105]. Transplantation of EPCs understanding EPC biology, their potential clinical role is still largely limited by lack of a consensus on the phe- notypic and functional definition of endothelial precur- sors [108]. At present, a single term ‘EPC’ refers to a very diverse group of cells of different lineages which appear to have some angiogenic potential, but not necessarily the ability to differentiate into functional endothelial cells, as expected from their name. Accord- ingly, a booming number of new publications on ‘EPCs’ may have limited scientific impact without a clear understanding what type of cell is actually being analysed. Although different factors may impair EPCs-mediated vascular repair (for example, abnormality in their mobilisa- tion from bone marrow and homing to the damaged vas- cular tissues, and exhaustion of their bone marrow niche), the available data strongly indicate functional characteris- tics of circulating endothelial progenitors are relevant. Accordingly, appropriate genetic modification of EPCs before their implantation may be a way to improve their angiogenic potential. The feasibility of this approach has been demonstrated by genetic inhibition of glycogen synthase kinase-3b signaling in human EPCs that was associated with significant enhancement of their angio- genic properties in an animal model of ischaemia [106]. Additionally, the angiogenic potential of EPCs can be improved by non-genetic ex-vivo stimulus (for example, by exposure to hypoxia). However, the clinical relevance of these approaches needs further investigation [107]. y Currently only so-called ‘outgrowth endothelial cells’ (or ‘late’ EPCs) are known to uniformly give origin to functional endotheliocytes. Indeed, CD34 or CD133 and alone or in different combinations (often with KDR) are the most popular markers used to define ‘circulating EPCs’. This popularity stems from initial reports show- ing that CD34+/KDR+ cells could form endothelium- like cells in vitro [4,109]. Although many studies have employed CD34+/CD133+/KDR+ cells as their defini- tion of ‘true EPCs’ their ability to generate endothelial cells has never been reliably proved. Furthermore, these cell populations probably represents a subset of CD45+ haematopoietic progenitors but do not form endothelial cells in vitro [110]. Inclusion of any additional markers Understanding of EPCs - where we are now? Understanding of EPCs - where we are now? Despite more that a decade of very intensive research and many studies devoted to the problem of Despite more that a decade of very intensive research and many studies devoted to the problem of Page 9 of 13 Page 9 of 13 Siddique et al. Journal of Angiogenesis Research 2010, 2:6 http://www.jangiogenesis.com/content/2/1/6 Association of European Society of Cardiology. CV is UK national coordinating investigator for the TRIAS programme. GL and CV are both investigators in the TRIAS trials. Association of European Society of Cardiology. CV is UK national coordinating investigator for the TRIAS programme. GL and CV are both investigators in the TRIAS trials. in the definition of ‘EPC’ may hamper a holistic approach towards EPC analysis even further and should only be based on robust data confirming the functional identity of the cells analysed. Received: 8 December 2009 Accepted: 22 February 2010 Published: 22 February 2010 The problem with the identification of functional EPCs is also present with approached based on cell cul- ture. For example, markers previously used to prove endothelial identity of putative progenitors, such as CD31, lectin binding or LDL accumulation are now known to be non-specific for the endothelial lineage, but also characteristic for cells of haematopoietic origin] [111,112]. In fact, the majority of so called ‘early’ EPCs represent populations of monocytes and lymphocytes which co-express ‘endothelial’ markers and possess some degree of angiogenic capacity [36]. 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N Engl J Med 2006, 355:1210-21. doi:10.1186/2040-2384-2-6 Cite this article as: Siddique et al.: Endothelial progenitor cells: what use for the cardiologist?. Journal of Angiogenesis Research 2010 2:6. results from a randomized controlled clinical trial. Circulation 2006, 114: I101. 122. Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Hölschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Süselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM, REPAIR-AMI Investigators: Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 2006, 355:1210-21. doi:10.1186/2040-2384-2-6 Cite this article as: Siddique et al.: Endothelial progenitor cells: what use for the cardiologist?. Journal of Angiogenesis Research 2010 2:6. doi:10.1186/2040-2384-2-6 Cite this article as: Siddique et al.: Endothelial progenitor cells: what use for the cardiologist?. Journal of Angiogenesis Research 2010 2:6. doi:10.1186/2040-2384-2-6 Cite this article as: Siddique et al.: Endothelial progenitor cells: what use for the cardiologist?. Journal of Angiogenesis Research 2010 2:6. 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
https://openalex.org/W4309561670	https://bmcpulmmed.biomedcentral.com/counter/pdf/10.1186/s12890-022-02228-z	English	null	A register-based study: cough - a frequent phenomenon in the adult population	BMC pulmonary medicine	2,022	cc-by	7,645	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Chronic cough, more than 8 weeks, can either be without co-morbidity called unexplained chronic cough (UCC) or with co-morbidity called refractory chronic cough (RCC). Using datasets from the Danish National Prescription Registry (Prescription Registry) and Danish National Patient Registry (Patient Registry) we wanted to inves‑ tigate the prevalence and factors of importance of cough in a Nationwide registry. Material and methods: Inclusion criteria were patients 18–90 years with at least one final cough diagnosis (ICD-10 DR05/DR059) in Patient registry or patients who have redeemed ≥2 prescriptions for relevant cough-medication within a 90-day harvest in the Prescription registry from 2008 to 2017. To validate this study’s chosen proxy on chronic cough an analysis of the Patient registry sub-population with a contact of ≥8 weeks and then final diagnosis code DR05/DR059 was also performed. The population was divided into UCC and RCC. Results: Of the 104,216 patients from the Prescription registry, 52,727 were classified as having UCC and 51,489 were classified with RCC. From the Patient registry 34,260 were included, of whom 12,278 had UCC and 21,982 had RCC. Cough were frequently found among females (p < 0.0001). Both genders were around 2 years older in RCC than UCC (p < 0.0001) Spirometry was performed in 69 and 57%, X-ray in 73 and 58% and asthma challenge test performed in 13 and 5% (UCC and RCC, respectively, p < 0.0001). The frequency of co-morbidities such as heart failure, rheumatologic disease, pulmonary embolism, and diabetes was < 10%. Conclusion: Many patients suffer from chronic cough or cough requiring medications, with or without co-morbidity; frequently found among menopausal women. Most patients had a substantial work-up performed. The high fre‑ quency and the resources consuming work-up program call for systematic coding of disease, systematic patient evaluation and more specific treatment options. The study was approved (ID: no. P-2019-191). Keywords: Cough, Unexplained chronic cough, Refractory chronic cough, Register-based study, Nat Unexplained chronic cough, Refractory chronic cough, Register-based study, Nationwide cohort latter may be very disturbing for the patients and difficult to treat for the medical staff. A register‑based study: cough ‑ a frequent phenomenon in the adult population Vibeke Backer1,2, Andreas Porsborg3, Victor Hansen3, Tina Skjold3, Johannes Martin Schmid3, Mette Kehlet4, Christian Torp‑Pedersen5,6,7 and Kristian Aasbjerg7 Backer et al. BMC Pulmonary Medicine (2022) 22:426 https://doi.org/10.1186/s12890-022-02228-z Open Access Correspondence: backer@dadlnet.dk 1 Department of otorhinolaryngology Head & Neck surgery, Rigshospitalet, Copenhagen University, Blegdamsvej 9, DK‑2100 Copenhagen, Denmark Full list of author information is available at the end of the article 1 Department of otorhinolaryngology Head & Neck surgery, Rigshospitalet, Copenhagen University, Blegdamsvej 9, DK‑2100 Copenhagen, Denmark Full list of author information is available at the end of the article Background Cough is one of the most common reasons for contact with the primary health sector [1]. Cough may be present as an acute, subacute or a chronic condition, where the f Coughing is a normal physiological process; it is a protective reflex that clears debris and secretions from the airways. The cough reflex consists of an afferent sensory limb and the central processing centre, activat- ing the efferent limb. The afferent nerves involved are the vagal nerve and it’s sensory branches. These cough important sensory nerves are found from the pharynx to the terminal bronchioles; most are located in the © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 2 of 10 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 2 of 10 Backer et al. BMC Pulmonary Medicine (2022) 22:426 larynx, carina and bifurcation of the larger bronchi [2]. The signal that irritants are present in the bron- chial system is mediated to the cough centre in the brainstem. The efferent pathways are then mediated back to the respiratory tract through the vagal, phrenic and spinal motor nerves [3]. This causes activity in the respiratory muscles, with closure of the vocal cords and activation of the bronchial smooth muscles. This mechanism is present in cough as a protective mech- anism, in cough as a symptom of underlying condi- tions, and as a reflex which has become dysregulated. Chronic cough is often, but not always, associated with different co-morbidities both in the respiratory area, but also outside the thorax [4]. The most common underlying conditions in patients with chronic cough are upper airway cough syndrome, chronic eosino- philic bronchitis, asthma and gastro-esophageal reflux disease (GERD) [5]. Coughing is a well-known side effect of treatment with ACE-inhibitors as up to 20% of the patients treated with an ACE-inhibitor reported coughing as a side effect, which can lead to treatment termination [6]. Material and methods Design This is a descriptive retrospective observational data- base-registry study, using data from the Danish National Patient Registry (Patient Registry) and The Danish National Prescription Registry (Prescription Registry). All inhabitants in Denmark are assigned a unique social security number (CPR number) at birth or immigration. It is not possible to select Danes born in Denmark and immigrants, they all have the same Unique social num- ber. This unique social security number is used in all public health registries such as the Patient Registry and the Prescription Registry and for registration of all con- tacts with the health system, i.e. general practitioners (GP), specialist care out-side hospital, private hospitals as well as examinations and treatment performed in hospi- tal settings. The importance of this, is that we gain data from both primary and secondary health care. The Dan- ish Data Protection Agency have approved the use of the Danish National Health Data for use in this study, in a anonymized form. The use of anonymized registry data does not require ethical approval in Denmark. The study was approved by the Danish Data Protection Agency (ID: no. P-2019-191). The true prevalence of chronic cough in the clini- cal setting, either as an unexplained symptom or as a refractory symptom in relation to a co-morbidity, is not known, since cough seldom is registered in a patient registry as a secondary diagnosis or a co-mor- bidity, when other diseases are present [7]. Study population l Inclusion criteria were patients ≥18 of age and below 90 with at least one final ICD-10 Patient registry diagno- sis code of DR05/DR059 (cough/cough with no further specification) in the Danish National Patient Registry – and/or – having redeemed ≥2 prescriptions for relevant cough-medication within a 90-day period in the Dan- ish National Prescription Registry in the 10-year period from January 1st 2008 to December 31st 2017. Two or more prescriptions within 90-days support that the need of medication is a chronic use. ATC Code: Anatomical Therapeutic Chemical Classification System and Inter- national Classification of Diseases (ICD) codes of version 10 was used (ICD-10).h Cough in population studies is often registered as part of the questionnaire based respiratory symptoms of chronic bronchitis [8], where cough might be the only symptom present. In a Danish population survey, self-reported chronic cough has been found to have a prevalence of 4% among never-smokers and 8% in a subgroup of current smokers [9]. The frequency of cough in a European study was estimated to be 12.7%, indicating geographic differences but possibly also a difference in methodology of recording and definition of cough) [10]. In the present paper, chronic cough is defined as coughing for more than 8 weeks. Chronic cough potentially related to other co-morbidities is defined as Refractory chronic cough (RCC), whereas chronic cough without any other diseases is defined as Unex- plained chronic cough (UCC) [11]. Yet, knowledge about the exact awareness of cough, use of cough treat- ment, co-morbidity treatment, and evaluation schemes of cough among adults is limited. In this registry study, we aim to examine the frequency of chronic cough, co-morbidities, treatment, factor of importance and demographics in a Nationwide study. The study consists of three populations from the two national validated databases: Prescription registry population – group 1 (Fig. 1 a) Included in the prescription registry population are patients who have redeemed ≥2 prescription medica- tions within 90 days to treat cough (Prescription registry ATC code R05) and including ATC codes for Codeine (R05DA04), Noscapine (R05DA07), Dextromethorphan (R05DA09), Pectyl (R05FA02), Opium drops (A07DA02) and all mucolytics (R05CB). Furthermore, for the iden- tification of co-morbidities possibly relating to cough, a Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 3 of 10 Fig. 1 a Patient-flow in population-based prescription registry. b Patient-flow in patients-based registry Fig. 1 a Patient-flow in population-based prescription registry. b Patient-flow in patients-based registry Fig. 1 a Patient-flow in population-based prescription registry. b Patient-flow in patients-based registry Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 4 of 10 Page 4 of 10 Over the counter medications are not recorded in the prescription registry and are therefore not possible to include in this study. search for upper and lower respiratory drugs (Prescrip- tion registry ATC codes R03 and R06) and reflux (ATC Patient registry: A02) were identified. The Prescription registry population represents primary care praxis since most cough related medications will be prescribed by general practitioners. y The Prescription registry population (group 1) is allo- cated from the first event of ≥2 prescriptions redeemed within 90 days in the 10-year study period. The 90 days were selected, due to the content of the package of cough medication and the devices in respiratory medicine. Tab- lets towards cough are most often prescribed with 40 tab- lets per package, and 2 prescriptions are needed within less than 90 days. In case of 3 tablets per day, a package of 100 tablets would last 33 days and in case of 2 tablets daily the amount will last 50 days. Mixture of cough medica- tion would be prescribed in 200 mL per bottle, with the lowest doses of 5 mL, lasting 40 days. Respiratory devices containing 30 doses for treatment once daily and 60 doses for treatment twice daily, i.e. one device per month, some packages might include 3 devices, which cover treatment for 90 days, but not 91 days, therefore number 2 prescrip- tions would be needed within 90 days, for continuation of treatment and this cut-off was selected in the current survey. Patient registry population ‑ group 2 (Fig. 1 b) Included in the population based on diagnosis from the patient registry are patients who have been diagnosed with the cough-specific diagnosis (DR05/059) in the National Danish Patient Registry.hi The identification of co-morbidities possibly relating to cough were done by searching the diagnosis registry for chronic lower respiratory disease coded with DJ40–46, including COPD (DJ44) and asthma (DJ45). Acute lower respiratory diseases (ICD-10 Patient registry of DJ20– 22), upper respiratory illnesses (ICD-10: DJ30–39) and lastly, GERD was based on ICD-10 code DK21, and psy- chogenic cough on ICD-10 code DF453. Non-respiratory illness which might lead to cough, such as heart failure (DI24–25), different types of arthritis (DM051–52, and DM060–69), pulmonary embolism (DI26) and diabetes (DE10–11) were examined as well. The Patient registry population represents secondary health care from hospi- tal/specialist setting. Cough specification Ch i h i Chronic cough is defined as cough > 8 weeks [11]. In this study the definition used for chronic cough is ≥2 redeemed prescriptions within 90 days or having a con- tact with the healthcare system where final diagnosis of the contact being ICD-10 code DR05/DR059. A final diagnosis code of cough from a health care contact can be used as a proxy for chronic cough since the patient will be referred by a general practitioner after a longer period of coughing before the hospital contact. The other reason is the economic incentive from the hospital/specialist to use a ‘real’ ICD-10 diagnosis code for reimbursement and only use cough if no other diagnosis is present. To vali- date this study’s chosen proxy on chronic cough an analy- sis of the Patient registry sub-population with a contact of ≥8 weeks and then final diagnosis code DR05/DR059 was also performed. A merged population with patients present in both the Prescription Registry and Patient Registry databases using the above inclusion criteria which represents the group of patients who have been in contact with both pri- mary and secondary health care and furthermore, when prescription of anti-cough medication has been both pre- scribed and collected. Exclusion criteria was the use of ACE-inhibitors (C09AA) within 12 weeks of diagnosis date, antibiotics (J01) within 6 weeks of diagnosis date and any malignant respiratory disease in the larynx (ICD-10 C32), tracheus (C33) or bronchus/lung (C34). Prescription registry population – group 1 (Fig. 1 a) The Patient registry population (group 2) is based by first occurrence of a final ICD-10 codes from a contact in the patient registry for cough-diagnoses (DR05 and DR059) and therefore patients only occur once in either population. Registries usedh The Danish National Patient Registry records all ICD- 10 diagnoses given within the Danish healthcare system which enables tracking of all patient contacts [12]. There is a strong economic incentive for the hospital depart- ments/specialists to use the system, since the Danish National Patient Registry uses the ICD-10 and testing codes for reimbursement [12].h The included populations were identified as having either Possible Unexplained Chronic Cough (UCC) or Possible Refractory Chronic Cough (RCC). Possible UCC being defined as not having a cough-relevant co-diagno- sis within 12 months before and after the point at which the diagnosis date was registered and possible RCC being defined as having a cough-relevant co-diagnosis within 12 months on either side of cough diagnosis-date. For the Prescription registry population co-morbidities were The Danish National Prescription Registry records all specific prescriptions’ ATC codes redeemed from Danish pharmacies also using the unique social security number. Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 5 of 10 identified through other prescriptions if they did not also have a contact in the Patient registry. Patient registry’s, co-diagnosis, co-medications, and rel- evant testing as well as examinations were performed for the population of the primary (UCC) and secondary out- come (RCC). A merge of the Prescription registry popu- lation and the Patient registry population within both UCC and RCC was performed. Statistical analysis (Chi square) has been performed between UCC and RCC, and a p value of < 0.003 are regarded as significant, corrected for multiple analysis (n = 15). Primary outcome Baseline characteristics and prevalence of individuals with possible UCC– including demographic composition and characteristics as well as relevant examinations in relation to diagnosis. Results Over a period of 10 years (2008–2017), 203,688 patients had redeemed ≥2 relevant prescriptions within 90 days, of which 99,472 were ineligible and 104,216 were included in the Prescription registry population (group 1) (Fig. 1 a). During the same period 57,754 patients were given diagnosis of cough in the Patient registry, of whom 23,494 were ineligible and 34,260 were included (group 2) (Fig. 1 b). 4004 (6.9%) patients were excluded due to ACE inhibitor initiated less than 12 weeks prior to the diagno- sis of cough. In the combined Prescription and Patient registry population 11,209 patients were registered to have both ≥2 redeemed prescriptions as well as the diag- nosis cough. Of these 8024 were ineligible and 3185 were included (group 3). Outcomes of the studyh The outcomes of the study were pre-defined and were as follows: Secondary outcomes Baseline characteristics of individuals with possible RCC – including demographic composition and character- istics, cough-related comorbidities, as well as relevant concomitant medications and relevant examinations in relation to diagnosis. Demographic data such as age, gender, and living region in Denmark (Capital city area, urban area, rural area) is registered in the Patient registry, whereas hos- pital-based prescription and hospital-based treatments such as biological drugs is not accessible through the Prescription Registry and is therefore not reported if the patients are only registered in the Prescription Registry. The cough-relevant medication redeemed at the pharma- cies for inclusion in the prescription-based population is reported (see below) [1]. Respiratory medication R03, antitussive and cold medication (R05), antihistamine (R06), and lastly, opium-drops (A07/DAO2). All patients included are sorted in Table 1 using the def- initions for UCC (no relevant co-morbidity ±12 months from diagnosis) and RCC (cough-relevant co-morbidity related to diagnosis) and divided into three groups (Pre- scription registry, Patient registry and the combination of the two). Of the 104,216 patients from the Prescrip- tion registry group, 52,727 were classified as having UCC (primary outcome) and 51,489 were classified with RCC (secondary outcome). Of the 32,260 patients from the Patient registry, 12,278 were classified as having UCC (primary outcome) and 21,982 were classified as having RCC (Table 1). There were 3185 patients present in both the Prescription registry and the Patient registry, where 529 were classified as having UCC and 2656 as having RCC. Examinations and tests For the Patient registry population a search for relevant examinations related to cough, such as lung function testing, asthma provocation, chest x-ray, CT-scan of tho- rax, HRCT of the lungs, gastroscopy, laryngoscopy, and bronchoscopy was performed (360 days on each side of diagnosis-date) to describe the relevant examinations in connection with cough diagnosis-date. It is not pos- sible to perform a search for relevant examinations for the Prescription registry population; therefore this is not reported. There was no access to patient records and blood tests. More females than males were diagnosed with cough in the group of possible UCC (Table 1, p < 0.0001), and an even more pronounced skewness towards females was observed in the group with possible RCC. The age varied between 50 and 66 years, with no age differences between females and males in all groups (Table 1). The mean age of females with UCC 56.88 and RCC 58.60 (p < 0.0001), and among males 57.70 and RCC 59.86 (p < 0.0001). Furthermore, the data have been grouped into age-related intervals. There was a skewed distribu- tion of patients with chronic cough (data not shown), point prevalence with an overweight among the bigger Statistics Data analysis was performed using SAS for Windows (SAS, Cary, NY, US) version 77.1. Categorical variables are described as absolute numbers as well as percent- age where possible. All data generated or analysed dur- ing this study are included in this published article. The analysis for demographic data included all inhabitants from Denmark, based on prescription lists and ICD-10 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 6 of 10 Table 1 Demographic data 2008–2017 (10 years) Table 1 Demographic data 2008–2017 (10 years) differences was found between the number of patients who had laryngoscopy performed (Table 2). cities, however an exact calculation is not possible due to the move of patients around in the area. Other co-morbidities such as heart failure, rheuma- tologic disease, pulmonary embolism, and diabetes are also shown in Table 1, and the frequency is low and with similar distributions in both UCC and RCC. Similar, cardiac co-morbidities were less frequent in the group of UCC (between 4 to 6%), compared to RCC (between 6 to10%), whereas no such differences were found among the other selected co-morbidities (Table 2, p < 0.0001). In the entire population, those selected through the Patient registry with the diagnose of cough, where dif- ferent testing possibilities was coded as well, the most frequently performed examination was spirometry (n = 16,407) followed by X-ray (n = 15,951), CT-thorax (n = 2020) and HRCT of the lungs (n = 2769) and lastly bronchial provocation (n = 2546). When dividing the total number of examined participants into UCC and RCC cohorts, 57% had spirometry, 73% X-ray, and 5% had bronchial challenge performed among those with UCC (Table 1) and 69% had spirometry, 58% had X-ray, and 13% had bronchial challenge test performed among RCC (Table 1).h Lastly, an extra analysis has been performed in patients who have had attachment to the department for 8 weeks or more followed by a diagnosis of cough or a prescrip- tion of the selected medication. This included patients diagnosed as UCC and RCC, 2442 and 5204, respectively, and when including the prescription and the diagnosis, it was 114 and 637 patients, respectively. The distribution of all the variable included, was similar as in the main group, thus fewer (data not shown). Statistics The number of patients with UCC having X-ray per- formed was higher than RCC (p < 0.0001), whereas all other tests (FEV1, asthma test, CT, HRCT; gastroscopy and bronchoscopy (Table 2)) was more frequently per- formed in patients with RCC than UCC. Whereas no Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 7 of 10 Table 2 Analysis of significant differences between UCC and RCC and both different tests performed and co-morbidities (% of Table 2 Analysis of significant differences between UCC and RCC and both different tests performed and co-morbidities (% o column participants) demographics of patients with cough [13–16]. Patients with RCC were a little older than patients with UCC, independent of gender. Importantly, no differences in age and gender were found based on the selection crite- ria being by the Prescription or Patient registry. Indicat- ing that the selection is unbiased and independent of the methods used.h Discussion This indicates that the cur- rent study might underestimate the frequency of cough in the society when using registries to identify patients, and a possible explanation being that cough is often viewed as a symptom rather than a medical condition and is used accordingly less frequent than ‘proper’ diagnoses such as pharyngitis, possible asthma, reflux, but not COPD, as that would need low level of lung function. In a survey by Weiner et al. [15], they demon- strated a similar low frequency of coded chronic cough, while the frequency of cough described in electronic patients files and data capturing was substantially higher, indicating, that many patients are complaining of cough, but neither a coding of the cough diagnose, due to lack of specific ICD.-10 codes, or prescription of relevant medication has been performed. These find- ings of diminished registration of cough, most likely both in our study and in other surveys, are relevant in the future, when further development in the area might come. In the future this might change as, we in Den- mark have developed an formal ICD-10 code called 05.97. Studies of chronic cough have shown low level of quality of life in those with the highest level of cough complains, indicated by a high Leicester Cough Ques- tionnaire score and a low Cough Quality of Life Ques- tionnaire score [CQLQ]), with a correlation coefficient of minus 0.80 [13].h The use of examinations and tests performed in both the UCC and RCC groups are numerous and pointing towards a wide variety of specialties such as pulmonol- ogy, rhinology, gastroenterology, and cancer diagnostics. Asthma provocation test was performed in 4.8% of UCC versus 12.6% in RCC. The most serious cause of chronic cough is lung cancer, and therefore all with cough should have had an X-ray performed, but the findings in the study was only 73% in the population with UCC. These findings are higher than in similar studies by Zeiger et al. [14], whom found that 62% have had an X-ray taken, where fewer had spirometry performed. Furthermore, since the most frequent cause of chronic cough is COPD or asthma, spirometry should also have been performed in all patients and data show the frequency of spirom- etry performed was even lower than performed X-rays. (Table 1). Discussion These findings suggest that although guidelines exists they might not always be followed when examin- ing a patient with cough and chronic cough [19], and this paper including the work-out of patients suffering of possible chronic cough suggest the need for system- atic evaluation. There are several different clinical path- ways, when evaluating chronic cough and symptoms of cough calls for collaboration between various special- ties, as e.g. upper airway illness with post-nasal drip and gastro-esophageal reflux also leads to cough [20–22]. A multidisciplinary approach might reduce the time spent for both patients and hospital and this is important on a national level since currently differences in culture exists between urban and rural areas. The skewness of cough towards inner city cough with higher frequency (data not shown), might also be related to environmental factors, such as pollution which is known to increase the level of symptoms [23]. The merged Prescription and Patient registry popu- lation with UCC showed a relatively small number of patients within the UCC-group whereas those with RCC were relatively more frequent, indicating that a sub- stantial group of patients suffers from chronic cough on top of their underlying main disease. The merged group might be a more trustfully group of patients, as they have both the symptoms and the prescription of relevant med- ication and has been seen in both primary and secondary care. The reason for the imbalance between the patients prescribed relevant medication and not given the diagno- sis of cough in hospital setting, might be a lack of refer- ral to hospital or that patients are given another diagnosis than cough due to a lack of proper ICD-10 code. The study set-up does not make any possibility of explanation. In patients with persistent cough despite treatment for an underlying cause (ie the RCC-population) the persistent cough might be viewed and treated as an uncontrolled disease with larger doses asthma- or reflux medication, whereas persistent cough of unknown cause is more dif- ficult to evaluate and treat. New approaches with other Discussion In this nationwide study and over a period of 10 years from 2008 and 2017, we have shown that cough encoun- ters are numerous and both patients with unexplained chronic cough (UCC) and refractory chronic cough (RCC) are frequently found. When using the Patient reg- istry to identify the frequency of UCC versus RCC, it is found that there are twice as many patients with RCC, which correlates well with other studies on the ratio on UCC vs RCC found in a specialist setting [10]. However, when examining the frequency of possible chronic cough based on the use of cough medications through the Pre- scription registry, the ratio between UCC and RCC were similar in the two groups, the latter of which might be explained by the reduced likelihood of coding for cough in hospital setting due to reimbursement policy or that the captured number of cough patients by the prescrip- tion list is too difficult. This is to our knowledge one among few studies, examining chronic cough from both primary care using the Prescription registry, and secondary care using the Nationwide ICD-10 Patient registry. The Prescrip- tion registry’s list of cough relevant medications leads to a set of data with more than 100.000 patients over 10 years. However, this large group only represents around 3% of the adult Danish population and when analyzing the group of patients coded with cough using the patient registry it only represents 0.9% of the Dan- ish population. These frequencies of cough are low compared with other studies [14]. In a population study, it was found that 4% suffered of self-reported chronic cough among the entire Danish population, thus only fi In this study we found an overweight of females (62%) and in general the patients were above the age of 50 years, which is similar to findings in other studies reporting Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 8 of 10 treatment options focusing more on the coughing reflex might alter this phenomenon. 3% suffered of chronic cough among never smokers [9]. The frequency of chronic cough was higher in popula- tions of COPD patients (10%) [17], as well as patients with asthma (8%) [18]. These frequencies are thus rela- tive low, as the frequency of cough are found to vary between 4 to 33% [9, 19]. Funding We have received an unrestricted research grant, from MSD, to support the time used for the analysis. The primary investigator, the first author, have administrated the grant through a research account at the main university hospital and written the entire manuscript. The last author has performed all analysis, based on a scientific analysis plan, which have been followed. In the Patient registry the diagnosis of cough is dependent on physician performing the right coding, and since cough being a symptom diagnosis and not a distinct medical condition it could be skewed due to the re-imbursement policy, but it will not be a source of bias to the data reported but only result in underreporting. In the future, it would be helpful to have specific disease code for cough and not only a code for symptoms such as cough, as a large evaluation program are used, with high cost and many visits, which should be relevantly re-imbursed, and furthermore, when new treatment pos- sibility develops, it might be of importance to follow the flow of patients ensure the quality of treatment. Conclusion Many patients suffer from chronic cough or cough requiring medications, with or without co-morbidity; frequently found among menopausal women. Most patients had a substantial work-up performed. The high frequency and the resources consuming work-up pro- gram call for systematic coding of disease, systematic patient evaluation and more specific treatment options. This calls for a multi-specialty approach at specialized Abbreviations RCC R f RCC: Refractory chronic cough; UCC: Unexplained chronic cough; GERD: Gastro-esophageal reflux disease; GP: General practitioners; CPR number: Social security number; ATC Code: Anatomical Therapeutic Chemical Clas‑ sification System; ICD-10: International Classification of Diseases (ICD) codes of version 10.. Strengths and limitations The number of patients, with the strengthen criteria, attachment to the department of 8 weeks and more, fol- lowed by a diagnosis of cough were reduced in number of included patients, but the distribution was similar. We therefore consider the main group as the final diagnosis and analysis. centers and illustrates the need for future therapeu- tic options. The new ICD-10 code covering cough might increase the validity of these studies, which will increase the possibility for further treatment of the patients and even better research. obese patients, which are known reasons for cough and tobacco cessation has been shown to reduce cough sub- stantially [9, 24]. It is a limitation in this study that sur- rogate measures are used to define chronic cough, both from the Prescription database and the Patient registry, even though measures were taken to eliminate reasons for acute cough through exclusion of antibiotics, cough due to ACE-inhibitors and diagnosed cancers as well as not having the exact time frame of the cough prior to final diagnosis in the Patient registry, even though in the Danish clinical setting the final diagnosis can be defined as a contact lasting for ≥8 weeks. One could argue that patients treated with ACE develop chronic cough, whereas only 7% had the diagnosis of cough less than 12 weeks before and were deleted from the cohort. From a clinical point of view, patients with cough, and treat- ment with ACE, are discontinued with the treatment to test the relationship between the two. As it is a register- based study, we cannot examine this, which might be a limitation of the study. Furthermore, another issue could be Codeine selected as a cough suppressant drug, which also have pain killer effect. We believe that this is only of minor importance, as the doses used in the analysis are equivalent to the prescribed doses for cough treatment. The number of patients, with the strengthen criteria, attachment to the department of 8 weeks and more, fol- lowed by a diagnosis of cough were reduced in number of included patients, but the distribution was similar. We therefore consider the main group as the final diagnosis and analysis. Availability of data and materials The data is on the National database, we have performed analysis and devel‑ oped a syntax file so we could do the same analysis again. No one is allowed to take data out of the National database, but you need specific allowance to analyze the data (ID: no. P-2019-191), which we had. Ethics approval and consent to participate The Danish Data Protection Agency have approved the use of the Danish National Health Data for use in this study, in a anonymized form. The use of anonymized registry data does not require ethical approval in Denmark. The study was approved by the Danish Data Protection Agency (ID: no. P-2019-191). Authors’ contributions VB: Developed the protocol, and wrote the main manuscript text. AP and VH: prepared the figures. KAa: performed all the analysis. KAa: developed the Tables. JS and TS: read the manuscript. CTP: Head of database system, read the manuscript. MK: Have read the manuscript. No part of the analysis, have not written the manuscript, have not performed the figures and have not done any of the tables. All authors: reviewed the manuscript. Consent for publication These data are anonymized – it is a true register-based study of 5–6 mio inhabitants of Denmark - and we have never met a patient, never performed examinations, we only have data from the National diagnosis registry with ICD-10 codes and the prescription registry with all ACT codes which is without any relation to persons. Acknowledgements We have a great thanks to the unrestricted grant support form MSD, which have covered the protocol performed prior to the study, including the analysis plan, the cost of the access to the National registries, the data analysis, the time spend when performing the manuscript which have been performed by the first author entirely, and proofread by the entire research group. Mette Kehlet, MD, is employed at 4) MSD Denmark, Havneholmen 25, 1561 Copenhagen V, Denmark. MSD have read the manuscript before submission, but I have had the responsibility for the manuscript, the research, the analysis, the figures, and the tables. Strengths and limitations One of the strengths of this study is that both the Pre- scription and Patient registries are validated on a national level, giving them a high level of credibility and there is a strong economic incentive to use the system, since the ICD-10 coding, given at the final visit, leads to reim- bursement and payment of the hospital department or specialist practice. In the country involved in this study, we have one number each, same number use in Medi- care, social welfare, education, medication and use of hospital facilities. All Medicare is free of charge for all inhabitants, independent of income or not. This support, that the data could be generalized, although it is a one nation study.h The weakness in this registry-based study is, that it is not possible to identify and exclude smokers or Backer et al. BMC Pulmonary Medicine (2022) 22:426 Page 9 of 10 obese patients, which are known reasons for cough and tobacco cessation has been shown to reduce cough sub- stantially [9, 24]. It is a limitation in this study that sur- rogate measures are used to define chronic cough, both from the Prescription database and the Patient registry, even though measures were taken to eliminate reasons for acute cough through exclusion of antibiotics, cough due to ACE-inhibitors and diagnosed cancers as well as not having the exact time frame of the cough prior to final diagnosis in the Patient registry, even though in the Danish clinical setting the final diagnosis can be defined as a contact lasting for ≥8 weeks. One could argue that patients treated with ACE develop chronic cough, whereas only 7% had the diagnosis of cough less than 12 weeks before and were deleted from the cohort. From a clinical point of view, patients with cough, and treat- ment with ACE, are discontinued with the treatment to test the relationship between the two. As it is a register- based study, we cannot examine this, which might be a limitation of the study. Furthermore, another issue could be Codeine selected as a cough suppressant drug, which also have pain killer effect. We believe that this is only of minor importance, as the doses used in the analysis are equivalent to the prescribed doses for cough treatment. References 1. Morice AH. Epidemiology of cough. Pulm Pharmacol Ther. 2002;15:253–9. 2. Ryan NM, Vertigan AE, Birring SS. An update and systematic review on 2. Ryan NM, Vertigan AE, Birring SS. An update and systematic review on drug therapies for the treatment of refractory chronic cough. 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Chronic cough in adults: make the diagnosis and make a dif‑ ference. Pulm Ther. 2019;5:11–21. 6. Smith JA, Woodcock A. Chronic cough. N Engl J Med. 2016;375:1544–51. 7. Morice AH, Fontana GA, Sovijarvi ARA, Pistolesi M, Chung KF, Widdicombe J, O’Connell F, Geppetti P, Gronke L, De Jongste J, Belvisi M, Dicpinigaitis P, Fischer A, McGarvey L, Fokkens WJ, Kastelik J. ERS task force. The diagnosis and management of chronic cough. Eur Respir J. 2004;24:481–92. Competing interests All authors, except from MD Mette Kehlet from MSD, have no conflict of interest. But all analysis and Tables are performed individually by the several of the authors and reviewed by the last author who have re-calculated all data analysis. The first author has written the manuscript from start to the end. Page 10 of 10 Page 10 of 10 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Backer et al. BMC Pulmonary Medicine (2022) 22:426 Mette Kehlet, MD, is employed at 4) MSD Denmark, Havneholmen 25, 1561 Copenhagen V, Denmark. MSD have read the manuscript before submission, but I have had the responsibility for the manuscript, the research, the analysis, the figures, and the tables.” 17. Landt E, Çolak Y, Lange P, Laursen LC, Nordestgaard BG, Dahl M. Chronic cough in individuals with COPD: a population-based cohort study. Chest. 2020;157:1446–54. 18. Çolak Y, Afzal S, Lange P, Laursen LC, Nordestgaard BG, Dahl M. Role and impact of chronic cough in individuals with asthma from the general population. J Allergy Clin Immunol Pract. 2019;7:1783–1792.e8. Author details 1 f 19. Løkke A, Dahl R. [New guidelines for diagnosis and staging of chronic obstructive pulmonary disease]. Ugeskr Laeger 2013;175:1257–1260. 1 Department of otorhinolaryngology Head & Neck surgery, Rigshospitalet, Copenhagen University, Blegdamsvej 9, DK‑2100 Copenhagen, Denmark. 2 Center of physical activity Research (CFAS), Rigshospitalet, Copenhagen University, Copenhagen, Denmark. 3 Department of Respiratory Medicine and Allergy, Aarhus University hospital, Aarhus University, Aarhus, Denmark. 4 MSD Denmark, Havneholmen 25, V, 1561 Copenhagen, Denmark. 5 Depart‑ ment of Cardiology, Nordsjaellands Hospital, Hillerød, Denmark. 6 Department of Cardiology, Aalborg University Hospital, Aalborg, Denmark. 7 Department of Public Health, University of Copenhagen, Copenhagen, Denmark. 20. Fokkens WJ, Lund VJ, Hopkins C, Hellings PW, Kern R, Reitsma S, Toppila- Salmi S, Bernal-Sprekelsen M, Mullol J, Alobid I, Terezinha Anselmo-Lima W, Bachert C, Baroody F, von Buchwald C, Cervin A, Cohen N, Constanti‑ nidis J, De Gabory L, Desrosiers M, Diamant Z, Douglas RG, Gevaert PH, Hafner A, Harvey RJ, Joos GF, Kalogjera L, Knill A, Kocks JH, Landis BN, et al. European position paper on rhinosinusitis and nasal polyps 2020. Rhinol J. 2020;0:1–464. 21. Fokkens WJ, Lund V, Bachert C, Mullol J, Bjermer L, Bousquet J, Canonica GW, Deneyer L, Desrosiers M, Diamant Z, Han J, Heffler E, Hopkins C, Jankowski R, Joos G, Knill A, Lee J, Lee SE, Mariën G, Pugin B, Senior B, Seys SF, Hellings PW. EUFOREA consensus on biologics for CRSwNP with or without asthma. Allergy. 2019;74:2312–9. Received: 5 December 2021 Accepted: 29 September 2022 Received: 5 December 2021 Accepted: 29 September 2022 22. Bachert C, Han JK, Wagenmann M, Hosemann W, Lee SE, Backer V, Mullol J, Gevaert P, Klimek L, Prokopakis E, Knill A, Cavaliere C, Hopkins C, Hell‑ ings P. EUFOREA expert board meeting on uncontrolled severe chronic rhinosinusitis with nasal polyps (CRSwNP) and biologics: definitions and management. J Allergy Clin Immunol. 2021;147:29–36. Publisher’s Note S i N t i Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 8. Thomsen RW, Lange P, Hellquist B, Frausing E, Bartels PD, Krog BR, Hansen A-MS, Buck D, Bunk AE. Validity and underrecording of diagnosis of COPD in the Danish National Patient Registry. Respir Med. 2011;105:1063–8. 9. Çolak Y, Nordestgaard BG, Laursen LC, Afzal S, Lange P, Dahl M. Risk factors for chronic cough among 14,669 individuals from the general popula‑ tion. Chest. 2017;152:563–73. 10. Song W-J, Chang Y-S, Faruqi S, Kim J-Y, Kang M-G, Kim S, Jo E-J, Kim M-H, Plevkova J, Park H-W, Cho S-H, Morice AH. The global epidemiology of chronic cough in adults: a systematic review and meta-analysis. Eur Respir J. 2015;45:1479–81. 11. Levine BM. Systematic evaluation and treatment of chronic cough in a community setting. Allergy asthma Proc. 2008;29:336–42. 12. Ribeiro M, De Castro Pereira CA, Nery LE, Beppu OS, Silva COS. A prospec‑ tive longitudinal study of clinical characteristics, laboratory findings, diagnostic spectrum and outcomes of specific therapy in adult patients with chronic cough in a general respiratory clinic. Int J Clin Pract. 2006;60:799–805. 13. Zeiger RS, Schatz M, Hong B, Li Q, Stern JA, Takhar HS, Weaver JP, Bali V, Schelfhout J, Chen W. Patient-reported burden of chronic cough in a managed care organization. J Allergy Clin Immunol Pract. 2021;9:1624– 1637.e10. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Publisher’s Note S i N t i Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 14. Zeiger RS, Xie F, Schatz M, Hong BD, Weaver JP, Bali V, Schelfhout J, Chen W. Prevalence and characteristics of chronic cough in adults identified by administrative data. Perm J. 2020;24:1–3. 15. Weiner M, Dexter PR, Heithoff K, Roberts AR, Liu Z, Griffith A, Hui S, Schelf‑ hout J, Dicpinigaitis P, Doshi I, Weaver JP. Identifying and characterizing a chronic cough cohort through electronic health records. Chest. 2020. https://doi.org/10.1016/j.chest.2020.12.011. 16. Meltzer EO, Zeiger RS, Schatz M, Dicpinigaitis P, Bernstein DI, Oppenhe‑ imer J, Way N, Li V, Weaver J, Doshi I, Urdaneta E, Boggs R. P351 chronic cough in AMERICA: prevalence and patient characteristics. Ann Allergy Asthma Immunol. 2019;123:S54.
https://openalex.org/W4301390543	https://www.bjcasereports.com.br/index.php/bjcr/article/download/conais22_64_69/conais22_64_69	Portuguese	null	EXTENSÃO CURRICULARIZADA NO CURSO DE FARMÁCIA: RELATO DE EXPERIÊNCIA	Brazilian Journal of Case Reports	2,022	cc-by	1,987	EXTENSÃO CURRICULARIZADA NO CURSO DE FARMÁCIA: RELATO DE EXPERIÊNCIA EXTENSÃO CURRICULARIZADA NO CURSO DE FARMÁCIA: RELATO DE EXPERIÊNCIA EXTENSÃO CURRICULARIZADA NO CURSO DE FARMÁCIA: RELATO DE EXPERIÊNCIA Palavras-chave: Extensão; Curricularização, Formação Profissional. Área temática: Inovações em Ensino e Educação em Saúde Modalidade: Comunicação Oral E-mail do autor: brunacarolinacosta@hotmail.com Área temática: Inovações em Ensino e Educação em Saúde Modalidade: Comunicação Oral E-mail do autor: brunacarolinacosta@hotmail.com E-mail do autor: brunacarolinacosta@hotmail.com RELATO DE EXPERIÊNCIA 1 Bruna Carolina Costa Rafael; 2 Átila Santana Barboza; 3 Déborah Krízia dos Santos Fonseca; 4Luiz Gustavo Martins de Sena; 5 Maria Eduarda Dias de Luna de Brito Pereira; 6Janaína Gonçalves da Silva Melo 1 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 2 Graduando em Farmácia pela Faculdade Pernambucana de Saúde - FPS; 3 Graduanda em Farmácia pela Faculdade 4 1 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 2 Graduando em Farmácia pela Faculdade Pernambucana de Saúde - FPS; 3 Graduanda em Farmácia pela Faculdade 1 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 2 Graduando em Farmácia pela Faculdade Pernambucana de Saúde - FPS; 3 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde - FPS; 4 Graduando em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 5 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 6 Docente em Farmácia da Faculdade Pernambucana de Saúde FPS; pela Faculdade Pernambucana de Saúde FPS; Graduanda em Farmácia pela Faculdade Pernambucana de Saúde - FPS; 4 Graduando em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 5 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 6 Docente em Farmácia da Faculdade Pernambucana de Saúde – FPS; Pernambucana de Saúde - FPS; 4 Graduando em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 5 Graduanda em Farmácia pela Faculdade Pernambucana de Saúde – FPS; 6 Docente em Farmácia da Faculdade Pernambucana de Saúde – FPS; RESUMO INTRODUÇÃO: As diretrizes para a extensão nos cursos de graduação constam normatizadas pelo Ministério da Educação através da Resolução CNE/CES Nº 7, de 18 de dezembro de 2018. A extensão curricularizada no curso de Farmácia da Faculdade Pernambucana de Saúde – FPS está contemplada nos períodos ímpares da matriz curricular como módulo Práticas Integradas da Extensão (I, II, III, IV e V), com carga horária diferenciada em cada um deles. Esta atividade extensionista objetiva desenvolver a formação do profissional crítico, reflexivo, comprometido com os problemas de saúde e educação da comunidade, visando contribuir para a adoção de estratégias de promoção da saúde e prevenção de doenças, considerando o olhar integral da atuação interprofissional. OBJETIVO: Relatar a experiência de estudantes do terceiro período do curso de farmácia da FPS nas atividades de extensão curricularizada. MÉTODO: Através das atividades extensionistas utilizamos metodologias ativas para as discussões sobre os fundamentos de farmacobotânica e farmacognosia; noções de morfologia e anatomia vegetal; coleta, armazenamento, secagem e herborização de amostras vegetais; plantas medicinais e fitoterapia na atenção básica e noções de farmacotécnica; com o intuito de garantir fundamentos para a elaboração de formulações que foram distribuídas em uma Instituição de Longa Permanência (ILP). RESULTADOS: Nas atividades práticas foram elaboradas formulações base de sabonete líquido glicerinado e loção polawax incorporados a extratos de camomila e alecrim, respectivamente. Os produtos manipulados foram entregues aos idosos com orientações dadas pelos estudantes sobre as propriedades terapêuticas e modo de usar, proporcionando o incentivo do autocuidado. CONCLUSÃO: A extensão curricularizada proporcionou a cada estudantes refletir acerca da responsabilidade profissional dentro do contexto social e intercultural, planejando e desenvolvendo ações educativas resultando num momento de troca valiosa de conhecimento com idosos em uma ILP. alavras-chave: Extensão; Curricularização, Formação Profissional. 1 INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO 1 O curso de Farmácia da Faculdade Pernambucana de Saúde – FPS usa o método Aprendizado Baseado em Problemas – ABP, o qual é baseado nos princípios ativos da aprendizagem, configurando-se como alternativa e estratégia capazes de mediar novas relações pedagógicas no contexto ensino-aprendizagem promovendo a possibilidade de uma efetiva interação teoria-prática, de modo a explorar questões que se encontrem além dos limites convencionais da organização curricular tradicional. Destaca-se o uso de um contexto clínico para o aprendizado, promovendo o desenvolvimento da habilidade de trabalhar em grupo, estimulando também o estudo individual. A matriz curricular do curso de Farmácia desta instituição foi referenciada pela Resolução nº 06, de 19 de outubro de 2017, que define as Diretrizes Curriculares Nacionais (DCN) para formação do farmacêutico generalista, que direciona a carga horária mínima para os eixos norteadores, os quais contemplam todas as áreas de integração do conhecimento com vistas à compreensão indissociável do ser humano e das suas relações e intervenções no contexto biopsicossocial (BRASIL, 2017). As diretrizes para a extensão nos cursos de graduação constam normatizadas pelo Ministério da Educação através da Resolução CNE/CES Nº 7, de 18 de dezembro de 2018 – que “Estabelece as Diretrizes para a Extensão na Educação Superior Brasileira e regimenta o disposto na Meta 12.7 da Lei 13.005/2014, que aprova o Plano Nacional de Educação – PNE 2014-2024 e dá outras providências” (BRASIL, 2018). A extensão curricularizada está contemplada nos períodos ímpares da matriz curricular do nosso curso como módulo Práticas Integradas da Extensão (I, II, III, IV e V), com carga horária diferenciada em cada um deles. Esta atividade extensionista objetiva desenvolver a formação do profissional crítico, reflexivo, comprometido com os problemas de saúde e educação da comunidade, visando contribuir para a adoção de estratégias de promoção da saúde e prevenção de doenças, considerando o olhar integral da atuação interprofissional. Dentre os princípios do conhecimento sobre utilização de plantas medicinas pela comunidade, contribuindo desta forma para o seu uso adequado, considerando as principais espécies de interesse do SUS e de ocorrência na região. Assim sendo, este trabalho tem como objetivo relatar a experiência de estudantes do terceiro período do curso de Farmácia da FPS nas práticas da extensão curricularizada. 2 MÉTODO MÉTODO MÉTODO 2 Trata-se de um relato de experiência vivenciado pelos estudantes do terceiro período do curso de Farmácia da Faculdade Pernambucana de Saúde, localizada em Recife - Pernambuco durante os meses de maio a julho de 2022, no módulo Práticas Integradas da Extensão II, contemplando uma carga horária de 60 horas tendo como tema central: Manipulação das Plantas Medicinais. Todo processo das atividades correspondentes à extensão curricularizada neste período acadêmico foi orientado pela docente responsável pelo módulo no formato presencial. No qual discutimos e trabalhamos os objetivos de aprendizagem através de metodologias ativas, utilizando ferramentas como fórum de discussão on-line, construção de jogo de memória, sala de aula invertida e apresentações de seminários. Estes objetivos abordaram os seguintes assuntos: fundamentos de farmacobotânica e farmacognosia; noções de morfologia e anatomia vegetal; coleta, armazenamento, secagem e herborização de amostras vegetais; plantas medicinais e fitoterapia na atenção básica e noções de farmacotécnica. Após as discussões e consolidação do componente teórico desta extensão curricularizada estava estabelecido no cronograma visita a uma Instituição de Longa Permanência (ILP) que abriga 110 idosos, localizada na região metropolitana de Recife. A visita em questão objetivou conhecermos os idosos institucionalizados e verificarmos a possibilidade de manipulação de alguma formulação fitoterápica para eles. Subsequente à visita ficou decidido que a turma iria manipular duas formulações para presentear os idosos num momento posterior, uma loção calmante e hidratante com extrato glicólico de camomila (Matricaria chamomilla) e sabonete líquido antisséptico com extrato glicólico de alecrim (Rosmarinus officinalis). Escolhemos a forma farmacêutica loção por apresentar fácil aplicação na pele proporcionando propriedades benéficas para os idosos e o sabonete antisséptico após por termos identificado vários idosos com autonomia de realizar a higienização de suas mãos quando necessário. A turma foi dividida em dois grupos A e B contendo seis e sete estudantes, respectivamente. C d fi á l l i l ã d f l ã A turma foi dividida em dois grupos A e B contendo seis e sete estudantes, respectivamente. Cada grupo ficou responsável pela manipulação de uma formulação. 3 RESULTADOS E DISCUSSÃO RESULTADOS E DISCUSSÃO RESULTADOS E DISCUSSÃO 3 A primeira etapa de execução prática no laboratório de Farmacotécnica foi o desenvolvimento das formulações base, ou seja, o sabonete líquido base glicerinada e a loção polawax base. Quanto à escolha dos extratos a serem utilizados, levou-se em consideração as ações farmacológicas comprovadas cientificamente inerentes a cada planta. Após diversos testes chegou-se à validação das formulações abaixo apresentadas (tabelas 1 e 2). Tabela 1. Formulação Sabonete Líquido Antisséptico com Extrato de Alecrim INSUMOS PERCENTUAL Lauril éter sulfato de sódio 30,00 Dietanolamida do ácido graxo de coco 90% 5,00 Glicerina 10,00 Metilparabeno 0,15 Propilparabeno 0,02 Hidroxietilcelulose 0,80 Extrato Glicólico de Alecrim 5,00 Óleo essencial de Alecrim q.s. Água destilada q.s.p. 100 Fonte: Autores, 2022. Tabela 2. Formulação Loção Calmante com Extrato de Camomila INSUMOS PERCENTUAL FASE A Cera polawax 6,00 Vaselina líquida 2,00 Propilparabeno 0,05 FASE B Metilparabeno 0,15 Propilenoglicol 2,00 Extrato Glicólico de Camomila 5,00 Água destilada q.s.p 100,00 Fonte: Autores, 2022. Tabela 1. Formulação Sabonete Líquido Antisséptico com Extrato de Alecrim Em seguida foram manipuladas 60 unidades de loção calmante e 60 unidades do sabonete líquidos, os mesmos foram envasados em embalagem contendo 60 mL cada e devidamente rotulados. A segunda visita à ILP para entrega dos produtos manipulados foi realizada com os estudantes e docente responsável. No momento da entrega aos idosos, os estudantes explicaram sobre o autocuidado principalmente com a higiene pessoal, dando informações sobre as propriedades terapêuticas dos produtos e modo de usar. Além dos produtos desenvolvidos e manipulados, foram entregues também à instituição mudas de algumas plantas medicinais para serem plantadas na horta já existente na ILP. CONCLUSÃO A metodologia ABP em que o discente é estimulado a buscar, construir e compartilhar todos os conhecimentos adquiridos e consolidados durante o trajeto da graduação, sendo de suma importância para o desenvolvimento e crescimento do estudante. Além disso, aprendemos desde o início a ter um olhar reflexivo e crítico referente ao nosso dever como futuros farmacêuticos, levando informações sobre o uso seguro e racional de plantas medicinais promovendo assim a sustentabilidade e a valorização do meio ambiente. O trabalho em conjunto na atenção aos cuidados para com os idosos fornece a oportunidade do atendimento humanizado e mais acolhedor na atenção farmacêutica. A extensão curricularizada proporcionou a cada estudante refletir acerca da responsabilidade profissional dentro do contexto social e intercultural, planejando e desenvolvendo ações educativas resultando num momento de troca valiosa de conhecimentos com os idosos em uma ILP. REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS AMARAL, W.; DESCHAMPS, C.; MACHADO, M.P.; KOELER, H.S.; SCHEER, A.P.; CÔCCO, L.C. Desenvolvimento da camomila, rendimento e qualidade do óleo essencial em diferentes idades de colheita. Rev. Bras. Pl. Med., Campinas, p. 237, 2014. BRASIL. Resolução CNE/CES nº 6, de 19 de outubro de 2017 - Institui as Diretrizes Curriculares Nacionais do Curso de Graduação em Farmácia e dá outras providências. Publicada em DOU. Brasília, 20 de outubro de 2017, Seção 1, p. 30. BRASIL. Resolução CNE/CES nº 6, de 19 de outubro de 2017 - Institui as Diretrizes Curriculares Nacionais do Curso de Graduação em Farmácia e dá outras providências. Publicada em DOU. Brasília, 20 de outubro de 2017, Seção 1, p. 30. BRASIL. Resolução CNE/CES nº 7, de 18 de dezembro de 2018 – Estabelece as Diretrizes para a Extensão na Educação Superior Brasileira e regimenta o disposto na Meta 12.7 da Lei 13.005/2014, que aprova o Plano Nacional de Educação – PNE 2014-2024 e dá outras providências. Publicada em DOU. Brasília, 17 de dezembro de 2018, Seção 1, p. 33. BRASIL. Resolução CNE/CES nº 7, de 18 de dezembro de 2018 – Estabelece as Diretrizes para a Extensão na Educação Superior Brasileira e regimenta o disposto na Meta 12.7 da Lei 13.005/2014, que aprova o Plano Nacional de Educação – PNE 2014-2024 e dá outras providências. Publicada em DOU. Brasília, 17 de dezembro de 2018, Seção 1, p. 33. LORENZI, H. & MATOS, F. J. Plantas Medicinais no Brasil: Nativas e Exóticas Cultivadas/ Francisco José de Abreu Matos/ Primeira Edição/ Instituto Plantarum/ Nova Odessa/ 512 pp. 2006.
https://openalex.org/W1971181980	https://scholarworks.gsu.edu/cgi/viewcontent.cgi?article=1024&context=ssw_facpub	English	null	Taking the Perspective that a Depressive State Reflects Inflammation: Implications for the Use of Antidepressants	Frontiers in psychology	2,012	cc-by	20,621	Georgia State University Georgia State University ScholarWorks @ Georgia State University ScholarWorks @ Georgia State University SW Publications School of Social Work 8-17-2012 Taking the Perspective that a Depressive State Reflects Taking the Perspective that a Depressive State Reflects Inflammation: Implications for the Use of Antidepressants Inflammation: Implications for the Use of Antidepressants Jill Littrell Georgia State University, littrell@gsu.edu Georgia State University Georgia State University ScholarWorks @ Georgia State University ScholarWorks @ Georgia State University School of Social Work School of Social Work Follow this and additional works at: https://scholarworks.gsu.edu/ssw_facpub Part of the Mental Disorders Commons, Psychiatric and Mental Health Commons, and the Social Work Commons Part of the Mental Disorders Commons, Psychiatric and Mental Health Commons, and the Social Work Commons DEPRESSIVE BEHAVIORS INVOLVE INFLAMMATION or provoking inﬂammation in the periphery induces an equiv- alent state of systemic inﬂammation. According to Maier and Watkins, systemic inﬂammation correlates with changes in the brain. Regardless of the initiating event, whether an infection or a psychological stressor, behavioral manifestations of depression emerge that are driven by synonymous changes in the brain. Maier and Watkins speculated that during evolution, an immune system forﬁghtingpathogensemergedﬁrst.Whenmulti-celledorganisms needed a mechanism for responding to an external psychological threat, the body recruited the already existing immune system for responding to external threat. Similar ideas were proposed by Maes et al. (1999). With regard to the evolutionary connec- tion between psychological stressors and inﬂammatory activation, Pace et al. (2012) have suggested that given a psychological threat, such as attack by a predator, preparing the immune system to ﬁght infection resulting from a predatory attack could be adaptive. The association between stress,systemic inﬂammation,and behav- ioral signs of depression has been recognized for over a decade (Raison et al., 2006; Capuron et al., 2008; Dantzer et al., 2011). Depressed and anxious persons display elevations in blood lev- els of inﬂammatory cytokines [Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)] as well as other markers of an inﬂam- matory state [e.g., C-reactive protein (CRP), soluble intracellular adhesion molecule-1 (ICAM-1), and macrophage chemoattrac- tant protein-1; Rajagopalan et al., 2001; Zorrilla et al., 2001; Schiepers et al., 2005; Raison et al., 2006; Howren et al., 2009; Dowlati et al., 2010], and given a social stressor, depressed individ- uals exhibit greater elevations in inﬂammatory cytokines, such as IL-6 (Pace et al., 2006). Depressed persons also exhibit lower levels of anti-inﬂammatory cytokines (Li et al., 2011). In addition to the ﬁndings for persons exhibiting behavioral signs of depression, the immune system function of individuals undergoing stress has been evaluated. In examining the leuko- cytes from individuals who reported loneliness, Cole et al. (2007, 2011) found increased transcriptional activity of Nuclear Factor- κB (NF-κB), a marker of inﬂammatory activation. The spouses of Alzheimer patients and caregivers for cancer patients are char- acterized by high levels of inﬂammatory cytokines such as IL-6 (Kiecolt-Glaser et al., 2003; von Kanel et al., 2006) and higher levels of NF-κB activity (Miller et al., 2008). REVIEW ARTICLE published: 17 August 2012 doi: 10.3389/fpsyg.2012.00297 REVIEW ARTICLE Manipulating psychological stress Given the association between subjective distress and markers of systematic inﬂammation, researchers investigated the direction of a possible causal relationship. Learned helplessness is an animal model of depression. To induce learned helplessness, researchers subject animals to bouts of uncontrollable shock. The animal can- not escape or avoid the shock. Subsequent to this stressor, the animal appears depressed. The animal fails to drink sucrose, dis- plays less locomotion,fails to explore new environments,and,even when subsequently given an opportunity to avoid an aversive stim- ulus, fails to escape or avoid. Animals subjected to helplessness inductions also have elevated levels of IL-1, in their hippocampus and hypothalamus (Maier and Watkins, 1998). Moreover, when an antagonist for the IL-1 is placed in the hippocampi of animals exposed to uncontrollable shock, the animal no longer exhibits depressed behavior, and no longer fails to escape aversive stimuli Correspondence: University, P.O. Box 3995, Atlanta, GA 30302, USA. Keywords: major depression, inﬂammation, antidepressants efﬁcacy, antidepressants, neurogenesis and inﬂam- mation DEPRESSIVE BEHAVIORS INVOLVE INFLAMMATION Persons who are under stress at work exhibit elevations in ICAM-1, inﬂammatory cytokines, and acute phase proteins such as ﬁbrinogen (von Kanel et al., 2001; Steptoe et al., 2003; Ramachandruni et al., 2004; Hong et al., 2006). Jill Leslie Littrell School of Social Work, Georgia State University, Atlanta, GA, USA This paper reviews both the evidence that supports the characterization of depression as an inﬂammatory disorder and the different biochemical mechanisms that have been pos- tulated for the connection between inﬂammation and depression. This association offers credible explanation for the short term efﬁcacy of antidepressants, which have short term anti-inﬂammatory effects. Evidence for those anti-inﬂammatory effects is discussed. Evi- dence of the contrary long-term effects of antidepressants, which increase rather than decrease inﬂammation, is also reviewed. It is argued that this increase in inﬂammation would predict an increase in chronicity among depressed patients that have been treated with antidepressants drugs, which has been noted in the literature. A brief discussion of alternatives for decreasing inﬂammation, some of which have demonstrated efﬁcacy in ameliorating depression, is presented. Recommended Citation Recommended Citation Littrell, J.L. (2012). Taking the perspective that a depressive state reflects inflammation: implications for the use of antidepressants. Frontiers in Psychology, 3,Article 297. doi: 10.3389/fpsyg.2012.00297 Littrell, J.L. (2012). Taking the perspective that a depressive state reflects inflammation: implications for the use of antidepressants. Frontiers in Psychology, 3,Article 297. doi: 10.3389/fpsyg.2012.00297 This Article is brought to you for free and open access by the School of Social Work at ScholarWorks @ Georgia State University. It has been accepted for inclusion in SW Publications by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact scholarworks@gsu.edu. REVIEW ARTICLE published: 17 August 2012 doi: 10.3389/fpsyg.2012.00297 Taking the perspective that a depressive state reﬂects inﬂammation: implications for the use of antidepressants Jill Leslie Littrell* Edited by: Edited by: Gian Mauro Manzoni, Istituto Auxologico Italiano IRCCS, Italy Reviewed by: Gianluca Castelnuovo, Università Cattolica del Sacro Cuore, Italy Brandon Abbs, Partners Healthcare, USA Francisco Aboitiz, Pontiﬁcia Universidad Catolica de Chile, Chile *Correspondence: Jill Leslie Littrell, Georgia State University, P.O. Box 3995, Atlanta, GA 30302, USA. e-mail: littrell@gsu.edu Francisco Aboitiz, Pontiﬁcia Universidad Catolica de Chile, Chile Peripheral inﬂammation does impact the brain Since the brain is supposed to be impervious to the entry of cytokines,the question of how cytokines in the periphery commu- nicatewithbrainhasbeenasubjectof inquiry.Severalmechanisms have emerged. First, the sensory vagus nerve, which has receptors for IL-1 and prostaglandins (Ek et al., 1998), responds to local inﬂammation and results in activation in the Nucleus Tractus Solitarius of the medulla which relays information to its projec- tion areas (the parabrachial,paraventricular and supraoptic nuclei and ventromedial preoptic area of the hypothalamus, the central amygdala, and the BNST; Dantzer et al., 2000, 2007; Goehler et al., 2000; Konsman et al., 2000). Second, activated leukocytes in circu- lation can communicate with brain tissue at areas where the blood brain barrier is weaker: the choroid plexus and circumventricular organs, such as the median eminence, the organum vasculosum lamina terminalis,the subfornical organ,and the area postrema. In these areas,the junctions between the endothelial cells lining blood vessels are less tight and allow for diffusion of cytokines (Konsman et al.,1999; Dantzer et al.,2000). Moreover,inﬂammatory proteins in plasma, such as monocyte chemoattractive protein-1 (MCP- 1), can weaken the blood brain barrier (Stamatovic et al., 2005). Finally, there are active transport molecules on endothelial cells that allow for cytokines to be transported across the blood brain barrier (Dantzer et al., 2007; Shelton and Miller, 2010). Once in the CNS, the cytokines activate the microglia. When microglia get activated, they release MCP-1 which will bring other white blood cells to the brain (Shelton and Miller, 2010). INVESTIGATING CAUSAL CONNECTIONS Relatively early in these investigations, Maier and Watkins (1998) advanced the hypothesis that undergoing psychological stress August 2012 \| Volume 3 \| Article 297 \| 1 www.frontiersin.org Littrell Inﬂammation (Maier and Watkins, 1995; Johnson et al., 2004; Koo and Duman, 2008; Arakawa et al., 2009). Thus, IL-1 in the hippocampus seems to play a causal role in mediating behavioral manifestations of depression. (Maier and Watkins, 1995; Johnson et al., 2004; Koo and Duman, 2008; Arakawa et al., 2009). Thus, IL-1 in the hippocampus seems to play a causal role in mediating behavioral manifestations of depression. In addition to the animal work, similar work in humans is available. Several of these studies observed random-assignment to either peripheral induction of systemic inﬂammation or to a control condition. Those who received endotoxin administration displayed impairment in memory which correlated with inﬂam- matory cytokine levels (Reichenberg et al., 2001; Krabbe et al., 2005) and an elevation in negative mood which correlated with cytokine levels (Reichenberg et al., 2001). Vaccinating people with typhoid proteins is another reliable way to elevate inﬂammatory cytokines. Vaccinated individuals displayed elevations in negative mood which correlate with IL-6 levels (Wright et al., 2005). Another way to make an animal exhibit signs of depression is to expose the animal to repeated episodes of social defeat. After exposure to rounds of social defeat the animal avoids other ani- mals, displays reduced preference for sucrose, and appears more depressed on tests such as the forced swim test and the tail sus- pension test. Concomitant with behavioral signs of depression, animals display elevation in inﬂammatory cytokines in plasma (Merlot et al., 2004; Kinsey et al., 2007, 2008; Avitsur et al., 2009). ( , ; y , , ; , ) In addition to the work with animals showing that stressing the animal will induce signs of systemic inﬂammation, there is also experimental work in humans. Persons who are subjected to laboratory stressors exhibit activation of NF-κB (Bierhaus et al., 2003) as well as an increase in inﬂammatory cytokines such as IL-6 in plasma (Pace et al., 2006). In those individuals who are low on chronic stress, increases in NF-κB activity are correlated with perceived intensity of the laboratory stressor (Wolf et al., 2009). A study by Slavich et al. (2010) examined how a social stressors inducing elevations in inﬂammatory cytokines changes brain function. INVESTIGATING CAUSAL CONNECTIONS Following the social stressor, those with elevations in the soluble receptor for TNF-α exhibited greater activation in the dorsal anterior cingulate and the anterior insula, two areas involved in processing negative affect. Manipulating peripheral inﬂammation Studies established that stressing the animal in various ways results in behavioral manifestations of depression along with elevations in inﬂammatory cytokines in brain and in plasma. The next question was whether establishing systemic inﬂammation in the periphery would result in behavioral manifestations of depression. Placing lipopolysaccharide (LPS) molecules in the paw of a mouse is a reli- able way to elevate cytokines in the blood stream without inducing real tissue damage to the organism from the bacteria. Follow- ing elevations in cytokines in the blood stream, the mice exhibit a lack of preference for formerly preferred foods, low levels of activity, more social avoidance, less exploration of novel environ- ments, an increase in body temperature, and an increase in the production of cortisol (Dantzer, 2001; Nadjar et al., 2005). Con- comitant with the behavioral change, the following are observed: an increase in the threshold for self-stimulation in the medial forebrain bundle (Borowski et al., 1998), a diminution in activ- ity in brain areas associated with positively motivated behavior (Stone et al., 2007), an increase in activation as detected by eleva- tions in FosB/∆FosB in areas of brain associated with depression (viz., parts of the shell of the Nucleus Accumbens, bed nucleus of the stria terminalis (BNST), and the central nucleus of the amygdala; Frenois et al., 2007; Engler et al., 2011). Moreover, IL-1 levels in brain (the hippocampus and hypothalamus) are ele- vated. Again, when IL-1 antagonists are placed into the brain, the depressed behaviors disappear, although it is noted that timing for the administration of the IL-1 receptor antagonist (IL-1ra) and placement of the IL-1ra can alter the parameters of the response to LPS that are abrogated (Bluthe et al., 1995; Konsman et al., 2008). Forces that can restrain systemic inﬂammation h b d d h h f l y The body does have mechanisms for curtailing an inﬂammatory response following clearance of a pathogen. Following activation in response to a pathogen, T regulatory cells (Tregs) are also differentiated. Tregs are identiﬁed by their surface expression of CD25 and the expression of the transcription factor: FoxP3. Tregs release IL-10 and transforming growth factor-β (TGF-β), which are anti-inﬂammatory cytokines (Murphy, 2012). Data supporting the hypothesis that stress will increase the level of inﬂammatory cytokines has been presented. It is also the case that a laboratory stressor will decrease the level of IL-10 (Buske- Kirschbaum et al., 2007) as well as the number of circulating Tregs (Freier et al., 2010). Lower levels of Tregs are also observed in those who are chronically stressed as well. Stress will decrease the number of circulating Tregs; will downregulate FoxP3, the tran- scription factor for Treg differentiation; and decrease plasma levels of IL-10 and TGF-β (Raison et al., 2010c). August 2012 \| Volume 3 \| Article 297 \| 2 Frontiers in Psychology \| Psychology for Clinical Settings Littrell Inﬂammation A protective role for IL-10 in preventing distress has been estab- lished. Administration of IL-10 along with administration of LPS prevents the emergence of behavioral signs of depression (Bluthe et al.,1999). Mice have been genetically engineered to over-express IL-10 display less anxiety, while IL-10 knock outs display greater levels of anxiety and depressive behavior on a forced swim test, although the impact of IL-10 is more pronounced in females (Mesquita et al., 2008). In the human literature, under stressful conditions, those persons with higher levels of IL-10 report less anxiety (Maes et al., 1998). Following vaccination with an inﬂam- matory antigen,optimistic persons maintain higher levels of IL-10 (Costanzo et al., 2004). noted changes in dopaminergic structures. LPS administration in the periphery results in less activity in the ventral striatal area (the brain’s reward area) in response to monetary reward. GENETIC RISK Vulnerability to depression under stressful conditions is conferred by various alleles associated with proteins of the immune sys- tem. The short promoter in the gene for the serotonin transporter has received a great deal of attention as a risk factor for depres- sion (Lasky-Su et al., 2005). Interestingly, those carrying the short promoter version of the serotonin transporter release more IL-6 relative to IL-10 given exposure to a laboratory stressor (Fredericks et al., 2010). The less frequently expressed alleles for the serotonin transporter are at increased risk for exhibiting depression given exposure to IFN-α (Bull et al., 2009; Lotrich et al., 2009), although the serotonin transporter gene interacted with alleles for IL-6 only making a difference in those who carried the allele for the less easily expressed IL-6 gene in the Bull et al. study. Particular alleles for TNF-α and IL-1 are also associated with risk for depression or severity of symptoms (Jun et al., 2003; Yu et al., 2003; Raison et al., 2006; Traks et al., 2008). Thus, in looking for genetic vulnerability to major depression, proteins of the immune system may be as or more predictive than proteins associated with neurotransmitter function. p ( p , ) Some of the human research has examined how systemic inﬂammation alters brain function as observed through functional magnetic resonance imaging. Administration of IFN-α induces activation of the dorsal anterior cingulate-error detection area (Capuron et al., 2005). Inoculation with typhoid proteins, which increases systemic levels of IL-6, similarly enhances activity of the insula and right anterior cingulate which correlate with subjective fatigue and confusion (Harrison et al., 2009). Moreover, inoc- ulation with typhoid proteins results in diminished control of the subgenual anterior cingulate cortex by regulatory centers for reward, emotion, and social processing, a phenomenon which is correlated with levels of IL-6 (Harrison et al., 2009). In addition to inducing changes in areas associated with error processing such as the anterior cingulate cortex, systemic inﬂammation is correlated with changes in dopaminergic system, the system which under- girds motoric activity and motivation. Following typhoid protein inoculation, levels of IL-6 correlate with altered activity in the substantia nigra and behavioral measures of slower reaction times (Brydon et al., 2008). A study by Eisenberger et al. (2011) also INFLAMMATORY CYTOKINES MAY EXPLAIN DEXAMETHASONE NON-SUPPRESSION Another consistent ﬁnding in the literature is that the level of cor- tisol hormone is often elevated in depressed individuals, although 30–50% of depressed individuals may not display elevations in cortisol (Muller and Holsboer,2006; Pace and Miller,2009). Corti- cotropin releasing hormone (CRH) from the parvocelluar neurons in the paraventricular nucleus of the hypothalamus is the proxi- mal cause of elevations in cortisol.With regard to regulation of this system,given normal functioning,cortisol will act on receptors for cortisol in the hippocampus. The hippocampus, through connec- tions in the BNST,will then communicate with the paraventricular nucleus of the hypothalamus to inhibit the CRH release. Moreover, the BNST is probably more important in regulating CRH release in situations of chronic stress (Sapolsky,1985; Ulrich-Lai and Her- man, 2009). This is the negative-feedback system for dampening a response. But in depressed individuals, this inhibitory system does not work well. The problem with CRH not being suppressible in depressed individuals may be related to inﬂammatory states. It has been demonstrated in ﬁbroblast cells that inﬂammatory cytokines will interfere with glucocorticoid receptors function, although possible variation in cell types is acknowledged (Pariante et al., 1999). Thus, inﬂammation may be the mechanism creating resistance to control of the cortisol system in depressed individuals (Pariante et al.,1999; Pace et al.,2007). Not only will inﬂammatory cytokines interfere with regulation of cortisol release, but inﬂam- matory cytokines are also associated with less circadian variation in cortisol release (Raison et al., 2010a). Others have attempted to increase T regulatory cells by vacci- nating with antigens known to elicit a Tregs response. Vaccination with a heat killed preparation of Mycobacterium vaccae will evoke a Treg response (Rook and Lowry, 2008). Those who were vacci- nated with M. vaccae report better quality of life (Dalbeth et al., 2004; O’Brien et al., 2004). OTHER CONSISTENT FINDINGS Beyond the random-assignment controlled research, there are other studies consistent with the hypothesis that systemic inﬂam- mation is associated with depressive behaviors. Particular dis- eases (metastatic melanoma and hepatitis virus C) are treated with Interferon-α (IFN-α). IFN-α will induce systemic inﬂam- mation. A signiﬁcant number of persons (30–50%) undergoing such treatment subsequently develop clinical depression (Raison et al., 2009). In fact, in those being treated with IFN-α, feelings of guilt and suicidal ideation are increased, not just malaise and fatigue (Capuron et al., 2009). In addition to the data on behavioral impact of IFN-α, several conditions are associated with a rise in inﬂammatory cytokines. Both obesity and sleep deprivation are associated with elevations in blood levels of inﬂammatory cytokines (IL-1β, TNF-α). Both these conditions are risk factors for depression (Irwin et al., 2006; SheltonandMiller,2011).Metabolicsyndrome,astateof increased weight around the waist, is associated with high levels of IL-6 and CRP and is also a risk factor for depression (Capuron et al., 2008). QUESTIONS REMAIN Questions about the hypothesis that inﬂammation has a critical role in depression have been raised. The symptoms induced by August 2012 \| Volume 3 \| Article 297 \| 3 www.frontiersin.org Littrell Inﬂammation cytokines versus a psychological stressor may only partially over- lap. Clinically, Capuron et al. (2009) compared the phenomenon emerging in those who are treated with IFN-α with the symptoma- tology in those who are depressed for other reasons. Psychomotor retardation and weight loss predominated in the former whereas guilt was more predominant in the latter. In those treated with IFN-α,sickness behavior emerged immediately whereas depressive symptoms emerged a month after treatment initiation (Capuron and Miller,2011). In terms of malleability,some interventions may differentially affect sickness behaviors versus depressive behaviors. For example, O’Connor et al. (2009a) administered an inhibitor of indolamine 2,3 dioxygenase (IDO to be discussed later) along with inﬂammatory cytokines. The IDO inhibitor precluded depressive behaviors (more immobility on a forced swim test and tail suspen- sion test) induced by cytokines while failing to affect locomotor activity, weight change, or fever. decreasestheadaptiveimmunesystemaswellasdecreasingnatural killer cell activity. In contrast to the adaptive system, stress acti- vates the innate system, i.e., monocytes and macrophages which release inﬂammatory cytokines (Maier andWatkins,1998; Zorrilla et al., 2001; Raison et al., 2006). Blume et al. (2011) observed that few studies have measured both immune system activation (as evidenced by inﬂammatory cytokines) and immune system suppression (less proliferation of T and B cells,attenuated Natural Killer Cell Cytotoxicity,lower levels of wound healing) in the same study. However, there is evidence that chronic activity of the innate immune system is associated with less robust response from cells of the adaptive immune system (Maier and Watkins, 1998; Blume et al., 2011). Indeed, exposure of T cells to TNF-α, an inﬂammatory cytokine, will decrease T cell proliferation and production of cytokines (Lee et al., 2008). One of the early investigations in the literature on psychoneu- roimmunology addressed the question of whether persons under- going severe stress are more likely to “catch a cold.” The studies showed that persons undergoing major stressors produced more mucous and exhibited more symptoms after inoculation with a ﬂu virus (Cohen, 2005). However, the question of whether more symptoms are reﬂective of greater activation or lesser activation of the immune system is complicated. Cold symptoms (mucous pro- duction,inﬂamed nasal passages) are phenomena reﬂecting innate immune system activation. A recent study by Huang et al. QUESTIONS REMAIN However, with rega m, there was suggestion of a stro ose who remained asymptomat monstrated that activation of t ompany a less vigorous respon E IMPORTANCE OF CYTOKINE HAVIORAL CHANGES searchers have abrogated the be S in the periphery or learned he antagonist to IL-1β into brain ( n et al.,2004;Koo and Duman,2 dings imply that IL-1β, an in anges which eventually result i pression. Other researchers hav anges associated with an increas re proximal factors in driving t d b h i Di i f STRESS AND SUPPRESSION OF SOME COMPARTMENTS OF THE IMMUNE SYSTEM The ﬁnding that depressed behaviors are associated with indi- cators of systemic inﬂammation was surprising. During the late 1980s and 1990s,the ﬁeld of psychoneuroimmunology was rapidly developing. Animal work and human studies showed that stress depresses many compartments of immune system function. Sem- inal observations included the following: Epstein–Barr virus is morelikelytobereactivatedinmedicalstudentsduringﬁnalexams (Glaser et al., 1994). Wound healing is slower in dental students during ﬁnal exams and in Alzheimer patient caregivers (Kiecolt- Glaser et al., 1995; Marucha et al., 1998). Stressed individuals, such as caregivers of Alzheimer Disease patients, fail to develop a robust antibody response to vaccination (Kiecolt-Glaser et al., 1996; Glaser et al., 2000). Later, the ﬁndings that systemic inﬂam- mation characterized depressed and stressed individuals emerged. The idea that the body’s response to stress involved an immune activation as well as suppression of some compartments of the immune system seemed contradictory. Frontiers in Psychology \| Psychology for Clinical Settings QUESTIONS REMAIN (2011) provided further clariﬁcation on which arms of the immune sys- tem are activated or suppressed in those manifesting symptoms in response to a cold virus. Huang et al., as in the earlier Cohen stud- ies, inoculated research-participants with live inﬂuenza and then tracked research-participant responses over time. Those remain- ing asymptomatic had downregulated the NF-κB pathway of the innate system. However, with regard to the adaptive immune sys- tem, there was suggestion of a strong Th1 response to the virus in those who remained asymptomatic. The Huang et al. study thus demonstrated that activation of the innate immune system can accompany a less vigorous response from the adaptive system. y g munology addressed the question severe stress are more likely to ed that persons undergoing maj us and exhibited more sympto rus (Cohen, 2005). However, th toms are reﬂective of greater acti mmune system is complicated. Co on,inﬂamed nasal passages) are p une system activation. A recent s ded further clariﬁcation on whic re activated or suppressed in tho nse to a cold virus. Huang et al., oculated research-participants w ed research-participant response ymptomatic had downregulated e system. However, with regard t here was suggestion of a strong who remained asymptomatic. T nstrated that activation of the mpany a less vigorous response fr MPORTANCE OF CYTOKINES I AVIORAL CHANGES rchers have abrogated the behav n the periphery or learned helple tagonist to IL-1β into brain (Mai al.,2004;Koo and Duman,2008 ngs imply that IL-1β, an inﬂam ges which eventually result in b ssion. Other researchers have in Subtle variations in the response to psychological stressors versus systemic inﬂammation induced by infection are possible. Immunological symptoms emerge in stages after exposure to a stressor (Maier and Watkins, 2010). The response to cytokines can vary as a function of dosage and will also emerge in stages (Dantzer, 2004; Capuron and Miller, 2011). Moreover, there may be subtypes of depression in which inﬂammation plays a lesser role. However, the overlap between response to a psychological stressor and response to a pathogen is well established. ucous and exhibited more sym virus (Cohen, 2005). However mptoms are reﬂective of greater immune system is complicated ction,inﬂamed nasal passages) a mune system activation. A rece ovided further clariﬁcation on w m are activated or suppressed in ponse to a cold virus. Huang et inoculated research-participan cked research-participant respo asymptomatic had downregul nate system. THE INFLAMMATORY PATHWAY Microglia are resident white blood cells in brain. They are essen- tially the brain’s resident macrophages, which can engulf and destroy pathogens. When microglia detect LPS or the animal is stressed, the microglia become activated and can release the inﬂammatory cytokines:IL-1,TNF-α,and IL-6 (Monje et al.,2003; Perry et al., 2003; Blandino et al., 2006; Schwartz and Ziv, 2008b; Ekdahl et al., 2009; Molina-Holgado and Molina-Holgado, 2010; Tynan et al., 2010). This is effectively an inﬂammatory pathway. Blocking microglia’s inﬂammatory response subsequent to LPS administration will abrogate the emergence of depressed behav- iors.Henryetal.(2008)showedthatif adrug(minocycline),which prevents activation of microglia, is given prior to LPS administra- tion, then depressive behavior (decreased preference for sucrose) is precluded. Moreover, in the Henry et al. study, minocycline pre- vented the rise in inﬂammatory cytokines (IL-1β and IL-6) in the hippocampus as well as attenuating the increased expression of IDO (which is discussed the next section). Another hypothesis links IDO activity to lower levels of sero- tonin. Given IDO activity, tryptophan, the precursor to serotonin gets depleted in plasma. Low serotonin has been suggested to play a role in causing depressed behavior (Gal and Sherman, 1980; Miller, 2009; O’Connor et al., 2009a). Against the hypothesis that IDO inﬂuences depressive behaviors by lowering tryptophan levels are the ﬁndings that tryptophan depletion does not reli- ably induce depression in all people, although those at risk for depression may be more susceptible (Bell et al., 2001). More- over, as previously mentioned, kynurenine alone, without any reduction in tryptophan, can induce depression (O’Connor et al., 2009b). Another puzzling ﬁnding is that activation of the IDO enzyme in macrophages in the periphery induces differentiation of T reg- ulatory cells (Henry et al., 2008; Johnson et al., 2009), which as discussed previously, are believed to counter depressive behav- iors. At this point, the role of IDO in producing depression given systemic inﬂammation awaits clariﬁcation. Subsequent research has offered clues into phenomenon in brain that occur in response to inﬂammatory cytokines such as IL-1β that might constitute more proximal causes for changes in behavior. Koo and Duman (2008) showed that pretreatment with an IL-1β inhibitor will block the emergence of depressive behav- iors and prevent the decrement in neurogenesis in response to stress. (The signiﬁcance of decrements in neurogenesis in medi- ating depressive behaviors will be discussed in a later section.) Downstream of IL-1β is activation of the transcription factor, NF- κB. THE IMPORTANCE OF CYTOKINES IN BRAIN MEDIATING BEHAVIORAL CHANGES Researchers have abrogated the behavioral changes in response to LPS in the periphery or learned helplessness induction by placing an antagonist to IL-1β into brain (Maier and Watkins, 1995; John- son et al.,2004;Koo and Duman,2008;Arakawa et al.,2009). These ﬁndings imply that IL-1β, an inﬂammatory cytokine, induces changes which eventually result in behavioral manifestations of depression. Other researchers have investigated the downstream changes associated with an increase in IL-1β that might operate as more proximal factors in driving the behavioral manifestations of depressed behavior. Discussion of downstream events subsequent to an increase in IL-1βilluminates the signiﬁcance of brain derived neurotrophic factor (BDNF), a protein which has been featured in explanations of the mechanisms behind the efﬁcacy of antidepres- sants, as well as the signiﬁcance of IDO, which has been purported to link to the serotonergic system. The immune system is complex and capable of making many types of responses. A big division of the compartments of the immune system is innate versus adaptive immunity. Innate immu- nityincludesthoseleukocytesthatrespondpromiscuously,activat- ing in response to many pathogens and other foreign molecules. Many of the inﬂammatory cytokines are products of the innate system. In contrast, the adaptive immune system refers to those leukocytes (T and B lymphocytes) that respond to only one type of molecule and whose responding takes a period of days to acti- vate. Adaptive immunity is evoked with vaccination. A consensus is beginning to emerge regarding the differential impact of stress on the innate versus the adaptive immune systems. Stress reliably Immunologists view inﬂammation as a mechanism for ﬁght- ing pathogens. Most immunology texts view the primary job of leukocytes as providing protection against pathogens. However, a Frontiers in Psychology \| Psychology for Clinical Settings August 2012 \| Volume 3 \| Article 297 \| 4 \| 4 Littrell Inﬂammation new role for cells of the immune system has been posited by Ziv and Schwartz who have shown that activated leukocytes play a vital role in maintaining the health of the brain (Schwartz and Ziv, 2008b). The “type of immune activation critically determines the outcome” (Schwartz and Shechter, 2010a, p. 343). The emerging view is that white blood cells, if activated along non-inﬂammatory pathways, can contribute to brain health and result in behavioral resilience in the face of external stressors. Growth factors, such as BDNF, are featured in the health-promoting activation research as well. THE IMPORTANCE OF CYTOKINES IN BRAIN MEDIATING BEHAVIORAL CHANGES In the literature on brain health, there has also been a focus on systemic immunological factors that inﬂuence the behavior of brain leukocytes. After examining speciﬁcs regarding the state of brain inﬂammation, a discussion of how the immune system can support brain health and behavioral resilience is presented. Some have suggested a causal role for the IDO enzyme in the depressive behaviors associated with inﬂammation (Miller, 2009; Raison et al., 2010b). The strongest evidence for IDO playing a causal role in LPS or bacille Calmette–Guréin induced depres- sion is the ﬁnding that inﬂammation fails to induce depressed behaviors in mice lacking the indolamine 2,3 dixoxygenase gene or when an inhibitor of the enzyme is given (O’Connor et al., 2009a,b; Raison et al., 2010b). Interestingly, inhibiting the IDO did not decrease the rise in inﬂammatory cytokines in response to vaccination (O’Connor et al., 2009a). The product of IDO is kynurenine. Systemic administration of kynurenine will induce depression (O’Connor et al., 2009b) and kynurenine can pass through blood brain barrier. In terms of potential ways that kynurenine might impact neurotransmission,kynurenine does get converted to quinolinic acid and kynurenic acid,both of which can affect neurotransmitters signaling such as glutamate signaling and dopamine signaling (Amori et al.,2009;Miller,2009;Sublette et al., 2011). THE INFLAMMATORY PATHWAY The changes in behavior induced by IL-1β can be abrogated by blocking the activity of the transcription factor NF-κB, whose activity is increased by IL-1β (Nadjar et al., 2005; Koo et al., 2010). NF-κB serves to further enhance inﬂammation. The signiﬁcance of neurogenesis and BDNF In the literature on neuroprotection, the state of the microglia and whether microglia foster or dampen the release of BDNF has received a great deal of attention. In fact, administration of IL-1β, an inﬂammatory cytokine, into the hippocampus will decrease the expression of BDNF (Barrientos et al., 2004). The role of BDNF and neurogenesis in depression has also been a focus of attention in the animal literature on depression. The hypotheses that loss of BDNF and downstream diminution of brain growth manifests as depression and conversely that increasing BDNF prevents depres- sion have been investigated. The ﬁndings suggest a more complex reality than represented in the initial hypotheses. The indolamine 2,3 dioxygenase connection Depressed animals and animals subjected to stress do exhibit low levels of BDNF in the hippocampus. Animals subjected to stress also exhibit less neurogenesis in the dentate gyrus of the hippocampus (Lucassen et al., 2011). BDNF induces the growth of dendrites from neurons so that they can make new connec- tions with other neurons (Lucassen et al., 2011). While BDNF is not necessary for neurogenesis, BDNF increases the survival of new cells (Sairanen et al., 2005). Consistent with the possibility Indolamnine 2,3 dioxygenase has been a focus of much discussion in the literature on inﬂammation mediating depressive behavior. This enzyme is present in macrophages, as well as other cell types, and operates when a macrophage is activated. It is also active in brain under conditions of chronic inﬂammation. IDO con- verts tryptophan to kynurenine and thereby depletes the local environment of tryptophan. Tryptophan is the substrate for the production of serotonin (Miller, 2009). August 2012 \| Volume 3 \| Article 297 \| 5 www.frontiersin.org Littrell Inﬂammation that BDNF is diminished in the hippocampus of depressed indi- viduals, depressed individuals have lower hippocampal volume (Bremner et al., 2000; Videbech and Ravnkilde, 2004). Moreover, placing BDNF into the hippocampus (Shirayama et al., 2002), or intracerebroventricular administration of IGF-1, which increases BDNF levels (Carro et al., 2000; Cotman and Berchtold, 2002), exert antidepressant effects in animals (Hoshaw et al., 2005). that BDNF is diminished in the hippocampus of depressed indi- viduals, depressed individuals have lower hippocampal volume (Bremner et al., 2000; Videbech and Ravnkilde, 2004). Moreover, placing BDNF into the hippocampus (Shirayama et al., 2002), or intracerebroventricular administration of IGF-1, which increases BDNF levels (Carro et al., 2000; Cotman and Berchtold, 2002), exert antidepressant effects in animals (Hoshaw et al., 2005). At least in some species, antidepressant drug efﬁcacy seems to require growth factors and the capacity for neurogenesis. The previous ﬁndings raise the possibility that a lack of BDNF or a lack of neurogenesis in the hippocampus might cause depression. Researchers have investigated this hypothesis. To investigate these questions researchers have used conditional knock outs. Using a conditional knock outs, researchers can control the timing for the loss of the capacity to express a particular protein. The indolamine 2,3 dioxygenase connection Irradiating the hippocampus to block neurogenesis does not increase the animals level of depression as evidence by longer latency to feed in a novel situation or immobil- ity on a forced swim test (Santarelli et al., 2003; Airan et al., 2007), nor does blocking neurogenesis in the hippocampus increase the animal’s level of distress as evidenced by failure to groom after uncontrollable stress (Santarelli et al., 2003). Others have looked at whether growth factors are required for antidepressant efﬁcacy. Monteggia et al. (2004) examined the efﬁcacy of antidepressants in blocking the emergence of depressed behavior after exposure to stress. Without an intact gene for expressing BDNF, antidepressants exhibited no efﬁcacy in abrogating the impact of chronic stress (Monteggia et al., 2004). While impaired capacity of cell proliferation in the hippocam- pus seems to be ruled out as a cause of depression at least under baseline conditions, problems with cell division elsewhere in the brain might play a causal role in producing behavioral depression. Pharmacological destruction of astrocytes cells in the prefrontal cortex (PFC) will induce depressed behavior in an animal. A simi- lar reduction of astrocytes in the PFC occurs in animals exposed to uncontrollable stress. Astrocyte cell death and behavioral signs of depression co-occur whether the astrocyte cell death results from exposure to uncontrollable stress or to a drug which is speciﬁ- cally toxic to astrocyte cells (Banasr and Duman, 2007). Astrocytes do inﬂuence neurons. They are a major source of neurotrophins in brain which seems to be induced by dopamine (Miklic et al., 2004). Moreover, the loss of glial cells (both oligodendrocytes and astrocytes) in the subgenual region of the anterior cingulate, the dorsolateral PFC, the supragenual cingulate cortex, and the orbitofrontal cortex, has emerged as marker for major depression (Ongur et al., 1998; Rajkowska and Miguel-Hidalgo, 2007). Nestler’s group has identiﬁed the mechanism through which stress alters the expression of BDNF and the mechanism through which antidepressants increase the expression of BDNF. When an animal is subjected to a stressor such as uncontrollable foot- shock or restraint stress, the BDNF gene is harder to express. Two methyl groups are added to the histone 3 at lysine 27 in the pro- moter region of the gene for BDNF making it difﬁcult to express the BDNF. Antidepressants do not reverse the effects of chronic stress on the BDNF gene; rather, they affect BDNF expression through another mechanism. The indolamine 2,3 dioxygenase connection Through the use of conditional knock outs,researchers know they are looking at the impact of the loss of functional presence of the protein product of the gene rather than the impact of the loss of the protein during development. Antidepressant drugs do increase neurogenesis in the dentate gyrus of the hippocampus (Santarelli et al., 2003), although this may be limited to adult males (Hodes et al., 2009). Research ask- ing whether the efﬁcacy of antidepressant drugs requires an animal with the capacity for generating new neurons in the dentate gyrus of the hippocampus has yielded inconsistent ﬁndings. Irradiation of the subgenual zone of the dentate nucleus of the hippocampus, blocked the efﬁcacy of antidepressants in precluding the behav- ioral effects of stress in one rat strain (Santarelli et al., 2003). Consistent with this, Airan et al. (2007) found the irradiation of the dentate gyrus precluded the efﬁcacy of antidepressants both on behavioral measures of depression and in increasing neuronal signaling output from the dentate gyrus. However, subsequent work in another rat species did not ﬁnd that irradiation abro- gated the efﬁcacy of the antidepressant ﬂuoxetine in exerting an antidepressant effect nor did those animals displaying lower lev- els of depressed behavior exhibit increased neurogenesis in the dentate gyrus (Holick et al., 2008). Moreover, some drugs (for example antagonists to melanin concentrating hormone) decrease depressed behavior without inﬂuencing neurogenesis (Sahay and Hen, 2007). Monteggia et al. (2007) created a conditional knock out mouse for BDNF. Male BDNF knock outs were more hyperactive. Female BDNF knockouts displayed deﬁcits on a forced swim task consid- ered to be a measure of depression and deﬁcit sucrose preference, a measure of anhedonia. Thus, in the Monteggia et al. study, loss of BDNF in increasing depressive behavior is dependent upon gender. Consistent with Monteggia et al.’s male knockouts, Ito et al. (2011) found that their conditional knockout of BDNF in the hippocampus, displayed increased levels of aggression toward conspeciﬁcs and did not differ from controls on the forced swim test, a commonly used measure of depression. Thus, the condi- tional knock out studies fail to support the hypothesis that loss of BDNF in male animals results in behavioral manifestations of depression. Other researchers have investigated whether blocking neuro- genesis induces depression. THE HEALTH-PROMOTING PATHWAY As previously discussed, when microglia are activated along an inﬂammatory pathway,depression is observed. In addition to acti- vation along an inﬂammatory pathway, microglia can also become activated along several other pathways as well (Michelucci et al., 2009; Derecki et al., 2011). Microglia can assume a form of activa- tion through which they maintain the health of neurons (Butovsky et al., 2006; Schwartz and Ziv, 2008b; Ekdahl et al., 2009; Kosloski et al., 2010). When in the growth promoting mode, microglia release Insulin-like growth factor-1 (IGF-1; Butovsky et al., 2005; Ekdahl et al., 2009; Kosloski et al., 2010; Schwartz and Shechter, 2010a; Ron-Harel et al., 2011). IGF-1 slows inﬂammation and decreasesproductionof inﬂammatorycytokines(PonsandTorres- Aleman, 2000; Venters et al., 2001; Park et al., 2011). In addition to being anti-inﬂammatory, IGF-1 will induce neurons to release BDNF (Carro et al., 2000; Cotman and Berchtold, 2002). Support for the importance of T cells in maintaining brain health comes from studies in which all T cells or T cells with TCRs speciﬁc for myelin basic protein have been eliminated. Mice that are genetically engineered to lack T cells display severe inability to learn. Replenishing T cells from normal mice into mice which lack T cells can restore the ability to learn and reverse memory deﬁcits (Yirmiya and Goshen, 2011). The importance of T cells has also been demonstrated for the capacity for resilience in the face of stress. Given a stressor such as a predator odor, the expression of ICAM-1 on endothelial cells lining blood vessels in the hippocam- pus and epithelial cells in the choroid plexus increases to facilitate and guide the migration of T cells, speciﬁc for brain proteins, into the brain (Lewitus et al., 2008). When this fails to occur because T cells speciﬁc for brain protein have been depleted, more behav- ioral signs of distress are observed subsequent to stressor exposure (Cohen et al., 2006). Activation of microglia along the inﬂammatory pathway versus the growth promoting pathway are mutually inhibitory. TNF-α,an inﬂammatory cytokine, is inversely correlated the release of IGF- 1 from microglia (Butovsky et al., 2006; Schwartz and Shechter, 2010b).IGF-1candecreaseNF-κBtranscriptionalactivityinastro- cytes and in TNF-α exposed cells (Pons and Torres-Aleman,2000). If exogenous IGF-1 is injected into the lateral ventricle along with LPS, then there is less transcription of the genes associated with an array of inﬂammatory factors including TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS; Park et al., 2011). The interaction between T cells and the microglia The interaction between T cells and the microglia Schwartz and Ziv have focused on the role that T cells play in deter- mining the behavior of microglia. Schwartz and Ziv (2008a) have proposed that self-reactive T cells with receptors (TCRs) targeted toward central nervous system (CNS) proteins are continually playing a surveillance role. When activated in pro-neurogenesis pathways, they maintain the health of the CNS. T cells, proba- bly by inﬂuencing microglia, are vital to the brain’s repair after stroke, for precluding a reduction in levels of BDNF following a psychological stressor, for avoiding symptoms of post traumatic stress disorder (PTSD) after psychological stress, for spatial learn- ing, for neurogenesis stimulated by exercise (Cohen et al., 2006; Ziv et al., 2006; Ziv and Schwartz, 2008a,b; Schwartz and Shechter, 2010a) and for capacity to detoxify reactive alpha-oxoaldehydes (Ron-Harel et al., 2011). T cells are recruited subsequent to activ- ity and/or stress (Schwartz et al., 2009) to the choroid plexus, the meninges, and the Virchow–Robin spaces from which they are able to inﬂuence the function of the microglia (Schwartz and Ziv, 2008b; Derecki et al., 2010; Schwartz and Shechter, 2010a,b). The presence of T cells is required for the efﬁcacy of environmental manipulations, such as an enriched environment, to increase the number of microglia releasing IGF-1 in the hippocampus (Ziv et al., 2006; Ziv and Schwartz, 2008a,b; Schwartz and Shechter, 2010a,b). T cells must also be present to witness an increase in IGF-1 in brain given physical activity (Schwartz and Shechter, 2010a). Summary Thus far, it has been argued that inﬂammation in the brain is sufﬁcient to create behavioral depression. Investigators have attempted to identify causal mechanisms through which inﬂam- matory cytokines in brain result in depressed behavior. Dimin- ished neurogenesis and/or reductions in growth factors such as BDNF were raised as possible mediating factors of inﬂamma- tory cytokines. Inﬂammation in brain does suppress neurogenesis and the expression of BDNF in the hippocampus. However, given that blocking neurogenesis in the hippocampus or preventing the expression of BDNF fails to create depression, suggests that lack of capacity for neurogenesis in the hippocampus is not sufﬁcient to cause depressed behavior. However, the possibility that inﬂam- mation creates depression by blocking cell division in astrocytes in the PFC remains a possibility. The indolamine 2,3 dioxygenase connection Nestler has shown that antidepres- sants decrease the expression of histone deacetylase 5 (HDAC5) in the hippocampus in stressed mice. Histone deacetylases remove acetyl groups from promoter sites so that a gene is less likely to be used to transcribe mRNA for producing a protein (Tsankova et al., 2006; Krishnan and Nestler, 2008). With a lesser amount of a particular HDAC5 present in the hippocampus, the expression of BDNF is increased (Tsankova et al., 2006). However, the story on HDAC5 is complex. Mice with global reductions in HDAC5 are more vulnerable to social defeat and the impact of antidepres- sants on HDAC5 levels is region speciﬁc. Contrary to decreasing HDAC5 expression in the hippocampus, imipramine will increase HDAC5 expression in the Nucleus Accumbens (Tsankova et al., 2006; Krishnan and Nestler, 2008). Location of BDNF expression determines the impact on behavior Placing BDNF into the hippocampus of an animal will exert an antidepressant effect on a forced swim test and on escape deﬁcit following exposure to inescapable shock (Shirayama et al., 2002). Frontiers in Psychology \| Psychology for Clinical Settings August 2012 \| Volume 3 \| Article 297 \| 6 Littrell Inﬂammation However, an opposite pattern is found in the Nucleus Accumbens. High levels of expression of BDNF in the Nucleus Accumbens released from neurons projecting from theVentral Tegmental Area will result in depressed behavior (Eisch et al., 2003). In terms of the function of BDNF in the NucleusAccumbens,Nestler and Car- lezon (2006) postulate that expression of BDNF in the Nucleus Accumbens is required for learning connections between exter- nal stimuli and negative or positive valences. Thus, the impact of BDNF on ameliorating depressive behaviors is brain area speciﬁc. Keeping microglia in a health-promoting state is important. Neurons and astrocytes release factors such as fractalkine and Jagged 1 to keep microglia in a quiescent state (Sestan et al., 1999; Elyaman et al., 2007; Michelucci et al., 2009) and fractalkine will downregulate microglia production of IL-1β in response to LPS (Mizuno et al., 2003; Wynne et al., 2010). However, researchers have also focused on the role of T cells in maintain brain health. The hygiene hypothesis The hygiene hypothesis attempts to explain why persons in the third world have much lower rates of disorders associated with inﬂammatory conditions: Parkinson’s disease, depression, anxiety disorders, asthma, and inﬂammatory bowel disease. According to the hygiene hypothesis, persons in the third world are exposed to more pathogens, such as helminthes (parasitic worms) and pathogens found in human feces, such as M. vaccae. These par- ticular pathogens are good at eliciting the differentiation of T cells into Tregs (Rook and Lowry, 2008, 2009; Raison et al., 2010c). (As previously discussed, Tregs will dampen inﬂamma- tion.) Downregulating inﬂammation systemically, will preclude inﬂammatory cytokines in brain, thereby decreasing depression and anxiety. There are other studies that suggest a salubrious role for T cells in promoting brain health. Reynolds et al. (2007, 2009a,b) have shown that co-culturing microglia with Tregs will attenu- ate reactive oxidative species (ROS) and NF-κB transcriptional activity subsequent to antigen exposure. Employing vaccination as a mechanism for increasing a T regulatory response has also been shown to yield neuroprotection. Researchers have vaccinated animals with vasoactive intestinal peptide. Vasoactive intestinal peptide elicits the differentiation of T cells into Tregs. Subse- quently the animals were exposed to 1-methyl1-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), a chemical which will cause degen- eration of neurons in the substantia nigra, creating symptoms of Parkinson’s disease. The animals that had been vaccinated showed resistance to the development of Parkinson’s disease (Kosloski et al., 2010; Reynolds et al., 2010). There are studies on how early life exposure to pathogens impacts resilience or vulnerability to stress. Consistent with the idea that early life exposure to pathogens is protective, Bilbo et al. (2008) found that early life exposure to E. coli protected mice from stress. As adults, when the mice were exposed to uncontrollable shock, they displayed less diminution of social exploration and less elevation in cortisol during the stressor. Contrary to the ﬁnd- ing that early life exposure to E. coli promotes resistance to stress, exposure to particular bacteria for example, type A2/Singapore inﬂuenza, may increase risk for later depression (Bale et al., 2010). So does early life exposure to infection promote resilience or vulnerability to stress? A testable hypothesis asserts that when a pathogen elicits Tregs, the pathogen promotes resilience; whereas when the pathogen fails to provoke Tregs, exposure promotes vul- nerability. What can be stated with conﬁdence? The mechanisms through which leukocytes play a role in main- taining the health of the CNS is an area of intense research having implications for degenerative diseases as well as mood disorders. The interaction between T effector cells, Tregs, microglia, and neurons requires more elucidation with recognition that ﬁnd- ings may not generalize across species or across strains within a species (Walsh and Kipnis, 2011). The research on whether Tregs are beneﬁcial or not has been particularly inconsistent. How Tregs impact brain inﬂammation may depend upon which cells the Tregs are inﬂuencing. Future work may further identify which types of T cells and which cytokines foster brain health versus inﬂammatory responses. However, in the human litera- ture, Tregs are prominently featured in work on the Hygiene Hypothesis. In the literature on the importance of T cells for the health of the CNS, the topic of which type of T cells exert a salubrious effect has not been a major focus of attention. However,given the various roles that cells of the immune system can play in the brain raises the issue of which particular cytokines and which particular type of T cells promote brain health.Yirmiya and Goshen,commenting on research by Derecki and others (Derecki et al., 2010; Yirmiya and Goshen, 2011) noted that T cells present in meninges secret- ing IL-4 were important for the capacity for learning. Presumably, these cells were Th2 differentiated a non-inﬂammatory pathway. Butovsky et al. (2006) showed that IL-4 induced microglia to release IGF-1. Some studies have asked about the impact of Tregs in inﬂuenc- ing brain inﬂammation. Findings on the impact of Tregs have been puzzling (Walsh and Kipnis, 2011). Kipnis et al. (2002) found that Tregs,in the choroid plexus and meninges of the brain,dampen the activity of other T cells so that less repair after injury occurs. When Tregs are depleted, the animal exhibits stronger recovery after crush injury of the optic nerve. Ziv et al. (2007) also found that for promoting neurogenesis in brain after stroke, downregulat- ing Tregs improves outcome. Moreover, subsequent work (Cohen et al., 2006) showed that depleting an animal of all Tregs while maintaining T cells speciﬁc for myelin basic protein precluded signs of distress following exposure to the odor of a predator. In some strains, the T effector cells and Tregs seem to cancel each other’s protective effects (Kipnis et al., 2004). THE HEALTH-PROMOTING PATHWAY Dantzer et al. (1999) concomitantly, administered LPS and IGF-1 into the intracerebroventricular area. The IGF-1 administration prevented the emergence of sickness behavior, viz., immobility and reduced exploration of a novel environment that otherwise occurs with LPS administration (Dantzer et al., 1999). Support for the importance of T cells speciﬁc to brain proteins also comes from vaccination studies. Lewitus et al. (2008, 2009) vaccinated animals with a protein found in the CNS, myelin basic protein, but which had been altered to make the myelin basic pro- tein less likely to induce a strong inﬂammatory response. Then the animalsweresubjectedtostressfulconditions,whichhadbeenpre- viously shown to induce depressive behavior as well as decreasing August 2012 \| Volume 3 \| Article 297 \| 7 www.frontiersin.org Littrell Inﬂammation neuroprotective effect. IL-10, a primary cytokine released by Tregs and microglia, blocks neuronal apoptosis after injury (Zhou et al., 2009). Interferon-γ (IFN-γ) and TGF-β also exert an inﬂuence of microglia which drives neuroprotection (Kipnis et al., 2004). Warner-Schmidt et al. (2011) argue that IFN-γ exerts an anti- depressant effect by increasing levels of p11, a protein involved in trafﬁcking serotonin receptors to the cell membrane. Thus, Warner-Schmidt et al. posited a role for IFN-γ beyond inﬂuencing further inﬂammation. Given the inconsistencies in impact across cytokines, it is important to replicate ﬁndings in various species to determine whether ﬁndings are species speciﬁc. the level of BDNF in the hippocampus. After exposure to the stressful conditions, the animals that had been vaccinated failed to display decreased sucrose preference, a measure of anhedonia. Moreover,the level of BDNF in the hippocampus remained at con- trol levels. Consistent with higher levels of BDNF, the immunized animals exhibited more neurogenesis in the hippocampus. Which type of T cell? In the Immunology literature, the identiﬁcation of the various paths along which T cells can be differentiated has been a vigorous area of investigation. Five major types of T cells have been identi- ﬁed: Th1, T follicular helper cells, Th2, Th17, and Tregs. Two types are germane here: Th2, which release IL-4, IL-3 and IL-13, and Tregs (Mimran and Cohen, 2005; Murphy, 2012). Frontiers in Psychology \| Psychology for Clinical Settings The hygiene hypothesis As ﬁndings accumulate identifying pathogens which Whereas the previous studies, examined the impact of Tregs, other studies have focused on identifying cytokines that exert a Frontiers in Psychology \| Psychology for Clinical Settings August 2012 \| Volume 3 \| Article 297 \| 8 Littrell Inﬂammation are powerful in eliciting Tregs, some of the discrepancies in the literature may be resolved. the microglia to be in a health and resilience promoting state have been asked. Studies examining the role of Tregs has yielded some contradictory ﬁndings. The future promises a more articulated story on how the behavior of microglia, which is inﬂuenced by speciﬁc types of T cells and the particular cytokines they release, can result in depressive behavior or behavioral resilience. Lowry has focused on those particular neuronal circuits that are activated when a T regulatory response occurs. Subsequent to elic- iting T regulatory response through vaccination with attenuated M. vaccae, serotonergic neurons in the interfascicular dorsal raphe were activated (Lowry et al., 2007). This resulted in a faster latency to swim on the forced swim test, a laboratory measure of resilience to depression. Inoculation with bacteria that failed to elicit a T regulatory response did not have this effect. Elsewhere, Hale and Lowry (2011) have differentiated the various serotonergic circuits in the raphe. Some of these serotonergic neurons (in the caudal, dorsal raphe) are involved in creating anxiety and learned helpless- ness (Maier et al., 2006). The interfascicular dorsal raphe neurons project to the medial PFC and are part of a feedback loop that will downregulate the caudal dorsal raphe serotonergic neurons to decrease learned helplessness. In terms of a mechanism for the antidepressant effect of vaccination with M. vaccae, the M. vaccae activate Tregs which activate the interfascicular dorsal raphe neu- rons that suppress the anxiogenic/depressogenic circuitry (Lowry et al., 2007, 2008). Lowry et al.’s research is important because it raises the possibility that Tregs not only confer resilience to stress by countering depressogenic inﬂammatory activation, but also may confer resistance through a direct impact on neuronal function. Lowry’s research suggests that Treg cytokines can induce activity in interfascicular dorsal raphe neurons. The interfascicular dorsal raphe neurons inhibit learned helpless behaviors. IMPACT OF ANTIDEPRESSANT DRUGS ON INFLAMMATION IMPACT OF ANTIDEPRESSANT DRUGS ON INFLAMMATION Antidepressantdrugshavebecomethestandardtreatmentforpeo- ple suffering from depression, often being used even when the patient is receiving psychological treatment. Given that inﬂam- mation is involved in depression, possibly causally, it is useful to examine the effects of antidepressant drugs on inﬂammatory state, and also on whether the drugs can counteract conditions leading to inﬂammation. Most of the studies that have been done on short term inﬂuences of the drugs,in both animals and people. However, it has become fairly standard for patients to remain on antidepres- sants for years at a time. Given that these drugs have deleterious side effects, it would be important to continue using them only as long as they are effective. Therefore we examine the effects of anti- depressants on inﬂammation in two sections: one on short term use, and a separate one to see what can be discerned from studies of people who have taken antidepressants for years. SUMMARY The case has been advanced that depression reﬂects an inﬂamma- tory state in the brain. An inﬂammatory state in the brain can be induced by inﬂammatory cytokines in the periphery or by psycho- logicalstressors.Whilesomehavefocusedonhowbraininﬂamma- tion results in manifestations of depression, others have examined resilience. Microglia are the source of inﬂammatory cytokines, associated with depressive behaviors, as well as the source of IGF-1, associated with resilience. Events downstream of microglia behavior have been investigated. Researchers have attempted to identify which consequences of inﬂammatory cytokines result in depressive behaviors. Inﬂammatory cytokines do decrease the expression of growth factors (e.g., BDNF), which in turn impact neurogenesis. However, ﬁndings fail to support the view that a simple lack of BDNF or an incapacity for neurogenesis in the hip- pocampus redounds in depression. Moreover,the impact of BDNF varies according to the structure in the brain under observations. Attempts to link cytokine levels in brain to a particular neuro- transmitter’s function to explain depressive behaviors or resilience have been made. Lowry’s work implied Treg anti-inﬂammatory cytokines inﬂuence particular serotonergic neurons to yield stress resilience. In fact, cytokines impact other neurotransmitter sys- tems as well (Pace et al., 2012). Thus, how cytokines impact neurotransmitters awaits further elucidation. The work on how microglia can protect the brain and confer behavioral resilience to stress has focused on identifying determinants of microglia behav- ior, that is whether the microglia release inﬂammatory cytokines or IGF-1. Questions regarding which particular cytokines, ema- nating from speciﬁc T cell types, are most impactful in inducing The hygiene hypothesis Although the research does not yet offer an articulated mech- anism for the ways in which inﬂammation mediates depressive mood and behaviors and conversely how healthy microglia confer resilience, it does appear that systemic inﬂammation is an inﬂu- ential link in the causal chain of depression. This, in turn, suggests that we would be well advised to consider a variety of ways to reduce systemic inﬂammation. The ways in which antidepressants impact the immune system are now considered. SHORT TERM STUDIES I h h li In the human literature, the efﬁcacy of antidepressants has been challenged (Kirsch, 2010). However, there is support for efﬁcacy in the animal literature. One causal factor in systemic inﬂam- mation is stress. Studies have examined whether antidepressants can abrogate the impact of stress. When animals are subjected to uncontrollable stress, the extent of their distress can be gaged by the reduction in their preference for sucrose consumption. Also, they will fail to escape from an aversive stimulus. It turns out that both of these stress responses can be abrogated by antidepressant treatment (Bessa et al., 2009). Another way to produce stress in animals is to put them in a novel environment. After exposure to a novel environment, stress is manifested on a number of mea- sures. Treatment with antidepressants will decrease immobility in a forced swim test, will increase exploration in a novel envi- ronment, and will preclude the decrement in food consumption produced by a novel environment (Dulawa et al., 2004). Questions about how selective serotonin reuptake inhibitors (SSRIs) achieve their efﬁcacy have been raised. Maes and col- leagues, using human subjects, have shown that if SSRIs are mixed with whole blood outside of the body, the production of anti-inﬂammatory cytokines is increased while the production of proinﬂammatory cytokines is decreased (Maes et al., 1999; Kubera et al., 2001a). Obuchowicz et al. (2006) also showed in cultured glial cells taken from animals, antidepressants will dampen the August 2012 \| Volume 3 \| Article 297 \| 9 www.frontiersin.org Littrell Inﬂammation release of inﬂammatory cytokines in response to LPS. In other studies, patients who were treated with antidepressants for a short period of time have undergone blood draws and then cytokines have been measured. Those persons taking antidepressants for 5– 8 weeks do exhibit lower levels of IL-6 levels in plasma (Sluzewska et al., 1995; Basterzi et al., 2005); lower TNF-α levels (Lanquillon et al., 2000; Tuglu et al., 2003); lower levels of monocytes and neu- trophils in plasma (Seidel et al., 1996; Tuglu et al., 2003); higher levels of IL-10 (Kubera et al., 2001b); and higher levels of Tregs (Himmerich et al., 2010). Moreover, when whole blood from per- sons taking antidepressants for 6 weeks are tested in response to a mitogen stimulus, the ratio of IFN-γ/IL-10 is lower (Maes et al., 1999; Kubera et al.,2001a). SHORT TERM STUDIES I h h li Thus,the case can be made that antide- pressants, taken for several weeks, exert anti-inﬂammatory effects as seen by speciﬁc changes cytokines, and thus offer a likely expla- nation for the initial positive effects ascribed to antidepressant drug use. of lymph nodes, acetylcholine from the post ganglion neuron of the parasympathetic nervous system will inhibit release of TNF-α, IL-1, IL-6, and IL-18 from macrophages. In the liver, greater vagal tone will inhibit NF-κB activation and thus the release of acute phase proteins (Pavlov and Tracey,2005; Rosas-Ballina and Tracey, 2009). Thus, increasing vagal tone can be expected to decrease systemic inﬂammation. Several meta-analyses and studies of non-medically ill patients treated with tricyclics for short periods of 6–16 weeks, have noted a decrease in heart rate variability (Tulen et al., 1996; van Zyl et al., 2008;Kempetal.,2010).ResultsforSSRIstakenoverashortperiod of time have been inconsistent with several small N studies noting a decrease in heart rate variability (Dawood et al., 2007) and oth- ers ﬁnding no impact (Davidson et al., 2005; van Zyl et al., 2008; Kemp et al., 2010). The question of whether treatment with antidepressants makes long-term outcomes less favorable requires investigation of how antidepressants impact HRV when taken for over a year. In con- trast to studies assessing short term impact of treatment, a study by Licht et al. (2008) examined the impact of antidepressants on persons with either remitted or current depression who were followed for 8 years. Two measures of heart rate variability were employed in the study: standard deviation of normal-to-normal (SDNN) beats and respiratory sinus arrhythmias (RSA). The study included 67 persons taking TCAs, 435 persons taking SSRIs, and 137 taking other antidepressants. There were 1,018 persons who were depressed or had been depressed but were not taking med- ications. The study also included 524 controls who had never been depressed. Thus, there was a control group of persons who were never depressed as well as persons who had been or were cur- rently depressed broken down according to medication status. The results were that those taking medications exhibited lower heart rate variability when compared to never-depressed controls (for SDNN, d = 0.207–0.849; for RSA, d = 0.413–0.862 across vari- ous medications). Comparing those remitted depressed not on medication or still depressed not taking medication did not dif- fer from controls on SDNN beats and did not differ as greatly from controls on RSA (d = 0.118). Licht et al. Weight gain b i i Obesity is considered to be an inﬂammatory state. Adipocytes are capable of releasing IL-6 and TNF-α. It is estimated that 30% of the IL-6 in circulation derives from adipose tissue. Moreover, omen- tal fat tissue (fat around the abdomen) is populated by activated macrophages releasing IL-6 (Mohamed-Ali et al., 1997; Hamer, 2007). Even in normal weight individuals, omental fat can be inﬂammatory (Shelton and Miller, 2010). Weight reduction can be expected to decrease systemic inﬂammation. SHORT TERM STUDIES I h h li noted a dose– response relationship between antidepressants and reduced HRV. Licht et al. concluded that the association between lowered HRV and depression (p. 1358)“appears to be mainly driven by the effect of antidepressants.” LONG-TERM STUDIES There have been studies examining the long-term impact of anti- depressants on systemic inﬂammation. Many of these studies have examined the impact of antidepressants on phenomena that are associated with increased systemic inﬂammation: diminished heart rate variability, weight gain, metabolic syndrome, type II diabetes, and atherosclerosis. These studies as well as the stud- ies examining the impact of long-term antidepressant use on the inﬂammatory marker of CRP are reviewed. C-Reactive protein C-Reactive protein is an acute phase protein released by the liver in response to IL-6. It is considered to be a marker for sys- temic inﬂammation (Blake and Ridker, 2002; Pearson et al., 2003). Dawood et al. (2007) noted that treatment with an SSRI for 12 weeks signiﬁcantly increased high sensitivity CRP. This ﬁnding was consistent with Hamer et al.’s (2011) ﬁndings from two data sets. In a data set of 4,584 research-participants from the White- hall studies of British civil servants, long-term use (10 years) of an antidepressant was associated with higher CRP after control- ling for psychological distress. In a large sample of 8,131 Scottish research-participants from another data set, an association was noted between higher CRP levels and treatment with tricyclic anti- depressants (TCAs) although no association was noted for those treated with SSRIs. The duration of medication use and dosage were not reported in the Scottish sample. However, Hamer et al. noted that in the Whitehall study antidepressant use at only one data point was not associated with CRP elevations. Thus, short exposure to antidepressants probably is insufﬁcient to raise CRP levels. Diabetes I ﬂ Inﬂammatory cytokines create insulin resistance. Type II diabetes, characterized by insulin resistance, is an inﬂammatory disease (Dandona et al., 2011). Thus, an increase in type II diabetes might be considered a proxy for an increase in systemic inﬂammation. In a case-controlled study, persons who were all depressed at entry into the study were followed for 2.8 years. Of those entering the study, 2,243 persons developed diabetes during the follow-up period. Those who developed diabetes were matched on age, gen- der, and time in the study with 8,963 comparison subjects, who had not developed diabetes. A comparison of long-term antide- pressant users versus short term users yielded no difference on their initial severity of depression. However, those developing dia- betes were more likely to have been treated with antidepressants at moderate to high doses for over 2 years prior to the onset of dia- betes. The increased risks obtained for amitriptyline, ﬂuvoxamine, paroxetine, and venlafaxine (Andersohn et al., 2009). Several other large-sample-size-studies have yielded ﬁndings consistent with the hypothesis that long duration of exposure antidepressants increase the risk of Type II diabetes. In a prospec- tive study of 9,197 participants followed for 4.8 years, treatment duration of over 6 months of an antidepressants (both the TCAs and the SSRIs), doubled the risk of Type II diabetes. Duration of exposure to the antidepressant was associated with elevated risk for diabetes in both those with mild and moderate depression (Kivimaki et al., 2010). In a third large N study (3,234 persons with risk factors for diabetes), with a follow-up of 10 years, Rubin et al. (2010) also found that continuous use of antidepressant use increased the probability of developing diabetes. In those treated with metformin, antidepressant use did not increase the risk of diabetes development. The previously discussed study by Raeder et al. (2006) also found that paroxetine was associated with higher rates of diabetes. Thepreviouslyreviewedliteraturesuggeststhatsustainedexpo- sure to antidepressants for over a year is associated with more sys- temic inﬂammation. The mechanisms through which long-term use antidepressants might increase systemic inﬂammation have not been investigated. Identifying these mechanisms is important for future research. The other lesson emerging from this literature is that evaluating drugs employing the usual 8 weeks of assessment customary in drug trials is not sufﬁcient for determining the efﬁ- cacy of pharmacological interventions. Metabolic syndrome Previous sections have advanced the case for depression as an inﬂammatory condition, with strong evidence for the conclusion that inﬂammation is an important link in the cause of depression. The evidence in this section points to the conclusion that short term use of current antidepressant medications reduces inﬂam- mation, and ameliorates depression. But additional data strongly suggest that the positive effects are reversed with prolonged use. That is, it is not merely the case that antidepressant medications appear to lose their efﬁcacy with prolonged use, but rather, they promote reverse effects, increasing inﬂammation. As noted above, weight around the abdomen is inﬂammatory. Weight around the abdomen is a component of metabolic syn- drome. In a cross-sectional, large N study (25,315 subjects), Raeder et al. (2006) found an association between abdominal obesity, general obesity, higher cholesterol levels (components of the metabolic syndrome), and the use of SSRIs after control- ling for symptoms of anxiety and depression. Associations were particularly pronounced among persons in their 40s who were taking paroxetine and were absent in those taking citalopram. The authors did not have data on duration of exposure, however, they remarked that they expected metabolic syndrome to take some time to develop. Previous sections have advanced the case for depression as an inﬂammatory condition, with strong evidence for the conclusion that inﬂammation is an important link in the cause of depression. The evidence in this section points to the conclusion that short term use of current antidepressant medications reduces inﬂam- mation, and ameliorates depression. But additional data strongly suggest that the positive effects are reversed with prolonged use. p g Contrasting data on the probability and frequency of depres- sive relapses prior to the advent of pharmacological treatments to the era after, Fava and Ofﬁdani (2011) along with an earlier paper by Fava (2003) raised the possibility that antidepressants create a chronic course in major depression. Littrell (1994) also contrasted the duration of wellness intervals between episodes of depression in persons before and after the advent of modern medications and also noted that antidepressants shorten the duration of wellness intervals. Fava and Ofﬁdani (2011) considered various possible explanations for the mechanism through which antidepressants contribute to chronicity of depression including tolerance, lower- ing the brain’s threshold for mood ﬂuctuations, and withdrawal phenomenon from antidepressants. Another possibility is that antidepressants,when takenforover aperiod of 6 months,increase systemic inﬂammation such that depression becomes chronic. Atherosclerosis There is an association between major depression and heart dis- ease (Frasure-Smith and Lespérance, 2006). Atherosclerosis, like depression, is an inﬂammatory disease and inﬂammation con- tributes to the calciﬁcation of the aorta (Kritharides et al., 2000). In a study of heart patients after controlling for depressive symp- toms, antidepressant use was found to be associated with greater aorta calciﬁcation (Shah, 2011). Heart rate variability Both the sympathetic and parasympathetic nervous systems inner- vate lymph nodes, as well as the heart. In terms of innervation of the heart, more parasympathetic control over the heart rate is manifested as greater heart rate variability (Porges, 2007, 2009). Greater heart rate variability is considered to reﬂect better coordi- nation between breathing and heart function. Moreover,decreased heart rate variability is a predictor of heart attacks and cardiovas- cular disease (Hayano et al., 1990, 1996). In terms of innervation A meta-analysis of the impact of antidepressants on weight gain suggested a strong effect for the TCAs and an effect for some SSRIs (paroxetine) in studies examining long-term exposure to antidepressants (over 6 months; Serretti and Mandelli, 2010). The meta-analysis was consistent with an early review by Fava (2000) Frontiers in Psychology \| Psychology for Clinical Settings August 2012 \| Volume 3 \| Article 297 \| 10 10 Littrell Littrell Inﬂammation and a review by Deshmukh and Franco (2003) which concluded that TCAs and paroxetine were particularly likely to be associated with weight gain. A study by Kivimaki et al. (2010) found a corre- lation between duration of treatment with antidepressants, both TCAs and SSRIs, and greater weight gain. Moreover, a study of persons treated with antidepressants for panic disorder for a year, noted signiﬁcant weight gain for all of the SSRIs (citalopram, ﬂu- oxetine, ﬂuvoxamine, and paroxetine) used in the study (Dannon et al., 2007). Diabetes I ﬂ When the assumption is that an individual will be exposed to a chemical over an extended period, years rather than weeks are required. Metabolic syndrome Evi- dence suggests that when taken over years,antidepressants increase systemic inﬂammation. 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(2001). WAY FORWARD Research implicates inﬂammation as a proximal causal factor in producing depressive symptoms. At present, psychological August 2012 \| Volume 3 \| Article 297 \| 11 www.frontiersin.org Littrell Inﬂammation treatments for depression such as interpersonal therapy and Cog- nitive Behavior Therapy have not been evaluated for their impact on inﬂammation. In evaluating interventions, psychologists may wish to include measures of inﬂammation, such as CRP, as depen- dent measures. If psychological interventions decrease systemic inﬂammation, then there would be evidence that psychologi- cal treatments not only decrease levels of depression through a direct pathway but also through the indirect pathway of impact on systemic inﬂammation. consumption of curcumin (Aggarwal, 2011) lower inﬂammation and lower the risk for depression. In animal work, curcumin has been shown to exert antidepressant effects of similar magnitude to antidepressant drugs (Wang et al., 2008). Interventions such as exercise (Hamer, 2007), yoga (Bernardi et al., 2001; Kiecolt- Glaser et al., 2010), meditation (Pace et al., 2009) have been shown to dampen inﬂammation. Meditation (Pace et al., 2009), exer- cise (Greenwood and Fleshner, 2008, 2011), and consumption of omega-3s (Kiecolt-Glaser et al., 2011) have been demonstrated to confer resilience to stress. Psychologists have a long tradition in strengthening behavioral compliance and have the skills for assist- ing individuals in adhering to life style changes. The recognition that depression is an inﬂammatory disease ushers in a wealth of new possibilities for treating and preventing mood disorders. Systemic inﬂammation does contribute to depression. Over the long run, antidepressants contribute to inﬂammation. If antide- pressants either lose efﬁcacy or contribute to inﬂammation in the long run, other mechanisms for decreasing inﬂammation must be relied upon for treating depression. 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Citation: Littrell JL (2012) Taking the perspective that a depressive state reﬂects August 2012 \| Volume 3 \| Article 297 \| 18 REFERENCES Copyright © 2012 Littrell. This is an open-access article distributed under the terms of the Creative Com- mons Attribution License, which per- mits use, distribution and reproduc- tion in other forums, provided the orig- inal authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. spatial learning abilities in adult- hood. Nat. Neurosci. 9, 268–275. inﬂammation: implications for the use of antidepressants. Front. Psychology 3:297. doi: 10.3389/fpsyg.2012.00297 Ziv, Y., and Schwartz, M. (2008a). Immune-based regulation of adult neurogenesis:implicationsforlearn- ing and memory. Brain Behav. Immun. 22, 167–176. Ziv,Y., Finkelstein,A., Geffen,Y., Kipnis, J., Smirnov, I., Shpilman, S., Vertkin, I., Kimron, M., Lange, A., Hecht, T., Reyman, K. G., Marder, J. B., Schwartz, M., and Yoles, E. (2007). 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https://openalex.org/W2596826687	https://www.crimejusticejournal.com/article/download/373/301/	English	null	Navigating Violence: Fear and Everyday Life in Colombia and Mexico	International journal for crime, justice and social democracy	2,017	cc-by	9,584	Abstract Violence and insecurity are often read as totalising narratives of communities in parts of Latin America, flattening the complexity of everyday life and the responses of occupants who suffer from fear. In this article we draw on ethnographic research undertaken in los Altos de Cazucá in Colombia and in San Luis Potosí in Mexico. While both sites are distinct locations with different historic, economic, social and political contexts they share features of communities affected by violence and insecurity: distrust of institutions of the state; rationalisations for managing violence in daily life; and narratives of fear that appear woven through the fabric of conversations. However, fear and violence are not all‐encompassing experiences and individuals in both these communities describe practices of navigation of violence that draw on positive communal experiences. This article explores how, in these communities where violence comes to be expected but never normalised, people navigate their everyday lives. Helen Berents, Charlotte ten Have Queensland University of Technology, Australia Helen Berents, Charlotte ten Have Queensland University of Technology, Australia Helen Berents, Charlotte ten Have Queensland University of Technology, Australia Please cite this article as: Berents H and ten Have C (2017) Navigating violence: Fear and everyday life in Colombia and Mexico. International Journal for Crime, Justice and Social Democracy 6(1): 103‐117. DOI: 10.5204/ijcjsd.v6i1.373. www.crimejusticejournal.com IJCJ&SD 2017 6(1): 103‐117 www.crimejusticejournal.com IJCJ&SD 2017 6(1): 103‐117 ISSN 2202–8005 Navigating Violence: Fear and Everyday Life in Colombia and Mexico This work is licensed under a Creative Commons Attribution 4.0 Licence. As an open access journal, articles are free to use, with proper attribution, in educational and other non‐ commercial settings. ISSN: 2202‐8005 Introduction Both Colombians and Mexicans have long experienced the effects of violence in their countries. While Colombia’s conflict has lasted more than half a century the parties to the conflict, both the leftist guerrillas and the right‐wing paramilitary organisations (as well as corrupt state officials) have also become involved in drugs and arms trafficking (Palacios 2006). The consequences of five decades of conflict and violence have been large scale displacement (approximately six million people (Edwards 2016) or approximately one in ten Colombians (Internal Displacement Monitoring Centre 2015) have been displaced due to the conflict), the wide scale abnegation of human rights, underdevelopment, poverty, and the undermining of the government’s authority and capacity to serve all its citizens (see, amongst others, Bouvier 2009; Pecaut 2006; Profamilia 2005; Richani 2002; Romero 2007). In parts of the country the state has lost its authority and illegal groups control communities. Since 2012 there has been a peace process underway between the government and the leftist guerrillas, the Revolutionary Armed Forces of Colombia (known by their Spanish acronym of FARC); however, however, even with a peace deal signed in late 2016 and being implemented, insecurity and inequality will persist in many places. In Mexico the self‐declared war on organised crime and drugs launched by President Felipe Calderón, newly elected in 2006, has seen an unprecedented wave of violence and insecurity convulse the nation (Morris 2009). Following the highly disputed elections Calderón dispatched tens of thousands of military troops and federal police to key narcotic‐trafficking states during his presidency, resulting in an estimated 60,000 to 70,000 deaths with a further 26,000 people missing (Gayón 2015; Rosen and Martínez 2014). In more recent years civilians, authorities and journalists have increasingly become the target of the growing insecurity with drug cartels branching out into extortion, human smuggling and kidnapping. This is to the exclusion of the extensive list of violations attributed directly to state forces (Beittel 2011; Human Rights Watch 2015). Similar to Colombia in the 1990s, the Mexican state has lost its monopoly over the use of force which is evidenced by the private armed groups, self‐defence groups, corrupt state forces and drug trafficking organisation which also routinely use violence as a tool. As a result Mexican citizens have become trapped in networks of extortion and coercion, in which both state forces and criminal organisations prey with impunity (Magaloni et al. 2015: 28). Keywords Violence; social navigation; Colombia; Mexico; insecurity; everyday life. This work is licensed under a Creative Commons Attribution 4.0 Licence. As an open access journal, articles are free to use, with proper attribution, in educational and other non‐ commercial settings. ISSN: 2202‐8005 © The Author(s) 2017 Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Online version via www.crimejusticejournal.com Introduction In both countries the violence has affected people unevenly but both situations are also characterised by widespread insecurity, the targeting of individuals such as human rights advocates and journalists (Molzahn et al. 2012; Human Rights Watch 2014; Tate 2007), the prevalence of stories of death, torture and violence in print and visual media (Brambila 2014), and the undermining of a sense of security and of rights. This article draws upon ethnographic research conducted by the authors, one working in an informal community on the outskirts of Colombia’s capital Bogotá, and the other in the metropolitan area of San Luis Potosí City, Mexico. While the sites differ in a range of significant ways including size and demographic features, occupants of both communities demonstrate the cost of living with fear and insecurity, the impact of violence, and the techniques people adopt to navigate their daily lives. We challenge both a flattened narrative of violence as totalising and an understanding of violence as ever normalised, which is sometimes implicit in engagements with the global South. Instead we propose a way of understanding the experiences of structural and direct violence that takes its starting point with the experiences of individuals who have learned to navigate their everyday lives despite, and because of, the insecurity and fear that impacts their lives. We offer this as a way of countering the ideas that these kinds of places that experience violence are inherently violent, or that sites of conflict and insecurity in the global South are intrinsically or totally sites of despair or danger, or that people are nothing more than passive victims in the face of violence in their daily lives. The forms of navigation – of agency and decision‐making – that we explore 104 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico here are generally mundane; they are practices in everyday life of moving through insecure and unpredictable terrains. The occupants of both communities recognise the challenges of living with violence and, as we discuss below, have developed strategies built on communication with other community members, on‐the‐spot decision making, and weighing of risk, to become skilled navigators in these contexts. Introduction We argue that attention to the often small, everyday agency exercised by individuals offers alternative perceptions of communities and circumstances provided by other research in similar locations and situations where those affected by violence collectively and openly organise against violence. We do not wish to suggest that the individuals in either community are passive; rather, their agency is shaped by their experiences of insecurity and violence and, in different ways, they navigate through and over these uncertain seas. To do this, we briefly introduce the two sites, explain our research processes and situate ourselves as researchers; we then proceeds in two parts. In the first part we explore notions of violence and fear to theorise them as complex and ever‐changing experiences. While violence can break individuals and communal bonds, it is not a normal or totalising experience. We also forward here a notion of a skilled navigator, drawing on Henrik Vigh’s (2009) work. In the second part we explore accounts of structural and direct violence experienced by participants in both Colombia and Mexico and the consequences for their lives. These experiences form the basis for understanding these individuals as skilled navigators of their everyday lives. In this article we explore how, in these sites, violence, fear and distrust are not extra‐ordinary conditions but part of everyday life. In acknowledging this, we do not seek to exoticise the communities presented in this research or reinforce a totalising narrative. Rather, we aspire to prompt more critical reflection on how these topics are described and understood, and argue for the capacity of individuals to navigate their everyday lives in ways that respond to and ameliorate the effects of violence and insecurity. Research process and positionality Original research for both sites was conducted independently for two different projects and is brought together in conversation here.1 In both cases the researchers spent several months in the community and adopted ethnographic methods. Such research is predicated on respect for those involved, recognition of expertise of the local community in issues that affect them, and commitment to reflexively engage in the complexity of lived experiences of participants. All names used here are pseudonyms. Both researchers speak Spanish fluently and research was conducted in Spanish in Mexico and Colombia. In Colombia Helen Berents spent time in the community of los Altos de Cazucá between September and December 2010 participating in daily life through a school in the community and a foundation, Fundacion Pies Descalzos, which supported students through school and community engagements. This original research was focused on young people’s responses to violence and construction of peace, and thus youth formed the bulk of the fieldwork data quoted here. Observation and, later, interviews with young people, their parents, teachers and guardians as well as other key community figures informed the research. In Mexico Charlotte ten Have conducted fieldwork between January and March 2013 with the use of participant observation and qualitative focus groups, and individual interviews. The first two months of fieldwork were conducted in San Luis Potosí and the last month in Guanajuato, with frequent visits back to the original fieldwork site. The Universidad Autónoma de San Luis Potosí, and the help of Dr Daniel Solís Domínguez were crucial in gaining access to respondents. The majority of the interviews were conducted at the university and two high schools in lower socio‐ economic suburbs of the city. The original research focused on coping mechanisms in relation to increasing violence. There are important considerations in undertaking research of this nature, which we acknowledge and respect. As two researchers from the global North (Australia and the Netherlands), conducting research in the global South requires recognition of perhaps uneven power relations and privileges that cannot be ‘solved’. There has been excellent research conducted by academics from within these countries about the situations we research, and avoiding extractive, disrespectful research was a key consideration for us both. Fieldwork sites As a result of this unprecedented increase, San Luis 105 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Potosí was added to the list of Mexico’s most dangerous cities in the 2013 National Social Plan of Prevention of Violence and Crime. During daytime, however, the only visible aspect of the violence were the posters of missing persons in the city and the enormous show of force by the many pick‐up trucks with masked municipal, state and federal police forces carrying automatic weapons. At a first glance life seemed to continue as normal in the vibrant colonial city centre with its many bookstores, busy public transport and many food carts but, despite this first appearance, the insecurity had seeped through into an underlying layer that deeply impacted everyday life in the form of fear and coping mechanisms. Violence was thus more often a visceral than a visual experience; nonetheless, one that was constantly present and constantly changing. Within this situation San Luis Potosí occupants, the Potosínos, had become knowledgeable and skillful in moving with and through this uncertain environment to continue their daily routines. Despite the differences between the two communities and their broader contexts, we argue that it is fruitful to bring together the experiences of these two communities to explore occupants’ navigation of everyday life in situations of insecurity and unpredictable violence. Fieldwork sites In Colombia, the community of los Altos de Cazucá (often referred to simply as Cazucá) is an illegally established community that forms part of Comuna 4 of the municipality of Soacha, on the periphery of Bogotá. There are over 60,000 inhabitants of Comuna 4 (Alcaldia Municipial de Soacha 2008: 8). The Comuna is one of the poorest in Colombia; it is also one of the largest recipient communities for people internally displaced by Colombia’s long‐running conflict. The poverty and neglect of the community is visible in the lack of paved roads, the ad‐hoc construction of houses, presence of rubbish, and lack of communal spaces. Most occupants do not have running water, and access to government services is often complex and fraught. Fear of violence by armed gangs is a constant concern of the occupants, and the violence and altercations between gangs and state forces paint a picture – for those outside the space of the community – of endemic violence and delinquent or threatening occupants. Despite these challenges, such a description is not a totalising account of a community that for the most part is comprised of families trying to live day‐to‐day, often displaced, negotiating poverty and the absence of state care (see Berents 2015). There is pride in the community and a desire to counter negative narratives. San Luis Potosí in Mexico is the state capital of San Luis Potosí State and has a population of approximately 772,604 inhabitants. The city is located halfway between Mexico City and the United States border and is in the middle of the triangle formed by Mexico City and the two other largest cities in Mexico, Guadalajara and Monterrey. While the city’s central location has made it an attractive location for legitimate investment, its good connections to the tumultuous northern regions, and ready access to the main railroad to the United States has also attracted criminal organisations. The region remained reasonably stable until the drug‐related violence increased by 49 per cent in the first four months of 2013, just before and during the fieldwork period (Overseas Security Advisory Council 2013). Complex notions of violence and fear Violence is complex and the ways in which people respond to violence are also complex. Scheper‐ Hughes and Bourgois (2003: 2) argue that ‘violence defies easy categorization’, and while violence performed on ‘grand’ scales such as war, genocide or state‐sanctioned oppression are ‘graphic and transparent’, it is the ‘everyday violence of infant mortality, slow starvation, disease, despair, and humiliation that destroys socially marginalized humans with even greater frequency [that] are usually invisible or misrecognized’. We acknowledge that both forms of violence – the ‘grand’ ‘graphic’ violence and the ‘usually invisible’ everyday violence – affect the lives of those in both communities. Violence, then, is not only deliberate and conscious but also arises from systems of oppression that are maintained and condoned through the maintenance of particular social orders (Farmer 2004: 307). Such an understanding is also linked to Galtung’s (1990) three pillars of violence. Galtung sees ‘direct violence’, such as murder or physical abuse, as one pillar. Secondly, ‘structural violence’ recognises that things such as inequality, repression and exclusion are part of structures that enable violence. Finally, ‘cultural violence’ for Galtung legitimises direct and structural violence through cultural norms. Galtung (1990: 294) argues that ‘direct violence is an event; structural violence is a process with ups‐and‐downs and cultural violence is an invariant or a permanence remaining essentially the same for long periods, given the slow transformations of basic culture’. Violence also has an emotional component. The threat or risk of violence creates an atmosphere of fear and insecurity. Fear spreads distrust and breaks communal bonds as people fear speaking up and thus suffer further. This self‐censorship not only occurs in the public space but also filters into the private sphere. Beyond the direct human suffering caused by conflict and drug‐related violence, violence thus also undermines people’s sense of security (Morris 2009). In such environments ‘fear, like pain, is overwhelmingly present to the person experiencing it, but it may be barely perceptible to anyone else and almost defies objectification. Subjectively the mundane experience of chronic fear wears down one’s sensibility to it’ (Green 1994: 230). The concept of ‘pain’, in this sense, is introduced as a bridging notion between violence and fear or, rather, as part of a relationship between violence and fear. In such situations pain can be emotional as well as physical. People hide their pain in response to fear and the silencing impact violence can have. Theorising violence, fear and navigation In this section we explore theorisations of violence and fear, understanding them as neither static nor simplistic but, instead, as complex, kinetic processes which impact people and to which people respond. Violence and fear can silence, debilitate and deconstruct people and relationships (Koonings and Kruijt 1999). However, in this section we also forward a notion of navigation. Building on Vigh’s (2009) work, we explore navigation as a way of understanding people’s everyday responses to violence and fear. Such a configuration challenges the passive construction implied by ‘coping’ and, alternatively, recognises agency and capacity. Through this theoretical exploration we arrive at the notion of a skilled navigator, a person who moves through experiences of violence and insecurity by drawing on their knowledge and expertise. Research process and positionality Engaging with important local research – not to assimilate it or extract that which is useful within our epistemological framework but to challenge our understanding, our assumptions – is part of an academic and creative practice that can perhaps respond to the inadequacies of North‐to‐South research that remains extractive and colonial.2 IJCJ&SD © 2017 6(1) 106 Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Ethnographic research can, to some extent, mediate these expectations as the researcher becomes better understood by the community, as they come to understand the community.3 As part of the social world under study, reflexivity allows us to acknowledge and question what role we play as researchers and beyond (see Gouldner 1971). Recognising our positionality we acknowledge we can never avoid it, and accept that we may be unable to fully move beyond understandings rooted in the traditions of the global North. However, we offer these explorations both in terms of methodological reflexivity here, as well as within our theoretical engagement by foregrounding the voices and experiences of those from Colombia and Mexico who shared parts of their experiences with us, and concede partiality in this endeavour. Complex notions of violence and fear Elaine Scarry notes that violence is a silencing act: ‘pain does not simply resist language but 107 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com elen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mex actively destroys it’ (1985: 4). The threat or previous experience of violence silences people who fear further violence. Fear of being a victim of violence pervades all the inhabitants of a city. However, the impact is not equally distributed throughout the city’s geographic area. The poor, for example, are disproportionately victimised (Briceño‐León and Zubillaga 2002). Green (1994: 227), in writing about Guatemala, argues that fear ‘penetrat[es] the social memory’ and, thus, rather than being an acute, individualised response, becomes a ‘chronic condition’. Such an understanding of fear as linked to violence evidences the complexity of the rubric of insecurity, violence and fear within which the occupants of San Luis Potosí and los Altos de Cazucá exist. In these communities, everyday life is uncertain; while occupants can anticipate violence and insecurity, they cannot predict it. Michael Taussig calls such situations ‘the nervous system’: ‘illusions of order, congealed by fear’ (1992: 2) where social life is defined by constant unease, and ‘attention towards change in the shape of possible acts of power and social forces’ (Vigh 2009: 422). Vigh (2009: 422) explains that the nervous system in Taussig’s configuration refers to both an unbalanced system (one that acts nervously) and a ‘sensory faculty, constantly focused on movement and necessarily feeling its way through unsettled environments’. Thus, the violences and insecurity of these environments and the fear and pain provoked are not experienced as acute or isolated episodes. Violence and fear here can be understood as a chronic condition, never normalised but as an expected part of daily life. It is important to emphasise violence is not a totalising experience – people find multiple ways of responding to and resisting the totalisation of violence, as we discuss below – but part of an ongoing, daily existence, to be managed like chronic pain or malady. Navigation of everyday violence The experience of multiple forms of violence and the accompanying fear create an insecure topography, one which people must navigate in their everyday lives. In this, we forward the specific idea of ‘navigation’ of violence, as distinct from ‘coping’ or ‘normalising’ violence. We take the idea from Vigh (2009: 420) who engages the idea of ‘social navigation’, understood as a concept that: … highlights motion within motion; it is the act of moving in an environment that is wavering and unsettled, and when used to illuminate social life it directs our attention to the fact that we move in social environments of actors and actants, individuals and institutions, that engage and move us as we move along. If we conceive of those impacted by violence and insecurity not as individuals fixed within a landscape in which they are battered by external forces of violence – structural, direct, cultural – but, rather, as individuals and collectives who move through and between an insecure, threatening topography, and who navigate social relationships, insecure terrain and unpredictable violence, we recognise that those impacted by violence are not passive. Although they lack structural power to effect large scale change, their everyday navigation is vital to maintaining daily life and resisting violence beyond their control. Navigation, as Vigh conceives of it, is not only topographical but also temporal. Social navigation ‘encompasses both the assessment of the dangers and possibilities of one’s present position as well as the process of plotting and attempting to actualise routes into an uncertain and changeable future’ (Vigh 2009: 425). This relies on a notion of radical interactivity, of ‘motion within motion’ that involves ‘implicating oneself in the ongoing life of the social and material world’ (Guyer 2007: 409). It is important to recognise that the landscape, the topography, in which people are navigating is similarly not fixed but constantly shifting. Navigation, in its Latin origins refers to wayfaring on the sea (Vigh 2009) and this gives us an analogy that is useful in 108 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico imagining the shifting, swelling, choppy, even stagnant, ocean of everyday life navigated by individuals and communities. Lefebvre (1991: 91‐92) argues for a similar conceptualisation of our social relations and spaces as ‘complex mobilities’ that form ‘convergence[s] of waves and currents’. Navigation of everyday violence This is not to minimise the crucial fact that, to extend the metaphor, some individuals have better vessels or find themselves on calmer seas. The ‘intensity and visibility of our navigational efforts’, argues Vigh, is dependent on ‘the speed and/or opacity of social change and our ability to control oncoming movement’ (2009: 430). Structural forces can limit this; however, knowledge of a local topography and temporality of violence and insecurity can enable better navigation. We forward this notion of navigation of everyday violence, adopted from Vigh (2009) combined with others, to recognise that those affected by violence and fear are not without capacity to respond, resist and reform their everyday ‘seascapes’. Using this notion, which builds from a scaffold of key work in the area of everyday violences and social suffering, we now turn to explore how this manifests in the communities of los Altos de Cazucá and San Luis de Potosí. Everyday expertise: San Luis Potosí and los Altos de Cazucá In both Colombia and Mexico participants have a fraught relationship with the institutions of the state and illegal actors. While the police are present in both communities, individuals expressed strong reservations about seeking their help and, in both sites, explained their distrust of agents of the state who frequently not only fail to provide security but often act in ways which exacerbate the insecurity of people. In los Altos de Cazucá most participants mentioned unpredictability of the police and, in San Luis Potosí, respondents tried to avoid having to interact or be confronted with state forces. In Cazucá, a conversation with two young women included the following exchange: Laura (aged 17 years): ... the police are useless here on the hill (la loma). Their only use is as decoration. That’s the truth. Helen: So the police are part of the problem? Charlotte: Do you feel that the government is taking care of you? Vanessa: Are you kidding, no! Just imagine, to me I am more ... and I live more ... Yes, it frightens me more and makes me more nervous to see a police officer than knowing someone is ‘‘bad’’ because of the abuse of power. Really the state does not give protection in any way, ever. When people come to visit I tell them to be careful, and to be even more careful with the police. There are thousands of cases of people who are in jail for things that never happened ... Or at least things that they passed. And that is what I fear most, because the narcos ... Well, as a woman I fear being raped, I guess ... But if they kill me, well at least then that’s it. Living in a society afflicted by violent crime is one thing; having to also cope with state institutions that neither deliver nor are transparent, nor can be minimally trusted, aggravates people’s predicaments to such an extent that they avoid protection from the state rather than seek reliable information or protection from it. In Cazucá the territory is contested by gangs associated with actors in the broader conflict and the security apparatus of the state. This creates severe insecurity for occupants (which oftentimes repeats the insecurity and violence from which they have fled). The police maintain a presence in the community via a van that is parked low on the hillside. Many occupants of Cazucá are deeply sceptical about police willingness to intervene, as indicated above. When the state does intervene, it is often sudden and violent, most often typified by night raids on suspected gang members’ houses that often spill over to violence on the street. In addition to the organised threat of gangs, violence against the property of people is common through robberies at gunpoint or, more commonly, knifepoint, both on the street and in the house, as well as through acts of vandalism. Specific manifestations of violence in an everyday context are a consequence of the ‘pervasive indifference, endemic oppression and sense of abjection that can make a person feel as though he or she is a mere object ... of no account ...’ (Jackson 2002: 44). Other research in Colombia notes that the police were identified as the main source of mistrust. Helen: So the police are part of the problem? Laura: Sometimes ... Because if you go to them with a problem they just say ‘so what do you want me to do about it?’ and do nothing. Rosa: (15): It is because the police now, are busy being thieves. They are just thieves dressed like police. Because they also rob you or say you need to pay for help from somewhere else. Luis, in San Luis Potosí made a similar observation: ‘When they become too old to be gang members and are not dead or in jail yet, they become police officers so they can legally fuck with people’. In San Luis Potosí, as in cities in other countries in the region, few crimes are reported to state officials as a result of the high levels of impunity (Briceño‐León and Zubillaga 2002). In general, only homicides and those crimes that require a judicial report to avoid accusation or to collect private insurance indemnities – as in the case of vehicle theft – are reported. The thousands of other crimes committed against persons are often not reported and do not ﬁnd their way into the statistics. Crimes are not reported because people consider it useless. It will not help them recover their goods and they do not expect the guilty to be punished (Briceño‐León and Zubillaga 2002). An important side effect of this is that, when official statistics are released, they only represent a fraction of the crimes that are committed and leave out the ones that are not reported. The assumption that crime statistics become totally unreliable in countries where law 109 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Online version via www.crimejusticejournal.com elen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mex enforcement is corrupt, inept or is itself prone to criminal acts is demonstrated here in an unmistakable way. The distrust of police and the state in San Luis Potosí is evident in this exchange with Vanessa: enforcement is corrupt, inept or is itself prone to criminal acts is demonstrated here in an unmistakable way. The distrust of police and the state in San Luis Potosí is evident in this exchange with Vanessa: Charlotte: Do you feel that the government is taking care of you? Charlotte: Do you feel that the government is taking care of you? Not only would they not provide ‘formal’ security (that they were meant to do as part of their job) but they would also extort people for bribes, cut deals with local gangs, and become complicit with violent occurrences (McIlwaine and Moser 2007: 131). An individual’s survival depends on their ‘ability to comprehend and manage the logics of bureaucratic processes and evade the official norm’ because the community runs via a ‘chain of legalities and illegalities’ that involve local authority figures and agents of the state (Picon, Arciniegas and Becerra 2006: 14). Such knowledge blurs the distinctions between Galtung’s (1990) forms of violence, with direct violence being enabled by the structural violence due to the abnegation of communities and corruption of officials. For occupants of both researched communities, violence is complex and this knowledge and management of the intertwined manifestations of violence is an aspect of the successful navigation of life by occupants in these communities. Expected, but never normal(ised) violence and it’s that, there are so many problems and people can’t realise what is normal, like they kill someone and it is ‘normal’. It is because of this we need to solve these problems, the actual situation, because it can change but we need to realise what is wrong to change it. Thus violence, both structural and direct, also has an emotional, fearful component. Occupants of both Cazucá and San Luis Potosí explain how those elements of insecurity and violence that they cannot control deeply affect their interactions in their daily lives and their understandings of self and community. It is crucial to recognise that violence and insecurity in both these sites is not normal or normalised. Narratives that tend towards an explanation of place as inherently violent or predisposed to insecurity create and perpetuate an illusion that violence can and should come to be expected as a normal part of life. We strongly reject this reading; while many of the participants in both Colombia and Mexico spoke about the everyday violence and how it is an expected part of life, none felt that this was something normalised or which should be accepted as normal within their everyday lives. Expected, but never normal(ised) violence These experiences of violence and insecurity prompt a sense of fear with which people live. As Green argues, fear becomes a ‘chronic condition’ (1994: 230). In Mexico Vanessa observes how violence comes to be expected but never normalised: There is a lot of indifference right now when it comes to violence, violence has become a part of us. One more corpse or murder, or to see the police on the street, it has become normal. I knew many friends who are now missing; many. But we IJCJ&SD 110 © 2017 6(1) IJCJ&SD © 2017 6(1) 110 Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico have become indifferent and have learned to live with it. Initially there was a lot of paranoia. Two years ago, for example, there was a motorcycle that made a noise and someone said that it was a shooting. Within seconds it was a mess, people were living in paranoia. We all have experiences, bad experiences. It has happened to us all directly or indirectly. The kind of situation that we are in is very ugly and I think that San Luis Potosí has changed a lot. Fear breaks the bonds of community and thus violence, which ‘occurs in the contested space of intersubjectivity, [and thus] its most devastating effects are not on individuals per se but on the fields of interrelationships that constitute their lifeworlds’ (Jackson 2002: 39, emphasis in original). This breakage of bonds is also translated in a telling Mexican proverb, which states: ‘trust is good but distrust is better’. Proverbs in this sense are the type of cultural products that are also reflected in Galtung’s three pillars of violence, in which direct and structural violence translate into cultural violence. Within a conflict area the experience of these multiple violences are both structural and symbolic, and have profound effects on the respondent’s negotiation of their environment, their relationships and their ‘self’. In Cazucá, Felipe (aged 16 years) feels the daily experience of violence impacts his ability to see himself as capable of effecting change: ‘The violence, well, I think it affects whoever it wants, no? ... I can’t do anything because the violence never leaves us’, while Brayan Alexander (aged 15 years) highlights how violence becomes normalised and thus difficult to change, despite its damaging consequences: ... Navigating everyday life So you leave anyway and you hope that you return home’. Such navigation requires knowing about the community; about why people might accuse you of doing something; about who, what and which locations to avoid; and how to present yourself. Activities might temporarily be suspended in the case of violent events that directly affect a community but daily life often resumes again promptly. This occurs not as a negation of risk or a lack of fear but rather the opposite; namely, the importance of the routines of everyday life. When Alejandro in los Altos de Cazucá was asked if he ever felt scared walking to or from school, he explained slowly and clearly – as if it should be self‐evident – that he ‘can’t just stop going to school; how else will things ever change if [he] gives up?’. As a result, young people and their parents used information from neighbours as well as their own expertise about their community to mitigate risk and attempt safe navigation. As Claudia in San Luis Potosí explained: You can’t just stop living your life, you have needs. You have to go to work, you have to continue your life. You have to ... I mean you can learn to live with it ... You have to accept that it can happen to anyone at any time, you can always be at the wrong place at the wrong time. Despite the threat of violence, Claudia recognises that individuals must continue to move through their everyday environments, and that requires certain recognition that even a skilled navigator can fail to escape violence; the oppressive and unpredictable nature of structural and direct violences in these places can frustrate individual efforts. In this way occupants of both locations sometimes restricted their own movements, avoiding going out. This seemed one of the most used navigation strategies and one of which respondents were consciously aware. Many of the respondents in San Luis Potosí, for example, argued that their lives had not changed, until they discussed precaution‐taking. However, avoidance of leaving the home seemingly had an emotional impact that led to awareness about the strategy adapted, and many of the respondents had their own reflections about this. Carlos, for example, stated: ‘San Luis is like the world upside down. Navigating everyday life We have discussed the concept of navigation as a counter to merely ‘coping’ or ‘normalising’ violence. This idea, developed from Vigh’s work (2009) affirms that individuals are not fixed points on a landscape battered by violences but, rather, individuals in situations of insecurity who navigate topographically and temporally to minimise harm, avoid violence and mitigate fear. Furthermore, we argue that the occupants of los Altos de Cazucá and San Luis Potosí are in fact skilled navigators, and have expertise in these topographical and temporal navigations of violence. Occupants must ‘involve themselves in their social worlds’ (Guyer 2007) and thus must embody ‘motion within motion’ to respond to situations as they arise. In los Altos de Cazucá, decisions by young people and their parents to take alternate paths to school because of information received by neighbours is an example of the skilled navigator at 111 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico work. Equally, the networks of information passed on from neighbour to neighbour in Colombia are evidence of sharing expertise to assist other’s navigation: ‘motion within motion’. While violence and fear can be oppressive, everyday life cannot cease. For Paola Andrea and Alejandro the most difficult thing about living in Cazucá is simply ‘to survive [sobrevivir]’. In San Luis Potosí respondents similarly identified adapting many daily activities in attempts to better navigate and predict violence. These included trading expensive cars for cheaper models so as not to draw attention; asking family members to pick up respondents rather than taking a bus or a taxi; avoiding eye contact with police or strangers; avoiding talking publicly about the problems or salary, whether high or low; prompted by fear of extortions or kidnappings, changing social media names into fictional names; and women starting to dress more conservatively. In doing so, respondents skilfully navigate their environment through informed, rationalised and weighted decision‐making. Adaptations to navigate violence so that the vital parts of daily life can continue thus occur in many small steps rather than a few big ones. Martha, in San Luis Potosí, explained: ‘you take precautions, right. Because you know that the violence is present but you cannot leave your routine because of fear. So you take your precautions and continue your life, even though something might happen. Navigating everyday life The murderers, kidnappers and criminals walk freely in the streets and the citizens are locked in the prisons made up by their own houses’. In Colombia, participants also spoke of feeling ‘stuck’ in their houses, at times unable to leave. In these stories they are navigating dangerous waters by avoiding being on the streets at times when it is potentially risky. But navigation has consequences: people can feel trapped by the violence. Navigation, then, is not a straightforward ‘solution’ to violence or fear, but rather merely a way of responding to the best of an individual’s ability. 112 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Although a large part of the responses by participants in Colombia were about the difficulties and dangers they had become experts in navigating, there was a concurrent series of stories about resilience and hope that challenged a totalising narrative. These efforts stemmed from frustration with the duration of the civil war and the absence of state assistance. One woman in Cazucá described how, since being in this community, she had become more aware of her rights as a person, and noted that, ‘because we are all in this [the uncertain situation of daily life] together,’ she has built networks of advocacy and resilience with her neighbours and friends. The community of Cazucá, and other communities in Soacha have a history of social organising despite (or sometimes because of) threats of violence (Rodriguez et al. 2009), in particular the Madres de Soacha who protested the kidnap and killing of their sons by government forces at great personal risk (Mateo Medina 2013). This is a more fraught form of navigation because it exposes individuals to risk and retribution but it is also one that these individuals are uniquely skilled in and able to navigate. In the case of San Luis Potosí, where the respondents had been experiencing a steady and shocking increase in violence rather than the long duration of insecurity of those in Cazucá, the sentiments were less optimistic. However, a growing resistance accompanied by a desire to counter the violence through breaking the self‐censorship was observed. Claudia, for instance, stated: Violence is also something that has kept us quiet, I think it is time to talk, to say: you know what, I do not agree. Navigating everyday life I do not agree with these people who have kept us living in fear; living in fear is an ugly thing. I have certain rules. I do not answer phone numbers that I don’t know on the phone or caller ID because they might extort you and sometimes when they ask my name, I give them another name because I have become very suspicious of people. It’s like if you go somewhere cool and someone wants to talk to you and you just almost say nothing, things like that. Well it is true that Potosínos are a bit closed, but due to this we have become more hermetic. But I think it is time to talk. Fear of violence can shut down individual’s capacity to respond. However, in both these communities individuals identify and navigate towards visions of life that affirm capacity for change, although to very different degrees. Such examples demonstrate that navigation can be more than avoidance strategies and can, in some cases, result in organised resistance to violence and insecurity. While navigation can allow an amelioration or avoidance of violence, it is undertaken because of pervasive and shifting waters that hold the promise of violence and involves risk to the individual. There are many ways in which occupants of both communities have become skilled at the topographical and temporal navigation of their daily lives. While some of these techniques allow for the mitigation of risk so that the vital aspects of daily life can continue to be carried out, at the same time individuals often feel trapped by fear, and avoid leaving the house or limit their movements. Navigation as a form of expression of resilience or hope was more evident in Cazucá because the duration of violence had meant occupants had built networks of capacity building in response to structural violence and state abnegation. In San Luis Potosí, however, respondents spoke less of resilience than of a fear that a new generation was growing up without ever having known peace and therefore not feeling a need to restore it. Fear and violence are not totalising narratives of either community, and skilled navigators wayfind with varying degrees of success on a day‐to‐day basis. Navigation enables individuals and communities to find ways of moving through and between the insecurity and fear. Conclusion Occupants of San Luis Potosí and Cazucá live in contexts of insecurity and risk. Their daily lives are impacted by experiences of insecurity and violence in different forms. Life in communities affected by violence is fraught and challenging but those who live daily in such places are not 113 IJCJ&SD © 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico inherently powerless victims. Instead they acquire knowledge from many sources – their own experiences; experiences of their neighbours, family and friends; news sources; and stories and rumours – and their ability to judge, ‘read’ the situation and use this information determines how they navigate their daily lives. This knowledge, vital in navigating the unpredictable waters of their communities, is a learned skill which enables manoeuvring in ways that minimise, avoid and mitigate risk and fear. For those outside these places, the environment may seem totalising and impossible to read. However, those who live there have learnt about the local topography and temporality of violence and insecurity through storytelling and experience. This has made them into skilled navigators who are able to read the currents, predict the weather, and set course to sail in these choppy waters. We suggested previously that such violence and fear is unable to be ‘cured’ but rather has to be managed like a chronic condition. It is this management that takes the form of temporal and spatial navigation through, at times, shifting unpredictable waters that makes the skilled navigator. Violence is complex; it can appear in direct, structural and cultural forms (Galtung 1990), all of which impinge on the lifeworlds of individuals. However, to render an area inherently violent or assumptively predisposed to violence and insecurity flattens and limits understandings of such communities. We have argued through this paper, drawing on Vigh’s (2009) concept of a social navigator, that attention to people’s everyday lives and navigation strategies crucially resists totalising narratives and instead opens new entry points to theorising and responding to violence and fear. Complex violences, including direct violence by organised criminal groups, structural violence of the abnegation of communities or the loss of monopoly of force by the state, and the cultural violence in which assumptions and stereotypes exist about communities and individuals, are reoccurring experiences throughout the global South. 2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality. 3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term ‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities g p pp p y g y g y 3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term ‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities in which they work. We see our own work reflected in this discussion of navigation and becoming skilled; however, in neither situation did we ever fully overcome these obstacles and our navigation was always conditional and choppy. It is useful to include this reflexivity here, and acknowledge the work done by Pearce (2009), Baird (2009) and Wheeler (2009) in drawing it out explicitly in the aforementioned edited volume. 2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality. p pp p y y 2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality. 3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term ‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities in which they work. We see our own work reflected in this discussion of navigation and becoming skilled; however, in neither situation did we ever fully overcome these obstacles and our navigation was always conditional and choppy. It is useful to include this reflexivity here, and acknowledge the work done by Pearce (2009), Baird (2009) and Wheeler (2009) in drawing it out explicitly in the aforementioned edited volume 1 Approval for research in Colombia was granted by the University of Queensland’s Behavioural and Social Science Review Board on 1 September 2010. Support for research in Mexico was provided by the VU University Amsterdam Conclusion We propose that the theoretical intervention we make here, illustrated by our work in Colombia and Mexico, is a starting point to think in more productive ways about violence, fear and the skilled navigation of everyday life. Recognising individuals as skilled navigators is crucial for understanding complex live experiences amidst violence. Correspondence: Dr Helen Berents, Lecturer, School of Justice, Faculty of Law, Queensland University of Technology, 2 George Street, Brisbane 4000 QLD, Australia. Email: helen.berents@qut.edu.au 3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term ‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities in which they work. 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DOI: 10.1111/j.1759‐ 5436.2009.00044.x. IJCJ&SD 117 © 2017 6(1) IJCJ&SD 117 © 2017 6(1) Online version via www.crimejusticejournal.com Online version via www.crimejusticejournal.com
https://openalex.org/W3094443244	https://zenodo.org/record/4781139/files/Social%20Drone%20Sharing%20to%20Increase%20the%20UAV%20Patrolling%20Autonomy%20in%20Emergency%20Scenarios.pdf	English	null	Social Drone Sharing to Increase the UAV Patrolling Autonomy in Emergency Scenarios	null	2,020	cc-by	1,876	Genova, Italy Email: ∗luca.morando@edu.unige.it, †carmine.recchiuto@dibris.unige.it, ‡antonio.sgorbissa@unige.it to a time of ﬂight of 15-30 minutes, thus resulting in a reduced operative area. to a time of ﬂight of 15-30 minutes, thus resulting in a reduced operative area. Abstract—Unmanned Aerial Vehicles (UAVs) popularity is increased in recent years, and the domain of application of this new technology is continuously expanding. However, although UAVs may be extremely useful in monitoring contexts, the operational aspects of drone patrolling services have not yet been extensively studied. Speciﬁcally, patrolling and inspecting with UAVs different targets distributed over a large area is still an open problem, due to battery constraints and other practical limitations. In this work, we propose a deterministic algorithm for patrolling large areas in a pre- or post-critical event scenario. The autonomy range of UAVs is extended with the concept of Social Drone Sharing: citizens may offer their availability to take care of the UAV if it lands in their private area, being thus strictly involved in the monitoring process. The proposed approach aims at ﬁnding optimal routes in this context, minimizing the patrolling time and respecting the battery constraints. Simulation experiments have been conducted, giving some insights about the performance of the proposed method. Our proposal lies in the context of pre-operative or post- operative intervention in emergency scenarios, where rescue teams or the civil protection need to periodically monitor or survey different critical targets located in various places taking as little time as possible. These targets could be, for instance, ﬁelds or woods whose dryness shall be periodically assessed to prevent ﬁres, soil slopes whose stability shall be monitored before or after ﬂoods, critical infrastructure (hospitals, schools, roads, bridges, etc.) whose risk shall be evaluated before or after earthquakes. In most cases the maximum drone ﬂight time, generally deﬁned by the drone technical speciﬁcation, is not sufﬁcient to visits all targets. For these reasons, the proposed approach aims to involve citizens in the monitoring process, by introducing the new concepts of Social Charging Stations (CS) and Social Drone Sharing (SDS). Index Terms—Autonomous UAV, Optimization, Social Robotics. More in detail we foresee a software architecture that is compose by different elements. A platform that permits to make in communication the authorities with the civil volun- teers, making also them able to verify the status of the drones involved in the patrolling activity and their ﬂight plans in real-time. Genova, Italy Email: ∗luca.morando@edu.unige.it, †carmine.recchiuto@dibris.unige.it, ‡antonio.sgorbissa@unige.it Finally, by a computational architecture based on a multiple deterministic approach that permits to ﬁrst maximize the coverage of the operational area given the availability of the resident volunteers and second to evaluate the optimal path for the UAVs taking into account the energy and the inspection constraints. I. INTRODUCTION Multirotor drone technology is available on a large scale on market and widely utilized in research. Indeed multirotors are endowed with several speciﬁc features such as vertical take-off and landing, the ability to hover freely in the air, the reduced size and the possibility to be programmed, that make them very interesting in different scenarios. Thanks to all these characteristics, drones are now used in mapping [1], in agriculture [2], in inspection sites [3] and soon they may be also used in delivery. Emergency scenarios are another domain where UAVs are becoming increasingly important [4]. Indeed there are various examples in literature, starting from the nuclear disaster of Fukushima [5] to the pandemic disease caused from the virus Sars-Cov-2, where UAVs have been successfully employed: in particular, drones have been utilized to control the body temperature of a multitude of people in the street during quarantine periods [6]. Despite their effectiveness, a signiﬁcant limit to the use of this technology lies in the absence of true autonomy. This problem is twofold: Civil authorities can interface with the platform to change the targets to survey depending on the need, and also visualize the CS available in the operative area. Resident volunteers give their availability through a mobile application, which alerts the user if their help is required to prepare the landing spot and to recharge the batteries if the drone lands on their property. The purpose is to actively involved residents volunteers in risk assessment and management areas, which may be important especially in areas with low population density where a community-based approach is repaid by a greater safety. • autonomous navigation behavior, i.e. the capability to autonomously coordinate a team of UAVs to perform a patrolling operations, is missing, thus requiring expert pilots also in the simpler operations. • autonomous navigation behavior, i.e. the capability to autonomously coordinate a team of UAVs to perform a patrolling operations, is missing, thus requiring expert pilots also in the simpler operations. Since resident volunteers are involved, which raises a num- ber of logistics and legal issues, it is important to minimize the number of the charging stations required and, at the same time, maximize the inspection time in each target area. Social Drone Sharing to Increase the UAV Patrolling Autonomy in Emergency Scenarios Luca Morando∗, Carmine Tommaso Recchiuto† and Antonio Sgorbissa‡ Laboratorium - Dibris, University of Genoa 2020 I-RIM Conference December 10-12, 2020, Rome, Italy (Virtual) ISBN: 9788894580518 DOI:10.5281/zenodo.4781139 2020 I-RIM Conference December 10-12, 2020, Rome, Italy (Virtual) ISBN: 9788894580518 DOI:10.5281/zenodo.4781139 2020 I-RIM Conference December 10-12, 2020, Rome, Italy (Virtual) ISBN: 9788894580518 DOI:10.5281/zenodo.4781139 Luca Morando∗, Carmine Tommaso Recchiuto† and Antonio Sgorbissa‡ Laboratorium - Dibris, University of Genoa Genova, Italy Email: ∗luca.morando@edu.unige.it, †carmine.recchiuto@dibris.unige.it, ‡antonio.sgorbissa@unige.it II. PROBLEM STATEMENT Our approach is based on the utilization of a single UAV, or a team of completely autonomous drones which are given by authorities to resident volunteers. The idea is that people give their availability in securing a recharging station spot in their property, such as a terrace or a garden, that a drone may use if it requires to be recharged. The availability of the resident volunteers are given as input to a ﬁrst optimization algorithm, based on a MILP implementation, that permits to maximize the covering area, minimizing at the same time the number of CS involved. An example of this optimization tool is given in Figure 1, where the operative area is discretized and the possible locations for the targets to be inspected are located on the map. As visible in the ﬁgure, only the charging stations that permit to reach the maximum number of targets keeping the path continuity are chosen. In fact the criterion of selection is also based on the energy autonomy of the UAV utilized, indicated in the map via a circle (which ray length is based on the distance covered by the UAV with half of a charge) centered on each of the selected charging station. The solution consist in an optimal selection of the charging stations, maxi- mizing the number of reachable targets keeping also the path continuity, as demonstrated by the overlap of different circles. The second optimization algorithm, expansively described in [7], take as input the targets and the charging station located in a better strategical position. The optimization procedure, based on a MILP algorithm deﬁned by only two decision variables, draw the best path that the UAV have to accomplish in order to visit all the inspection targets, respecting also the energy constraints imposed by the batteries charge. An example of the algorithm is visible in Figure 2, where the performance are exploited in a realistic simulation located in a rural area, III. CONCLUSION AND FUTURE WORK This paper proposes a novel approach for the optimal routing of UAVs based on the new concepts of Social Charging Stations and Social Drone Sharing. Indeed, involving citizens in the monitoring process may be vital for increasing UAVs patrolling capabilities. The approach has been preliminarily validated with simulation experiments, analysing computa- tional time and general performances, and eventually showing its feasibility. Future extensions of this work concern the possibility of using more drones that share the Targets and their inspection time. In any case, the possibilities to expand this work, to adapt it to a real patrolling scenario, are enormous, and all very in- teresting to explore, ultimately leading to a wider deployment of UAVs in critical contexts. [1] Patrik Schmuck and Margarita Chli, “Multi-UAV Collaborative Monoc- ular SLAM,” 2017 IEEE International Conference on Robotics and Automation (ICRA) Singapore, May 29 - June 3, 2017. I. INTRODUCTION To this aim, this work describes an approach to ﬁnd optimal routes • energetic autonomy, i.e., the capability to cover long distances without the need to be recharged, is limited • energetic autonomy, i.e., the capability to cover long distances without the need to be recharged, is limited Fig. 1. Maximization of the target covering area via an optimal selection of the charging station place. Fig. 2. One solution of the iterative realistic simulation obtained with a number of 14 charging station and an inspection time tisp = 1080s in each target. Fig. 2. One solution of the iterative realistic simulation obtained with a number of 14 charging station and an inspection time tisp = 1080s in each target. where the continuous patrolling of the agricultural ﬁeld is necessary. The charging stations are located near the house of the resident volunteers. In the evaluation of the optimal path, the energy autonomy (estimated in an empirical way) of a DJI Mavic 2 Pro is considered. Each target is associated to an inspection time required to complete the monitoring task. Figure 2 shows the ﬁnal solution obtained after an iterative optimization process, where the inspection time required in each target is increased until no more solutions can be found. At this point more charging stations in various location are added to the map and the optimization process is repeated, obtaining the maximum inspection time in each target with the maximum number of available charging stations, ﬁnally placed on the map. In the solution depicted in Figure 2, a maximum inspection time of tins = 1080s is obtained with a total number of 14 charging stations and 5 targets. The computational time required by the optimization algorithm, running on Matlab on a Apple MacBook Pro equipped with an Intel Core i5 and 8 Gb of RAM, is about topt = 5.9377s. Fig. 1. Maximization of the target covering area via an optimal selection of the charging station place. for autonomously ﬂying UAVs in patrolling contexts, taking into account the new concepts of Social Drone Sharing. REFERENCES [1] Patrik Schmuck and Margarita Chli, “Multi-UAV Collaborative Monoc- ular SLAM,” 2017 IEEE International Conference on Robotics and Automation (ICRA) Singapore, May 29 - June 3, 2017. [2] J. Gagoa, C. Doutheb, R.E. Coopmanc, P.P. Gallegoa, M. Ribas-Carbob, J. Flexasb, J. Escalonab, H. Medranob. (2015) ”UAVs challenge to assess water stress for sustainable agriculture”. Agricultural water management, 153, 9-19. [3] Nadeem M Shajahan, Arjun Sasikumar, Thomas Kuruvila and Dhivin Davis, “Automated Inspection of Monopole Tower using Drones and Computer Vision” 2019 International Conference on Intelligent Au- tonomous Systems (ICoIAS). [4] Recchiuto, C.T., Sgorbissa, A. Post-disaster assessment with unmanned aerial vehicles: A survey on practical implementations and research approaches (2018) Journal of Field Robotics, 35 (4), pp. 459-490. [5] https://www.neimagazine.com/features/featurefukushima-looks-to- drones-6728269/ [6] https://www.thelocal.it/20200410/hovering-police-drones-take-italians- temperature-and-issue-ﬁnes. [7] Morando, L., Recchiuto, C.T., Sgorbissa, A.. ”Social Drone Sharing to Increase the UAV Patrolling Autonomy inEmergency Scenarios”, (2020), 29th IEEE International Conference in Human Robot Interaction (RO- MAN), Naples, IT.
https://openalex.org/W2948572625	http://fulir.irb.hr/5408/2/ALICE_EurPhysJC_79_2019_857_arxive1905.07208.pdf	English	null	Charged-particle production as a function of multiplicity and transverse spherocity in pp collisions at $$ \sqrt{\mathbf{s}} =\mathbf{5.02}$$ and 13 TeV	European physical journal. C, Particles and fields	2,019	cc-by	20,275	EUROPEAN ORGANIZATION FOR NUCLEAR RESEARCH UROPEAN ORGANIZATION FOR NUCLEAR RESEARCH CERN-EP-2019-094 7 May 2019 ALICE Collaboration∗ ALICE Collaboration∗ c⃝2019 CERN for the beneﬁt of the ALICE Collaboration. Reproduction of this article or parts of it is allowed as speciﬁed in the CC-BY-4.0 license. c⃝2019 CERN for the beneﬁt of the ALICE Collaboration. ∗See Appendix A for the list of collaboration members c⃝2019 CERN for the beneﬁt of the ALICE Collaboration. Reproduction of this article or parts of it is allowed as speciﬁed in the CC-BY-4.0 license. Reproduction of this article or parts of it is allowed as speciﬁed in the CC-BY-4.0 license. Charged-particle production as a function of multiplicity and transverse spherocity in pp collisions at √s = 5.02 and 13 TeV v:1905.07208v2 [nucl-ex] 8 Nov 2019 1 Introduction Proton-proton collisions at the Large Hadron Collider (LHC) energies have unveiled features very simi- lar to the ones observed in heavy-ion collisions [1]. The previous consensus of the heavy-ion community was that the partonic system created in nuclear collisions needs a large volume to thermalize and to lead to phenomena like collective ﬂow. However, radial [2–4] and anisotropic ﬂow [5], as well as strangeness enhancement [6], are also observed in pp and p-A collisions when they are studied as a function of event multiplicity. Surprisingly, with the same level of precision, microscopic and macroscopic approaches describe qualitatively well the observed features in pp collisions. While macroscopic models incorporate hydrodynamical evolution of the system [7], the others include overlapping strings [8], string percola- tion [9], multi-parton interactions and color reconnection [10, 11]. The multiphase transport model [12], as well as the fragmentation of saturated gluon states [13, 14], is able to describe some features of data. The inclusive transverse momentum (pT) spectrum of charged particles carries information of the dy- namics of soft and hard interactions. On one hand, the high-pT (pT > 10 GeV/c) particle production is quantitatively well described by perturbative QCD (pQCD) calculations; on the other hand, the un- derstanding of particle production at low-pT has to resort to phenomenological QCD inspired models. Most of the new effects discovered in pp collisions have been unveiled in the low- (pT < 2 GeV/c) and intermediate- (2 ≤pT < 10 GeV/c) pT domains [2–6]. The present paper reports a novel multi-differential analysis aimed at understanding charged-particle production associated to partonic scatterings with large momentum transfer and their possible correlations with soft particle production. The transverse momentum distributions are reported for two multiplicity estimators which cover different pseudorapidity regions. The estimators are based on either the total charge deposited in the forward detector (covering the pseudorapidity regions 2.8 < η < 5.1 and −3.7 < η < −1.7) or on the number of tracks in the pseudorapidity region \|η\| < 0.8. The forward multiplicity estimator is commonly used by the ALICE collaboration to minimize the possible autocorrelations induced by the use of the mid- pseudorapidity estimator. One such examples is the “fragmentation bias” [15], which is the correlation between jet fragments and event multiplicity arising when the particle’s pT and event multiplicity are both measured within the same pseudorapidity interval [16]. Abstract arXiv:1905.07208v2 [nucl-ex] 8 Nov We present a study of the inclusive charged-particle transverse momentum (pT) spectra as a function of charged-particle multiplicity density at mid-pseudorapidity, dNch/dη, in pp collisions at √s = 5.02 and 13 TeV covering the kinematic range \|η\| < 0.8 and 0.15 < pT < 20 GeV/c. The results are presented for events with at least one charged particle in \|η\| < 1 (INEL> 0). The pT spectra are reported for two multiplicity estimators covering different pseudorapidity regions. The pT spectra normalized to that for INEL> 0 show little energy dependence. Moreover, the high-pT yields of charged particles increase faster than the charged-particle multiplicity density. The average pT as a function of multiplicity and transverse spherocity is reported for pp collisions at √s = 13 TeV. For low- (high-) spherocity events, corresponding to jet-like (isotropic) events, the average pT is higher (smaller) than that measured in INEL> 0 pp collisions. Within uncertainties, the functional form of ⟨pT⟩(Nch) is not affected by the spherocity selection. While EPOS LHC gives a good description of many features of data, PYTHIA overestimates the average pT in jet-like events. Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 2 The ALICE apparatus The main detectors used in the present work are the Inner Tracking System (ITS), the Time Projection Chamber (TPC) and the V0 detector. The ITS and TPC detectors are both used for primary vertex and track reconstruction. The V0 detector is used for triggering and for background rejection. More details concerning the full ALICE detector system can be found in Ref. [22]. The central barrel covers the pseudorapidity region \|η\| < 0.8 for full-length tracks. The main central- barrel tracking devices are the ITS and the TPC, which are located inside a solenoid magnet providing a 0.5 T magnetic ﬁeld allowing the tracking of particles from 0.15 GeV/c. The ITS is composed of six cylindrical layers of high-resolution silicon tracking detectors. The innermost layers consist of two ar- rays of hybrid Silicon Pixel Detectors (SPD) located at an average radial distance (r) of 3.9 and 7.6 cm from the beam axis and covering \|η\| < 2 and \|η\| < 1.4, respectively. The SPD is also used to reconstruct tracklets, which are track segments built using the position of the reconstructed primary vertex and two hits, one on each SPD layer. The number of tracklets gives an excellent estimate of the charged-particle multiplicity at mid-pseudorapidity (Nch). The outer layers of the ITS are composed of silicon strip and drift detectors, with the outermost layer sitting at r = 43 cm. The TPC is a large cylindrical drift detector of radial and longitudinal size of about 85 < r < 250 cm and −250 < z < 250 cm, respectively. It is seg- mented in radial “pad rows”, providing up to 159 tracking points. The measurement of charged particles is based on “global tracks”, reconstructed using the combined ITS and TPC information. The V0 de- tector consists of two forward scintillator arrays (V0-A and V0-C) employed for triggering, background suppression, and event-class determination. They are placed on either side of the interaction region at z = 3.3 m and z = −0.9 m, covering the pseudorapidity regions 2.8 < η < 5.1 and −3.7 < η < −1.7, respectively. The data were collected using a minimum-bias trigger which required coincident signals in both V0-A and V0-C detectors . The events were recorded in coincidence with signals from two beam pick-up counters each positioned on either side of the interaction region to tag the arrival of proton bunches from both directions. 1 Introduction For each estimator, we deﬁned different multiplicity classes based on either the number of tracks at mid-pseudorapidity (\|η\| < 0.8) or the signal in the forward detectors. It is worth mentioning that a similar study has been performed by ALICE using p-Pb data; the results showed different modiﬁcations of the spectral shapes depending on the multiplicity estimators which were used [17]. To disentangle the energy and multiplicity dependence, for a given multiplicity class, the pT distributions are measured for pp collisions at √s = 5.02 and 13 TeV. Particle production from intermediate to high pT (> 4 GeV/c) is studied by ﬁtting a power-law function to the invariant yield, and studying the multiplicity and energy dependence of the exponent. This has been proposed in Ref. [18] as a way to characterize the high-pT tails of different systems and energies in a convenient way that may make the comparison for the different systems more straightforward. Finally, we explore a new approach, which has been proposed to study multi-parton interaction effects in pp collisions. Transverse spherocity [19], hereinafter referred to as spherocity, has been proven to be a valuable tool to discriminate between jet-like and isotropic events [20] associated with an underlying event activity which is either suppressed or enhanced. The previous measurement of average transverse momentum of inclusive charged particles as a function of event multiplicity [21] is now explored adding a new dimension: the event shape characterized by spherocity. The aim of this study is to investigate the importance of jets in high-multiplicity pp collisions and their contribution to charged-particle production at low pT. The paper is organized as follows: Section 2 describes the run conditions during the data taking and the main detectors used in the present analysis. Section 3 outlines the analysis details for the event and track selection, as well as the deﬁnitions of the different event classes. The correction procedures and the 2 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration estimation of the systematic uncertainties are summarized in Sections 4 and 5, respectively. Results and discussions are presented in Section 6. Finally, our summary and conclusions are reported in Section 7. 2 The ALICE apparatus Control triggers taken for various combinations of beam and empty buckets were used to measure beam-induced and accidental backgrounds. The contamination from background events was removed ofﬂine by using the timing information from the V0 detector, which has a time resolution better than 1 ns. Background events were also rejected by exploiting the correlation between the number of clusters of pixel hits and the number of tracklets in the SPD. 3 Analysis The results presented here were obtained from the analysis of about 105 and 60 million minimum-bias pp events at √s = 5.02 and 13 TeV, respectively. The interaction probability per single bunch cross- ing ranges between 2% and 14% for pp collisions at 13 TeV and from 0.3% to 6% for pp collisions at 5.02 TeV. The measurements have been obtained for events having at least one charged particle produced in the pseudorapidity interval \|η\| < 1 (INEL> 0). For the analysis, the events were furthermore required to have a reconstructed vertex located within \|z\| < 10 cm, where z is the position of the vertex along the beam axis, and z = 0 cm corresponds to the nominal center of the detector [22]. Events containing more than one distinct vertex were tagged as pileup and discarded from the analysis. The systematic uncer- tainty associated to pileup is between 3 – 4% and is not the dominant source of uncertainty for the pT spectra reported here. The corrections are calculated using Monte Carlo events from the PYTHIA 6 [23] (tune Perugia 2011 [24]) event generator with particle transport performed via a GEANT 3 [25] simula- tion of the ALICE detector. 3 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Only primary charged particles in the kinematic range \|η\| < 0.8 and 0.15 < pT < 20 GeV/c are consid- ered in the transverse momentum analysis. A primary charged particle is deﬁned as a charged particle with a mean proper lifetime τ larger than 1 cm/c, which is either produced directly in the interaction or from decays of particles with τ smaller than 1 cm/c, excluding particles produced in interactions with the detector material [26]. Only primary charged particles in the kinematic range \|η\| < 0.8 and 0.15 < pT < 20 GeV/c are consid- ered in the transverse momentum analysis. A primary charged particle is deﬁned as a charged particle with a mean proper lifetime τ larger than 1 cm/c, which is either produced directly in the interaction or from decays of particles with τ smaller than 1 cm/c, excluding particles produced in interactions with the detector material [26]. Transverse momentum distributions. The measurement of the pT spectra follows the standard pro- cedure already employed in several ALICE publications [27–29]. Tracks reconstructed using the infor- mation from the ITS and TPC detectors are used. 3 Analysis The track selection criteria have been optimised for best track quality and minimal contamination from secondary particles. Tracks are required to have at least two hits in the ITS detector, of which at least one is in either of the two innermost SPD layers. The geometrical track length L (in cm) is calculated in the TPC readout plane, excluding the information from the pads at the sector boundaries (∼3 cm from the sector edges). The number of crossed TPC rows has to be larger than 0.85L. The number of TPC clusters has to be larger than 0.7L. The ﬁt quality for the ITS and TPC track points must satisfy χ2 ITS/Nhits < 36 and χ2 TPC/Nclusters < 4, respectively, where Nhits and Nclusters are the numbers of hits in the ITS and the number of clusters in the TPC, respectively. Tracking information from the combined ITS and TPC track reconstruction algorithm is compared to that derived only from the TPC and constrained by the interaction vertex point. Then, the quantity χ2 TPC−ITS is derived as described in Ref. [30]. Only tracks with χ2 TPC−ITS < 36 are included in the analysis in order to improve the purity of primary track reconstruction at high pT. Tracks are rejected if their distance of closest approach to the reconstructed vertex in longitudinal and radial direction, dz and dxy, respectively, satisﬁes dz > 2 cm or dxy > 0.018 cm + 0.035 cm ×p−1.01 T , with pT in GeV/c. Multiplicity estimators. In order to study the multiplicity dependence of the inclusive charged particle pT distributions, the INEL> 0 sample is divided into event classes based on the total charge deposited in the V0 detector (V0M amplitude) and on the number of SPD tracklets (NSPDtracklets) in the pseudorapid- ity region \|η\| < 0.8. The event classes used in the analysis and the corresponding mid-pseudorapidity charged particle densities are summarized in Tables 1 and 2. The average charged-particle multiplicity densities for INEL> 0 collisions and for the multiplicity classes are obtained by integrating the corre- sponding fully corrected pT spectra (measured using ITS and TPC information). To this end, the pT spectra were extrapolated to pT = 0 with a Hagedorn function [31]. Different functions were used and the differences with respect to the reference values were considered in the systematic uncertainties. 3 Analysis For INEL> 0 pp collisions at √s = 5.02 TeV the mid-pseudorapidity (\|η\| < 0.8) charged-particle density is ⟨dNch/dη⟩= 5.91±0.45, while for √s = 13 TeV the corresponding value is 7.60±0.50. The compar- ison of results obtained with these estimators allows to understand potential biases from measuring the multiplicity and pT distributions in overlapping η regions. Spherocity. For the data analysis we followed a strategy similar to that already reported in Ref. [32]. Spherocity, S0, originally proposed here [33] is deﬁned for a unit vector ˆns which minimizes the ratio: S0 ≡π2 4 min ˆns ∑i \|⃗pT,i × ˆns\| ∑i pT,i 2 , (1) (1) where the sum runs over all reconstructed ITS-TPC tracks. At least three tracks are required within \|η\| < 0.8 and pT > 0.15 GeV/c in order to achieve a good spherocity resolution. The spherocity resolu- tion improves with the track-reconstruction efﬁciency, therefore the restrictions on the purity of primary charged particles can be relaxed. For spherocity we considered all tracks with at least 50 clusters in the TPC, which satisfy: dxy < 2.4 cm, dz < 3.2 cm, and χ2 TPC/Nclusters < 4. The exclusion of the ITS requirements guarantees a homogeneous azimuthal track-reconstruction efﬁciency. where the sum runs over all reconstructed ITS-TPC tracks. At least three tracks are required within \|η\| < 0.8 and pT > 0.15 GeV/c in order to achieve a good spherocity resolution. The spherocity resolu- tion improves with the track-reconstruction efﬁciency, therefore the restrictions on the purity of primary charged particles can be relaxed. For spherocity we considered all tracks with at least 50 clusters in the TPC, which satisfy: dxy < 2.4 cm, dz < 3.2 cm, and χ2 TPC/Nclusters < 4. The exclusion of the ITS requirements guarantees a homogeneous azimuthal track-reconstruction efﬁciency. It is worth mentioning some important features of spherocity: 4 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Table 1: V0M event multiplicity classes, their corresponding experimental deﬁnition and their corresponding ⟨dNch/dη⟩in \|η\| < 0.8. The uncertainties are the quadratic sum of statistical and systematic contributions. Statis- tical uncertainties are negligible compared to the systematic ones. 3 Analysis pp collisions at √s = 13 TeV Class name I II III IV V V0M percentile 0 – 1% 1 – 5% 5 – 10% 10 – 15% 15 – 20% ⟨dNch/dη⟩ 26.6±1.1 20.5±0.8 16.7±0.7 14.3±0.6 12.6±0.5 Class name VI VII VIII IX X V0M percentile 20 – 30% 30 – 40% 40 – 50% 50 – 70% 70 – 100% ⟨dNch/dη⟩ 10.6±0.5 8.46±0.40 6.82±0.34 4.94±0.28 2.54±0.26 pp collisions at √s = 5.02 TeV Class name I II III IV V V0M percentile 0 – 1% 1 – 5% 5 – 10% 10 – 15% 15 – 20% ⟨dNch/dη⟩ 19.2±0.9 15.1±0.7 12.4±0.6 10.7±0.5 9.47±0.47 Class name VI VII VIII IX X V0M percentile 20 – 30% 30 – 40% 40 – 50% 50 – 70% 70 – 100% ⟨dNch/dη⟩ 8.04±0.42 6.56±0.37 5.39±0.32 4.05±0.27 2.27±0.27 Table 2: Event multiplicity classes based on the number of tracklets (NSPDtracklets) within \|η\| < 0.8, their cor- responding experimental deﬁnition and their corresponding ⟨dNch/dη⟩in \|η\| < 0.8. The uncertainties are the quadratic sum of statistical and systematic contributions. Table 2: Event multiplicity classes based on the number of tracklets (NSPDtracklets) within \|η\| < 0.8, their cor- responding experimental deﬁnition and their corresponding ⟨dNch/dη⟩in \|η\| < 0.8. The uncertainties are the quadratic sum of statistical and systematic contributions. pp collisions at √s = 13 TeV Class name (percentile) I′ 0–0.006% II′ 0.006–0.058% III′ 0.058–0.177% IV′ 0.177–0.513% V′ 0.513–1.419% NSPDtracklets ≥51 41 – 50 36 – 40 31 – 35 26 – 30 ⟨dNch/dη⟩ 54.1±2.7 44.6±2.2 38.9±1.9 34.1±1.7 29.3±1.5 Class name percentile VI′ 1.419–3.699% VII′ 3.699–9.059% VIII′ 9.059–20.77% IX′ 20.77–45.25% X′ 45.25–100.0% NSPDtracklets 21 – 25 16 – 20 11 – 15 6 – 10 0 – 5 ⟨dNch/dη⟩ 24.5±1.3 19.5±1.2 14.4±0.9 9.03±0.58 2.91±0.29 pp collisions at √s = 5.02 TeV Class name (percentile) - II′ 0.009–0.088% III′ 0.088–0.253% IV′ 0.253–0.700% V′ 0.700–1.840% NSPDtracklets - 41 – 50 36 – 40 31 – 35 26 – 30 ⟨dNch/dη⟩ - 34.6±1.8 29.9±1.5 26.2±1.3 22.4±1.1 Class name (percentile) VI′ 1.840–4.573% VII′ 4.57–10.69% VIII′ 10.69–23.50% IX′ 23.50–49.48% X′ 49.48–100.0% NSPDtracklets 21 – 25 16 – 20 11 – 15 6 – 10 0 – 5 ⟨dNch/dη⟩ 18.5±1.0 14.6±0.9 10.6±0.7 6.58±0.43 2.21±0.24 – The vector products are linear in particle momenta, therefore spherocity is a collinear safe quantity in pQCD. amount of particles. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 S 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 m S 4 − 10 3 − 10 2 − 10 1 − 10 1 = 13 TeV s PYTHIA 6, pp c 0.15 GeV/ ≥ T p 0.8, ≤\| η\| =25.00 〉 η d m N d 〈 ) m S P( 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 S 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 m S 4 − 10 3 − 10 2 − 10 1 − 10 1 = 13 TeV s PYTHIA 6, pp c 0.15 GeV/ ≥ T p 0.8, ≤\| η\| =3.12 〉 η d m N d 〈 ) m S P( Fig. 1: The detector response for spherocity for two track multiplicity classes: ⟨dNm/dη⟩= 3.12 (left) and ⟨dNm/dη⟩= 25 (right). Proton-proton collisions were simulated using PYTHIA 6. The simulations include the particle transport performed via a GEANT 3 simulation of the ALICE detector. The markers (boxes around the points) indicate the average (RMS) of the measured spherocity distributions for each bin of spherocity at generator level. The spherocity binning varies with dNm/dη, because in this way, it allows the analysis of ten event sub- classes of equal size. The probability that an event with spherocity S0 be reconstructed with spherocity Sm is represented by P(Sm). Fig. 1: The detector response for spherocity for two track multiplicity classes: ⟨dNm/dη⟩= 3.12 (left) and ⟨dNm/dη⟩= 25 (right). Proton-proton collisions were simulated using PYTHIA 6. The simulations include the particle transport performed via a GEANT 3 simulation of the ALICE detector. The markers (boxes around the points) indicate the average (RMS) of the measured spherocity distributions for each bin of spherocity at generator level. The spherocity binning varies with dNm/dη, because in this way, it allows the analysis of ten event sub- classes of equal size. The probability that an event with spherocity S0 be reconstructed with spherocity Sm is represented by P(Sm). In order to study the spherocity dependence of the particle production for a given track multiplicity value, the sample is divided into ten event sub-classes of equal size (percentiles), based on the measured spherocity distribution. amount of particles. Since the goal of the present study is to separate jet events from isotropic ones, we study different sphe- rocity classes for a given multiplicity value. The multiplicity is measured by counting the number of tracks within \|η\| < 0.8. As explained later, we adopted the procedure used in the analysis of average pT as a function of multiplicity to correct the number of tracks for detector effects [21]. The detector response is represented by a two-dimensional distribution: reconstructed spherocity as a function of gen- erated spherocity, each bin of generated spherocity is normalized to unity. In this representation, the two-dimensional distribution gives the normalized response matrix R′(S0,Sm), which contains the proba- bility that an event with spherocity S0 is reconstructed with spherocity Sm. Figure 1 shows the spherocity response matrices for two track multiplicity (Nm). Tracking efﬁciency effects on the spherocity resolu- tion are relevant only for low-multiplicity events, therefore, the S0 resolution improves with increasing multiplicity. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 S 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 m S 4 − 10 3 − 10 2 − 10 1 − 10 1 = 13 TeV s PYTHIA 6, pp c 0.15 GeV/ ≥ T p 0.8, ≤\| η\| =3.12 〉 η d m N d 〈 ) m S P( 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 S 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 m S 4 − 10 3 − 10 2 − 10 1 − 10 1 = 13 TeV s PYTHIA 6, pp c 0.15 GeV/ ≥ T p 0.8, ≤\| η\| =25.00 〉 η d m N d 〈 ) m S P( Fig. 1: The detector response for spherocity for two track multiplicity classes: ⟨dNm/dη⟩= 3.12 (left) and ⟨dNm/dη⟩= 25 (right). Proton-proton collisions were simulated using PYTHIA 6. The simulations include the particle transport performed via a GEANT 3 simulation of the ALICE detector. The markers (boxes around the points) indicate the average (RMS) of the measured spherocity distributions for each bin of spherocity at generator level. The spherocity binning varies with dNm/dη, because in this way, it allows the analysis of ten event sub- classes of equal size. The probability that an event with spherocity S0 be reconstructed with spherocity Sm is represented by P(Sm). 3 Analysis – The lower limit of spherocity (S0 →0) corresponds to event topologies where all transverse mo- – The vector products are linear in particle momenta, therefore spherocity is a collinear safe quantity in pQCD. – The lower limit of spherocity (S0 →0) corresponds to event topologies where all transverse mo- mentum vectors are (anti)parallel or the sum of the pT is dominated by a single track. – The lower limit of spherocity (S0 →0) corresponds to event topologies where all transverse mo- mentum vectors are (anti)parallel or the sum of the pT is dominated by a single track. – The upper limit of spherocity (S0 →1) corresponds to event topologies where transverse momen- tum vectors are “isotropically” distributed. S0 = 1 can only be reached in the limit of an inﬁnite 5 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 4.1 Transverse momentum distributions as a function of particle multiplicity The transverse momentum spectrum for a speciﬁc event class is obtained by correcting the track yields Nrec reconstructed in each (∆η,∆pT) interval for all detector effects that either inﬂuence the event recon- struction or the track reconstruction. The transverse momentum distribution is obtained as follows: 1 Nev d2Nch dηdpT ≡Nrec(η, pT)C(η, pT) Nrec ev ∆η∆pT εev.classεvz. (2) (2) The event selection (for a speciﬁc event class) and vertex reconstruction efﬁciencies are represented by εev.class and εvz, respectively. The number of events of a given event class is represented by Nrec ev . For the lowest multiplicity class selected using the V0M amplitude and for √s = 5.02 TeV (√s = 13 TeV) they reach 66% and 95% (75% and 95%), respectively, while for the highest multiplicity class the detector is fully efﬁcient. The track-level correction factors, C(∆η,∆pT), are obtained for events which satisfy the selection criteria; they include acceptance, efﬁciency, purity, and pT resolution. The estimation of the four terms will be explained in detail in the following. A data-driven method has been developed to reduce the systematic uncertainty related to incorrect de- scription of the particle composition in Monte Carlo. The tracking efﬁciency is determined using the re-weighting procedure which is discussed for the ﬁrst time in Ref. [29] and which is employed also in the present paper. The method uses the knowledge of the particle composition at LHC energies, i.e. the abundances of the different particle species within a speciﬁc interval of pT and for a speciﬁc event class. To correct the distributions for secondary-particle contamination, i.e. the products of weak decays of kaons and Λ baryons, and the particles originating from interactions in the detector material, we used the dxy distributions of particles in data and Monte Carlo simulations. Exploiting the differences of the dxy distributions between primary and secondary particles, especially in the tails, the measured distributions were ﬁtted by a linear combination of dxy distributions (templates) for primary and secondary particles obtained from Monte Carlo simulations in different pT bins. For INEL> 0 pp collisions at √s = 13 TeV the contamination ranges from 8.5% at pT = 0.2 GeV/c to 1% for pT > 2 GeV/c. The contamination exhibits a small multiplicity dependence, which is below 2%. For pp collisions at √s = 5.02 TeV, the correction factors reach similar values. 4 Corrections All the measurements presented in this paper are fully corrected for acceptance and tracking efﬁciency, contamination from secondary particles, event and signal loss, as well as multiplicity and spherocity resolution. Details of these corrections are presented below. Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 4.1 Transverse momentum distributions as a function of particle multiplicity The transverse momentum spectra are also corrected for pT resolution; the correction factor is calculated using the covariance matrix of the Kalman ﬁt [35]. The pT (multiplicity) dependence of the correction factor is negligible [29] (below 1%). Finally, the pT spectra are corrected for the amount of signal which is missing from the yield due to the event selection (signal loss). This correction is negligible for high-multiplicity events and reaches 13% (4%) at pT = 0.2 (pT = 1) GeV/c for the lowest multiplicity class based on NSPDtracklets. amount of particles. From now on, the most jet-like and isotropic events will be referred to as 0 – 10% and 90 – 100% spherocity event class, respectively. It has been reported that the evolution of several observables as a function of center-of-mass energy can be factored out to be due to the changes in charged-particle multiplicities which in turn depend on the energy. For example, the particle production sensitive to the underlying event for different √s exhibits approximate scaling properties connected to changes in ⟨Nch⟩[34]. Moreover, within uncertainties, the average pT as a function of multiplicity exhibits a small energy dependence [21]. Therefore, the spheroc- ity dependent average pT as a function of charged-particle multiplicity is only presented for pp collisions at √s = 13 TeV. The physics message is valid for other center-of-mass energies, this was veriﬁed using data from pp collisions at √s = 5.02 TeV. 6 6 4.2 Spherocity studies The measurement of the average transverse momentum as a function of charged-particle multiplicity and spherocity is performed following a strategy close to that used in earlier publications [21, 36]. The trans- verse momentum spectra for different multiplicity and spherocity classes are fully corrected as described in the previous section. The average transverse momentum is then calculated from the corrected spectra as the arithmetic mean in the kinematic range 0.15 < pT < 10 GeV/c and \|η\| < 0.8. 7 7 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration To extract the correlation between ⟨pT⟩and the number of primary charged particles (Nch) in \|η\| < 0.8 and for the spherocity class S0, the following re-weighting procedure is applied to account for the exper- imental resolution of the measured event multiplicity (Nm) and spherocity (Sm): ⟨pT⟩(Nch,S0) = ∑ Nm∑ Sm R(Nch,Nm)⟨pT⟩(Nm,Sm)R′(S0,Sm). (3) (3) (3) This method is an extension to the one developed for the previous ⟨pT⟩analysis [36]. It exploits the nor- malized response matrices R and R′ which encode the multiplicity, and spherocity detector resolutions, respectively. The average pT for the S0 event class is encoded inside the inner sum, where the weights R′(S0,Sm) are explicitly applied to ⟨pT⟩values. The resulting ⟨pT⟩(Nm,S0) is then corrected for multi- plicity resolution. It is worth mentioning that the spherocity-integrated class (0 – 100%) only requires the multiplicity correction. The Monte Carlo non-closure, discussed in the next section, is assigned as systematic uncertainty. 5.1 Transverse momentum spectra The relative systematic uncertainties on the pT spectra are summarized in Table 3. They include the ef- fect of the event selection based on the vertex position, which is studied by comparing the fully corrected pT spectra obtained with alternative vertex selections: \|z\| < 5 cm and \|z\| < 20 cm. The corrections due to trigger and vertex selection were determined using the EPOS LHC [37] event generator and the devia- tions with respect to the nominal values, i.e. those obtained with PYTHIA 6, were assigned as systematic uncertainties. The same procedure was employed for the estimation of the systematic uncertainty associ- ated to the signal loss correction. The systematic uncertainty related to the track selection was studied by varying the track cuts for which we used the variation intervals described in Ref. [29]. We also studied the systematic effects related to the uncertainty on the primary particle composition which is assumed for the efﬁciency correction. This uncertainty takes into account the extrapolation of the spectra to low pT, the relative particle abundances at high pT, the uncertainties of the measured particle spectra, and the Monte Carlo assumptions on the Σ±/Λ spectra ratios. The systematic uncertainties of the correction for secondaries contamination is estimated by varying the ﬁt model using two templates, i.e. for primaries and secondaries, or three templates, i.e. primaries, secondaries from interactions in the detector material, and secondaries from weak decays, as well as varying the ﬁt momentum ranges. Since we are using the same event selection and track cuts as those used in Ref. [29], the systematic uncertainties associated with matching efﬁciency, pT resolution and material budget, are identical. 5.2 Average transverse momentum A summary of the systematic uncertainties for three multiplicity values and for different spherocity classes is shown in Table 4. In order to estimate the systematic uncertainties of ⟨pT⟩, the results of the data analysis and of the evaluation of the corrections from Monte Carlo simulations were studied considering cut variations and Monte Carlo assumptions, within reasonable limits. The effect of the track cuts on ⟨pT⟩was found to be spherocity independent and of the order of 1%. The efﬁciency correction is another spherocity independent contribution and it is found to be ∼1%. This contribution takes into account the different particle composition in data and models, as well as the multiplicity dependence of the correction. We also studied the multiplicity dependence of the purity correction; the effect was found to be smaller than 0.5%. The most relevant spherocity independent contribution is related to the re-weighting procedure to correct for the detector multiplicity resolution. This was quantiﬁed from the Monte Carlo non-closure, it amounts to ∼1.36%, ∼0.86% and ∼1.26% for dNch/dη = 1.88, 6.25, 25.00, respectively. 8 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Table 3: Main sources and values of the relative systematic uncertainties of transverse momentum spectra for pp collisions at √s = 5.02 TeV. The maximum values of the uncertainties, among all the multiplicity classes, are reported for low-, intermediate-, and high-pT intervals. Systematic uncertainties for pp collisions at √s = 13 TeV are shown inside the parentheses. The systematic uncertainty due to trigger and event selection is pT independent and therefore it is not included in the table. It reaches ∼7.6% (∼6.3%) for the lowest multiplicity class in pp collisions at √s = 5.02 TeV (√s = 13 TeV), and it is smaller than 0.5% for the other multiplicity classes. 5.2 Average transverse momentum pp collisions at √s = 5.02 TeV (√s = 13 TeV) pT (GeV/c) 0.15 3.0 10 Pileup 3.5% (3.5%) 4.5% (4.5%) 4.5% (4.5%) Vertex selection 0.5% (0.5%) 0.5% (0.5%) 0.5% (0.5%) Signal loss 2.3% (1.1%) 1.2%(0.5%) 0.4% (0.7%) Track selection 1.6% (1.7%) 3.1% (1.7%) 4.0% (4.0%) Secondary particles 1.3% (1.4%) 1.0% (1.0%) 1.0% (1.0%) Particle composition 2.0% (2.0%) 2.5% (2.5%) 2.0% (2.0%) Tracking efﬁciency 1.0% (1.0%) 4.2% (4.2%) 4.2% (4.2%) pT resolution 0.0% (0.0%) 0.0% (0.0%) 0.1% (0.1%) Material budget 1.5% (1.5%) 0.5% (0.5%) 0.2% (0.2%) Total 5.4% (5.1%) 7.5% (7.0%) 7.7% (7.7%) Total (Nch-dependent) 4.1% (3.5%) 5.8% (5.5%) 5.9% (6.5%) The set of track cuts used to measure spherocity was also varied compared to those used for the pT spec- tra analysis. The effect on the results amounted to 1%. The most relevant contribution to the systematic uncertainties originates from the re-weighting procedure method which is used to correct for the sphe- rocity resolution. The Monte Carlo non-closure is assigned as a systematic uncertainty. For the lowest multiplicity value, dNch/dη=1.88, the uncertainty reaches 3.23%, 4.55%, and 7.06% for the 0 – 10%, 40 – 50%, and 90 – 100% spherocity classes, respectively. For higher multiplicities, e.g. dNch/dη=25.0, the Monte Carlo non-closure amounts to 0.57%, 1.07%, and 2.01% for the 0 – 10%, 40 – 50%, and 90 – 100% spherocity classes, respectively. As expected from the detector response, the most relevant effects are observed for low-multiplicity events in particular for the isotropic classes. As will be seen later, in Monte Carlo jet-like events, the average pT shows a strong change with multiplicity at dNch/dη ∼7. This effect increases the size of the uncertainty (Monte Carlo non-closure) in that multiplicity interval. This is the dominant contribution to the systematic uncertainties and covers the largest variations observed between data and PYTHIA 8 (version 8.212) [10] (tune Monash 2013 [38]). The model dependence is also checked by using events simulated with PYTHIA 8 and EPOS LHC which include the particle transport through the detector. The corrections were calculated using these simulations and the maximum variation with respect to the nominal values (using PYTHIA 6 simulations) are below 1%. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity Table 4: Main sources and values of the relative systematic uncertainties on the average pT for different spherocity classes. The three quoted values (for each contribution) correspond to dNch/dη = 1.88, 6.25, and 25.0, respectively. INEL> 0 pT distribution exhibit two distinct behavior. While at low pT (< 0.5 GeV/c) the ratios exhibit a modest pT dependence, for pT > 0.5 GeV/c they strongly depend on multiplicity and pT. Figure 3 shows the multiplicity dependent pT spectra using a multiplicity selection based on the V0M am- plitude. Results for pp collisions at √s = 5.02 and 13 TeV are shown. The average multiplicity values are signiﬁcantly smaller than those reached with the mid-pseudorapidity estimator (based on NSPDtracklets). For example, in pp collisions at √s = 13 TeV, while the average charged-particle multiplicity density amounts to 56.55 for the highest NSPDtracklets class, it only reaches 27.61 for the highest V0M multiplic- ity class. We note that for similar average particle densities, e.g. the multiplicity classes II (V0M) and VII’ (SPD tracklets) in pp collisions at √s = 13 TeV, the ratios measured using the V0M amplitude and the NSPDtracklets are similar. The comparison of the pT spectra for these multiplicity classes is shown in Fig. 4. We observe that for transverse momentum below 0.5 GeV/c, the spectra exhibit the same shape. For transverse momenta within 0.5–3 GeV/c the spectra for the multiplicity class II is harder than that for the VII” class. At higher pT, the spectral shapes are the same, but the yield of the class II is ∼15% higher than that for the VII’ class. Similar results are obtained if we compare the multiplicity classes I and VI’ for pp collisions at 5.02 TeV. Commonly, the particle production is characterized by quantities like integrated yields, or any ﬁt parame- ter of the curve extracted from ﬁts to the data, for example, the so-called inverse slope parameter reported by ALICE in Ref. [39]. This facilitates the visualization of the evolution of the particle production as a function of multiplicity and the comparison among different colliding systems. Several publications have adopted this strategy for soft (pT < 2 GeV/c) [2, 6, 21] physics and others to describe the particle pro- duction for intermediate and high pT (2 ≤pT < 20 GeV/c) [40]. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity The pT distributions of charged particles, measured in \|η\| < 0.8 for pp collisions at √s = 5.02 and 13 TeV, are shown in Fig. 2 for the different multiplicity classes selected using the estimator based on NSPDtracklets. The bottom panels depict the ratios to the pT distribution of the INEL> 0 event class. The features of the spectra, i.e. the change of the spectral shape going from low- to high-multiplicity values, are qualitatively the same for both energies. The only signiﬁcant difference is the multiplicity reach which is higher at 13 TeV than that at 5.02 TeV. In the following we discuss the results for pp collisions at the highest energy. As shown in Fig. 2, the pT spectra become harder as the multiplicity increases, which contributes to the increase of the average transverse momentum with multiplicity. The ratios to the 9 9 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Table 4: Main sources and values of the relative systematic uncertainties on the average pT for different spherocity classes. The three quoted values (for each contribution) correspond to dNch/dη = 1.88, 6.25, and 25.0, respectively. Spherocity-dependent contributions Spherocity class 0 – 100% 0 – 10% 40 – 50% 90 – 100% Model dep. (%) 0.5, 0.7, 0.2 0.4, 0.4, 0.6 0.6, 0.5, 0.3 0.9, 0.8, 0.2 Sec. particles (%) 0.2, 0.3, 1.2 1.0, 1.5, 1.9 0.3, 0.3, 1.1 0.6, 0.3, 0.9 Ev. selection (%) 2.2, 0.0, 0.0 1.9, 1.4, 0.4 1.3, 0.4, 0.0 1.3, 0.1, 0.00 So res. corr. (%) na 3.2, 5.4, 0.6 4.6, 2.4, 1.1 7.1, 3.7, 2.0 So track cuts (%) 1.00 Spherocity-independent contributions Nch res. corr (%) 1.4, 0.9, 1.3 pT track cuts (%) 0.8, 0.9, 1.2 Efﬁciency corr. (%) 0.4, 0.2, 0.2 Particle composition (%) 1.0, 1.0, 1.0 Nch dep. eff. corr. (%) 0.5, 0.7, 0.9 Nch dep. sec. corr. (%) 0.2, 0.1, 0.1 So-dep. total (%) 2.2, 0.8, 1.2 4.0, 5.8, 2.3 4.9, 2.7, 1.9 7.3, 3.9, 2.4 So-indep. total (%) 2.0, 1.8, 2.2 Total (%) 3.0, 2.0, 2.5 4.5, 6.1, 3.2 5.3, 3.2, 3.0 7.6, 4.3, 3.3 INEL> 0 pT distribution exhibit two distinct behavior. While at low pT (< 0.5 GeV/c) the ratios exhibit a modest pT dependence, for pT > 0.5 GeV/c they strongly depend on multiplicity and pT. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to INEL>0 d 1 10 5 − 10 2 − 10 10 4 10 7 10 10 10 13 10 15 10 = 5.02 TeV s ALICE pp Stat. unc. Total syst. unc. ) 1 10 × X' ( ) 2 10 × IX' ( ) 3 10 × VIII' ( ) 4 10 × VII' ( ) 5 10 × VI' ( 1 10 SPD tracklets mult. classes = 13 TeV s ) 6 10 × V' ( ) 7 10 × IV' ( ) 8 10 × III' ( ) 9 10 × II' ( ) 10 10 × I' ( 1 10 1 − 10 1 10 2 10 Stat. unc. Uncorr. syst. unc. 1 10 1 10 0 5 10 15 1 10 Ratio to INEL>0 Fig. 2: Transverse momentum distributions of charged particles for multiplicity classes selected using SPD track- lets in \|η\| < 0.8. Results for pp collisions at √s = 5.02 and 13 TeV are shown in the left and right panels, respectively. Statistical and total systematic uncertainties are shown as error bars and boxes around the data points, respectively. In the middle panels, ratios of multiplicity dependent spectra to INEL> 0 are shown in logarithmic scale. In the bottom panels we show the ratios in a linear scale to illustrate the dramatic behavior of the ratios. The systematic uncertainties on the ratios are obtained by considering only contributions uncorrelated across multiplic- ity. The spectra are scaled to improve the visibility. Within uncertainties, going from low to high multiplicity n decreases taking values from 6 to 5, respec- tively. A similar behavior has been reported for heavy-ion collisions [41]. Moreover, the results using the two multiplicity estimators are consistent within the overlapping multiplicity interval. This result is consistent with that shown in Fig. 4. PYTHIA 6 and 8 simulations describe the trends very well, but a strong deviation between EPOS LHC and data is observed. In PYTHIA 8, it has been shown that the number of high-pT jets increases with event multiplicity. Moreover, for a given event multiplicity and ﬁxed jet pT, the high-pT tails of the charged-particle spectra are very similar in low- and high-multiplicity events [16]. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity It is interesting and important to deﬁne a common quantity to compare the shape of the high-pT part of the spectra of different particle species and collision systems. The natural choice is ﬁtting a power-law function (α × p−n T ) to the invariant yield and studying the multiplicity dependence of the exponent (n) extracted from the ﬁt. Figure 5 illustrates the results considering particles with transverse momentum within 6–20 GeV/c for pp at √s = 13 TeV. It is worth mentioning that within uncertainties the power-law function describes rather well the data in that pT interval. Similarly, the pT spectra simulated with the different generators are well described (within 2%) by the power-law function. 10 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to INEL>0 d 1 10 5 − 10 2 − 10 10 4 10 7 10 10 10 13 10 15 10 = 5.02 TeV s ALICE pp Stat. unc. Total syst. unc. ) 1 10 × X' ( ) 2 10 × IX' ( ) 3 10 × VIII' ( ) 4 10 × VII' ( ) 5 10 × VI' ( 1 10 SPD tracklets mult. classes = 13 TeV s ) 6 10 × V' ( ) 7 10 × IV' ( ) 8 10 × III' ( ) 9 10 × II' ( ) 10 10 × I' ( 1 10 1 − 10 1 10 2 10 Stat. unc. Uncorr. syst. unc. 1 10 1 10 0 5 10 15 1 10 Fig. 2: Transverse momentum distributions of charged particles for multiplicity classes selected using SPD track- lets in \|η\| < 0.8. Results for pp collisions at √s = 5.02 and 13 TeV are shown in the left and right panels, respectively. Statistical and total systematic uncertainties are shown as error bars and boxes around the data points, respectively. In the middle panels, ratios of multiplicity dependent spectra to INEL> 0 are shown in logarithmic scale. In the bottom panels we show the ratios in a linear scale to illustrate the dramatic behavior of the ratios. The systematic uncertainties on the ratios are obtained by considering only contributions uncorrelated across multiplic- ity. The spectra are scaled to improve the visibility. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity Therefore, based on PYTHIA 8 studies, the reduction of the power-law exponent with in- creasing multiplicity can be attributed to an increasing number of high-pT jets. 11 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to INEL>0 d 1 10 5 − 10 2 − 10 10 4 10 7 10 10 10 13 10 15 10 = 5.02 TeV s ALICE pp Stat. unc. Total syst. unc. ) 1 10 × X ( ) 2 10 × IX ( ) 3 10 × VIII ( ) 4 10 × VII ( ) 5 10 × VI ( 1 10 V0 mult. classes = 13 TeV s ) 6 10 × V ( ) 7 10 × IV ( ) 8 10 × III ( ) 9 10 × II ( ) 10 10 × I ( 1 10 1 − 10 1 10 2 10 Stat. unc. Uncorr. syst. unc. 1 10 1 10 0 5 10 15 1 10 Fig. 3: Transverse momentum distributions of charged particles for different V0M multiplicity classes. Results for pp collisions at √s = 5.02 and 13 TeV are shown in the left and right panels, respectively. Statistical and total sys- tematic uncertainties are shown as error bars and boxes around the data points, respectively. In the middle panels, ratios of multiplicity dependent spectra to INEL> 0 are shown in logarithmic scale. The systematic uncertainties on the ratios are obtained by considering only contributions uncorrelated across multiplicity. The spectra are scaled to improve the visibility. ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to INEL>0 d 1 10 5 − 10 2 − 10 10 4 10 7 10 10 10 13 10 15 10 = 5.02 TeV s ALICE pp Stat. unc. Total syst. unc. ) 1 10 × X ( ) 2 10 × IX ( ) 3 10 × VIII ( ) 4 10 × VII ( ) 5 10 × VI ( 1 10 V0 mult. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity classes = 13 TeV s ) 6 10 × V ( ) 7 10 × IV ( ) 8 10 × III ( ) 9 10 × II ( ) 10 10 × I ( 1 10 1 − 10 1 10 2 10 Stat. unc. Uncorr. syst. unc. 1 10 1 10 0 5 10 15 1 10 Ratio to INEL>0 Fig. 3: Transverse momentum distributions of charged particles for different V0M multiplicity classes. Results for pp collisions at √s = 5.02 and 13 TeV are shown in the left and right panels, respectively. Statistical and total sys- tematic uncertainties are shown as error bars and boxes around the data points, respectively. In the middle panels, ratios of multiplicity dependent spectra to INEL> 0 are shown in logarithmic scale. The systematic uncertainties on the ratios are obtained by considering only contributions uncorrelated across multiplicity. The spectra are scaled to improve the visibility. As pointed out above, the ratios to the INEL> 0 pT distributions for ⟨dNch/dη⟩≲25 exhibit a weak pT-dependence for pT > 4 GeV/c. This applies to both energies and to all multiplicity estimators. To illustrate better the behaviour of the yields at high momenta, we adopted a representation previously used for heavy-ﬂavour hadrons [42] to point out to the similarities between the two results. The trend at high-pT is highlighted in Fig. 6, which shows the integrated yields for three transverse momentum intervals (2 < pT < 10 GeV/c, 4 < pT < 10 GeV/c, and 6 < pT < 10 GeV/c) as a function of the aver- age mid-pseudorapidity multiplicity. Both the charged-particle yields and the average multiplicity are self-normalized, i.e. they are divided by their average value for the INEL> 0 sample. The high-pT (> 4 GeV/c) yields of charged particles increase faster than the charged-particle multiplicity, while the increase is smaller when we consider lower-pT particles. The trend of the data is qualitatively well reproduced by PYTHIA 8, but for pT > 6 GeV/c the model signiﬁcantly overestimates the ratio by a 12 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 1 10 4 − 10 3 − 10 2 − 10 1 − 10 1 10 ALICE = 5.02 TeV s pp 1.0) ± VI' SPD tracklets mult. class (18.5 0.9) ± I V0 mult. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity class (19.2 = 13 TeV s pp 1.2) ± VII' SPD tracklets mult. class (19.5 0.8) ± II V0 mult. class (20.5 1 10 0.6 0.8 1 1.2 1.4 Stat. unc. ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to V0 class d Fig. 4: Transverse momentum distributions of charged particles for high-multiplicity (⟨dNch/dη⟩≈20) pp col- lisions at √s = 5.02 (empty markers) and 13 TeV (full markers). Results for V0-based (squares) and SPD-based (circles) multiplicity estimators are shown. The bottom panel shows the pT spectrum obtained using the V0-based multiplicity estimator normalized to that using the SPD-based multiplicity estimator. Only statistical uncertainties are shown as error bars. 1 10 4 − 10 3 − 10 2 − 10 1 − 10 1 10 ALICE = 5.02 TeV s pp 1.0) ± VI' SPD tracklets mult. class (18.5 0.9) ± I V0 mult. class (19.2 = 13 TeV s pp 1.2) ± VII' SPD tracklets mult. class (19.5 0.8) ± II V0 mult. class (20.5 1 10 0.6 0.8 1 1.2 1.4 Stat. unc. ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to V0 class d 1 10 4 − 10 3 − 10 2 − 10 1 − 10 1 10 ALICE = 5.02 TeV s pp 1.0) ± VI' SPD tracklets mult. class (18.5 0.9) ± I V0 mult. class (19.2 = 13 TeV s pp 1.2) ± VII' SPD tracklets mult. class (19.5 0.8) ± II V0 mult. class (20.5 1 10 0.6 0.8 1 1.2 1.4 Stat. unc. ) c (GeV/ T p -1 ) c (GeV/ η d T p /d ch N 2 Ratio to V0 class d Fig. 4: Transverse momentum distributions of charged particles for high-multiplicity (⟨dNch/dη⟩≈20) pp col- lisions at √s = 5.02 (empty markers) and 13 TeV (full markers). Results for V0-based (squares) and SPD-based (circles) multiplicity estimators are shown. The bottom panel shows the pT spectrum obtained using the V0-based multiplicity estimator normalized to that using the SPD-based multiplicity estimator. Only statistical uncertainties are shown as error bars. factor larger than 1.5. Although the shapes of the spectra (characterized by n) are not well reproduced by EPOS LHC, the model gives the best description of the self-normalized yields. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity Comparisons with Monte Carlo generators predictions are shown in the middle and right panels. Statistical and total systematic uncertainties are shown as error bars and boxes around the data points, respectively. 6.1 Transverse momentum spectra as a function of charged-particle multiplicity Despite the large uncertainties, it is clear the data show a non-linear increase. 13 13 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 1 10 4.5 5 5.5 6 6.5 7 = 13 TeV s ALICE pp c < 20 GeV/ T p \|<0.8, 6 < η \| V0 mult. estimator SPD tracklets mult. estimator 1 10 4.5 5 5.5 6 6.5 7 Data PYTHIA 8 EPOS LHC PYTHIA 6 6.85 INEL>0 \|<0.5 η \|〉 η /d ch N d 〈 6.87 0.19 ± 6.46 6.69 1 10 4.5 5 5.5 6 6.5 7 Stat. unc, Total syst. unc. \|<0.5 η \| INEL>0 〉 η /d ch N d 〈 / η /d ch N d Exponent of power-law fit 1 10 4.5 5 5.5 6 6.5 7 = 13 TeV s ALICE pp c < 20 GeV/ T p \|<0.8, 6 < η \| V0 mult. estimator SPD tracklets mult. estimator 1 10 4.5 5 5.5 6 6.5 7 Data PYTHIA 8 EPOS LHC PYTHIA 6 6.85 INEL>0 \|<0.5 η \|〉 η /d ch N d 〈 6.87 0.19 ± 6.46 6.69 1 10 4.5 5 5.5 6 6.5 7 Stat. unc, Total syst. unc. \|<0.5 η \| INEL>0 〉 η /d ch N d 〈 / η /d ch N d Exponent of power-law fit Fig. 5: Evolution of the spectral shape of the transverse momentum distribution as a function of charged-particle multiplicity. The spectral shape is characterized by the exponent of the power-law function which ﬁts the high-pT part (pT > 6 GeV/c) of the invariant yields. Results for pp collisions at √s = 13 TeV are shown. A comparison of the two multiplicity estimators discussed in this paper is shown in the left panel. Comparisons with Monte Carlo generators predictions are shown in the middle and right panels. Statistical and total systematic uncertainties are shown as error bars and boxes around the data points, respectively. Fig. 5: Evolution of the spectral shape of the transverse momentum distribution as a function of charged-particle multiplicity. The spectral shape is characterized by the exponent of the power-law function which ﬁts the high-pT part (pT > 6 GeV/c) of the invariant yields. Results for pp collisions at √s = 13 TeV are shown. A comparison of the two multiplicity estimators discussed in this paper is shown in the left panel. 6.2 Double-differential study of the average transverse momentum The spherocity-integrated average pT as a function of dNch/dη for pp collisions at √s = 13 TeV is shown in Fig. 7. In accordance with measurements at lower energies [21], the ⟨pT⟩increases with dNch/dη. In PYTHIA 8 the effect is enhanced by color reconnection, which allows the interaction among partons originating from multiple semi-hard scatterings via color strings. The minimum-bias data are compared with analogous measurements for the most jet-like structure (0 – 10%) and isotropic (90 – 100%) event classes. Studying observables as a function of spherocity reveals interesting features. On one hand, for isotropic events the average pT stays systematically below the spherocity-integrated ⟨pT⟩over the full multiplicity range; on the other hand, for jet-like events the ⟨pT⟩is higher than that for spherocity- integrated events. Moreover, within uncertainties the overall shape of the correlation, i.e. a steep linear rise below dNch/dη = 10 followed by a less steep but still linear rise above, is not spherocity-dependent. Figure 8 shows that within uncertainties, PYTHIA 8 with color reconnection gives an adequate descrip- tion of the spherocity-integrated event class. It is worth mentioning that color reconnection was origi- nally introduced to explain the rise of ⟨pT⟩with multiplicity [43]. However, PYTHIA 6 shows a steeper rise of ⟨pT⟩with dNch/dη than that seen in data. The Perugia 2011 tune relies on Tevatron and SPS minimum-bias data, while the Monash tune was constrained using the early LHC measurements [38]. The comparison of data with EPOS LHC is also shown. Clearly, the quantitative agreement is as good as that achieved by PYTHIA 8. The EPOS LHC model uses a different approach in order to simulate the hadronic interactions. Namely, the model considers a collective hadronization which depends only on the geometry and the density [37]. For the 0 – 10% and 90 – 100% spherocity classes, Fig. 8 also shows comparisons between data and Monte Carlo generators (PYTHIA 6, PYTHIA 8 and EPOS LHC). It is worth mentioning that we also used spherocity percentiles in all the Monte Carlo event generators reported in this paper because their spherocity distributions do not differ much from those measured in data. For further Monte Carlo comparisons the spherocity binning which was used in the analysis is provided as HEP data. 6.2 Double-differential study of the average transverse momentum INEL>0 \|<0.5 η\|〉 η /d ch N d 〈 6.87 0.19 ± 6.46 6.85 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 \|<0.5 η\| INEL>0 〉 η /d ch N d 〈 / η /d ch N d INEL>0 〉 η /d ch N d 〈 / 〉 η /d ch N d 〈 Ratio Ratio Fig. 6: Self-normalized yields of charged particles integrated over different pT intervals: 2 < pT < 4 GeV/c, 4 < pT < 6 GeV/c, and 6 < pT < 10 GeV/c. The integrated yields for pp collisions at √s = 13 TeV are shown as a function of charged-particle density at mid-pseudorapidity. Statistical and uncorrelated (across multiplicity) systematic uncertainties are shown as error bars and boxes around the data points, respectively. Statistical uncer- tainties are negligible compared to systematic uncertainties. Data are compared with PYTHIA 8 (dashed lines) and EPOS LHC (solid lines). The dotted line is drawn to see the differences between data and the linear dependence. Deviations of data from the linear trend are shown in the bottom panel. nection) are smaller and larger respectively for the 0 – 10% and 90 – 100% spherocity classes than those seen for the 0 – 100% spherocity class. The effect could be a consequence of the reduction of color re- connection contribution in events containing jets surrounded by a small underlying event activity. For isotropic events the three models quantitatively describe the correlation. Even for PYTHIA 6, the size of the discrepancy which was pointed out for the spherocity-integrated event class is reduced. On the con- trary, for jet-like events both PYTHIA 6 and 8 exhibit a larger disagreement with the data. These models produce three distinct multiplicity regions, for dNch/dη ≲7 the models give a steeper rise of ⟨pT⟩than data. Within the intermediate multiplicity interval (7 ≲dNch/dη ≲25), the slope of ⟨pT⟩given by mod- els is more compatible with that seen in data, although the models overestimate the average pT. While in data the average pT increases at a constant rate with multiplicity for dNch/dη ≳7, PYTHIA 6 and 8 shows a third change of the slope of ⟨pT⟩, observed for dNch/dη ≳25. The data to model ratio indicates a discrepancy larger than 10%, which is larger than the systematic uncertainties associated to ⟨pT⟩in that multiplicity interval. 6.2 Double-differential study of the average transverse momentum In low- multiplicity events (dNch/dη < 10), the deviations between data and PYTHIA 8 (without color recon- 14 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 0 1 2 3 4 5 6 7 8 9 10 0 5 10 15 20 25 30 35 40 45 = 13 TeV s ALICE pp SPD tracklets mult. estimator c < 4 GeV/ T p 2 < c < 6 GeV/ T p 4 < c < 10 GeV/ T p 6 < Data PYTHIA 8 EPOS LHC Stat. unc. Uncorr. syst. unc. INEL>0 \|<0.5 η\|〉 η /d ch N d 〈 6.87 0.19 ± 6.46 6.85 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 \|<0.5 η\| INEL>0 〉 η /d ch N d 〈 / η /d ch N d INEL>0 〉 η /d ch N d 〈 / 〉 η /d ch N d 〈 Ratio Fig. 6: Self-normalized yields of charged particles integrated over different pT intervals: 2 < pT < 4 GeV/c, 4 < pT < 6 GeV/c, and 6 < pT < 10 GeV/c. The integrated yields for pp collisions at √s = 13 TeV are shown as a function of charged-particle density at mid-pseudorapidity. Statistical and uncorrelated (across multiplicity) systematic uncertainties are shown as error bars and boxes around the data points, respectively. Statistical uncer- tainties are negligible compared to systematic uncertainties. Data are compared with PYTHIA 8 (dashed lines) and EPOS LHC (solid lines). The dotted line is drawn to see the differences between data and the linear dependence. Deviations of data from the linear trend are shown in the bottom panel. 0 1 2 3 4 5 6 7 8 9 10 0 5 10 15 20 25 30 35 40 45 = 13 TeV s ALICE pp SPD tracklets mult. estimator c < 4 GeV/ T p 2 < c < 6 GeV/ T p 4 < c < 10 GeV/ T p 6 < Data PYTHIA 8 EPOS LHC Stat. unc. Uncorr. syst. unc. 6.2 Double-differential study of the average transverse momentum nection) are smaller and larger respectively for the 0 – 10% and 90 – 100% spherocity classes than those seen for the 0 – 100% spherocity class. The effect could be a consequence of the reduction of color re- connection contribution in events containing jets surrounded by a small underlying event activity. For isotropic events the three models quantitatively describe the correlation. Even for PYTHIA 6, the size of the discrepancy which was pointed out for the spherocity-integrated event class is reduced. On the con- trary, for jet-like events both PYTHIA 6 and 8 exhibit a larger disagreement with the data. These models produce three distinct multiplicity regions, for dNch/dη ≲7 the models give a steeper rise of ⟨pT⟩than data. Within the intermediate multiplicity interval (7 ≲dNch/dη ≲25), the slope of ⟨pT⟩given by mod- els is more compatible with that seen in data, although the models overestimate the average pT. While in data the average pT increases at a constant rate with multiplicity for dNch/dη ≳7, PYTHIA 6 and 8 shows a third change of the slope of ⟨pT⟩, observed for dNch/dη ≳25. The data to model ratio indicates a discrepancy larger than 10%, which is larger than the systematic uncertainties associated to ⟨pT⟩in that multiplicity interval. In order to study the details of the changes of the functional form of ⟨pT⟩(Nch) due to the spherocity se- lection, Fig. 9 shows the average pT of jet-like and isotropic events normalized to that for the spherocity- integrated event class. For jet-like events, the data exhibit a hint of a modest peak at dNch/dη ∼7, which is not signiﬁcant if we consider the size of the systematic uncertainties. Moreover, within uncertainties the ratio remains constant for dNch/dη ≳25. EPOS LHC describes rather well the high-multiplicity behavior, however, it overestimates the peak. PYTHIA 6 and 8 show the worst agreement with the data. In this representation, the three distinct regions, which were described before are highlighted. In 15 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 0 5 10 15 20 25 30 35 40 45 50 η /d ch N d 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 ) c (GeV/ 〉 T p 〈 = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.8, 0.15 ≤ \| η\| Spherocity class 0-100% 0-10% (Jetty) 90-100% (Isotropic) Fig. 6.2 Double-differential study of the average transverse momentum The ⟨pT⟩remains constant within uncertainties for 4 < pmax T < 10 GeV/c in data and for 6 < pmax T < 10 GeV/c in PYTHIA 8. For pmax T = 2 GeV/c the ⟨pT⟩decreases by 23% (29%) in data (PYTHIA 8) compared to pmax T = 10 GeV/c. The relative difference of ⟨pT⟩between data and PYTHIA 8 amounts to 9% (4%) for pmax T = 2 GeV/c (pmax T = 10 GeV/c). The results suggest that the power-law tail produces a smaller impact on data than in PYTHIA 8. A similar ratio for isotropic events shows a smaller structure at dNch/dη ∼7. This effect is well reproduced by all models. Finally, we also examined the evolution of ⟨pT⟩(Nch) going from the most jet-like to the most isotropic event classes. Figure 10 shows the spherocity-dependent ⟨pT⟩(Nch) in data and models, the data to model ratios are displayed in Fig. 11. The difference between the 0 – 10% and 10 – 20% spherocity classes is smaller for data and EPOS LHC than for PYTHIA 6 and 8. Moreover, within uncertainties PYTHIA 8 describes rather well the data for the 10 – 20% spherocity class. This contrasts with the disagreement between the model and data for the 0 – 10% spherocity class. Other features in PYTHIA 6 and 8 are the reduction of the bump at dNch/dη ∼7 and the disappearance of a third rise of the ⟨pT⟩for dNch/dη ≳25 when one goes from the 0 – 10% to the 10 – 20% spherocity classes. The agreement among models and data for the 20 – 100% spherocity classes is similar to that observed for the 10 – 20% spherocity class. Within uncertainties, PYTHIA 8 and EPOS LHC qualitatively describe the data for dNch/dη ≳10, while PYTHIA 6 overestimates the average pT. From previous LHC studies we know that the production cross section of jets in high-multiplicity pp collisions is smaller in data than predicted from the Monte Carlo generators [32, 44, 45]. Therefore, a possible interpretation is that the low-momentum partons, color connected with higher momentum ones (jets), would produce an overall increase of the hadron transverse momentum. This would affect more the low-pT part of the spectrum associated with jet-enriched samples, which are achieved by requiring low-spherocity values. 6.2 Double-differential study of the average transverse momentum 7: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for the spherocity-integrated case (0 – 100%) are contrasted with the measurements for the most jet-like (0 – 10%) and isotropic (90 – 100%) events. Statistical uncertainties (error bars) are negligible compared to systematic un- certainties (boxes around the data points). Fig. 7: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for the spherocity-integrated case (0 – 100%) are contrasted with the measurements for the most jet-like (0 – 10%) and isotropic (90 – 100%) events. Statistical uncertainties (error bars) are negligible compared to systematic un- certainties (boxes around the data points). PYTHIA 8, the peak (at dNch/dη ∼7) in jet-like events is caused by particles with transverse momen- tum above 2 GeV/c. The size of the peak is determined by particles with pT > 5−6 GeV/c. In contrast, data do not show a signiﬁcant peak structure for any speciﬁc transverse momentum interval. We also varied the upper pT (0.15 < pT < pmax T ) limit (pmax T = 10 GeV/c is the default) and studied the effect on the extracted ⟨pT⟩. The ⟨pT⟩remains constant within uncertainties for 4 < pmax T < 10 GeV/c in data and for 6 < pmax T < 10 GeV/c in PYTHIA 8. For pmax T = 2 GeV/c the ⟨pT⟩decreases by 23% (29%) in data (PYTHIA 8) compared to pmax T = 10 GeV/c. The relative difference of ⟨pT⟩between data and PYTHIA 8 amounts to 9% (4%) for pmax T = 2 GeV/c (pmax T = 10 GeV/c). The results suggest that the power-law tail produces a smaller impact on data than in PYTHIA 8. A similar ratio for isotropic events shows a smaller structure at dNch/dη ∼7. This effect is well reproduced by all models. PYTHIA 8, the peak (at dNch/dη ∼7) in jet-like events is caused by particles with transverse momen- tum above 2 GeV/c. The size of the peak is determined by particles with pT > 5−6 GeV/c. In contrast, data do not show a signiﬁcant peak structure for any speciﬁc transverse momentum interval. We also varied the upper pT (0.15 < pT < pmax T ) limit (pmax T = 10 GeV/c is the default) and studied the effect on the extracted ⟨pT⟩. 6.2 Double-differential study of the average transverse momentum The incorporation of these new observables into the PYTHIA 8 tuning could be a challenge because, on one hand, the color reconnection has to be reduced to describe the low-S0 data; on the other hand, the variation should not be too large because the good description of the spherocity- 16 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration η /d ch N d ) c (GeV/ 〉 T p 〈 Data/Model 10 20 30 40 50 0.4 0.6 0.8 1 = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.8, 0.15 ≤ \| η\| -integrated) o S 0-100% ( 10 20 30 40 50 PYTHIA 6 PYTHIA 8 PYTHIA 8 (rr=0) EPOS LHC 0-10% (Jetty) 10 20 30 40 50 90-100% (Isotropic) 10 20 30 40 50 0.8 0.9 1 1.1 1.2 10 20 30 40 50 10 20 30 40 50 Fig. 8: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for the spherocity-integrated case (0 – 100%), the most jet-like (0 – 10%) and isotropic (90 – 100%) events are compared with Monte Carlo predictions. Predictions of PYTHIA 8 with and without (null reconnection range, rr = 0) color reconnection, as well as PYTHIA 6 and EPOS LHC are displayed. Statistical uncertainties (error bars) are negligible compared to systematic uncertainties (shaded area around the data points). Data to model ratios are shown in the bottom panel. The color band around unity represents the systematic uncertainty. η /d ch N d ) c (GeV/ 〉 T p 〈 Data/Model 10 20 30 40 50 0.4 0.6 0.8 1 = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.8, 0.15 ≤ \| η\| -integrated) o S 0-100% ( 10 20 30 40 50 PYTHIA 6 PYTHIA 8 PYTHIA 8 (rr=0) EPOS LHC 0-10% (Jetty) 10 20 30 40 50 90-100% (Isotropic) 10 20 30 40 50 0.8 0.9 1 1.1 1.2 10 20 30 40 50 10 20 30 40 50 Fig. 8: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for the spherocity-integrated case (0 – 100%), the most jet-like (0 – 10%) and isotropic (90 – 100%) events are compared with Monte Carlo predictions. 6.2 Double-differential study of the average transverse momentum Predictions of PYTHIA 8 with and without (null reconnection range, rr = 0) color reconnection, as well as PYTHIA 6 and EPOS LHC are displayed. Statistical uncertainties (error bars) are negligible compared to systematic uncertainties (shaded area around the data points). Data to model ratios are shown in the bottom panel. The color band around unity represents the systematic uncertainty. integrated and isotropic classes could be affected. integrated and isotropic classes could be affected. 7 Summary and conclusions In this paper, we have reported the transverse momentum spectra of inclusive charged particles in pp col- lisions at √s = 5.02 and 13 TeV. The measurements were performed in the kinematic range of \|η\| < 0.8 and pT > 0.15 GeV/c. The particle production was studied as a function of event multiplicity quantiﬁed by two estimators, one based on the number of SPD tracklets within \|η\| < 0.8, and the second one based on the multiplicity in the V0 forward detector (V0M amplitude). For similar average charged-particle densities, the particle production above pT = 1 GeV/c is higher in pp collisions at √s = 13 TeV than at √s = 5.02 TeV. For a ﬁxed center-of-mass energy, particle production above pT = 0.5 GeV/c exhibits a remarkable multiplicity dependence. Namely, for transverse momenta below 0.5 GeV/c, the ratio of the multiplicity dependent spectra to those for INEL> 0 pp collisions is rather constant, and for higher momenta, it shows a signiﬁcant pT dependence. The behavior observed for each of the two multiplicity estimators are consistent within the ⟨dNch/dη⟩interval deﬁned by the V0M multiplicity estimator, which gives a ⟨dNch/dη⟩reach of ∼25. For the highest V0M multiplicity class, the ratio increases going from pT = 0.5 GeV/c up to pT ≈4 GeV/c, then for higher pT, it shows a smaller increase. The particle production at high transverse momenta is characterized by the exponent of a power-law function which is ﬁtted to the invariant yield considering particles with 6 < pT < 20 GeV/c. Within that pT interval, the power-law function describes rather well the pT spectra. In concordance to the ratios discussed above, within uncertainties, the functional form of n as a function of ⟨dNch/dη⟩is the same for the two multiplicity estimators used in this analysis. Moreover, n is found to decrease with ⟨dNch/dη⟩. Within uncertainties, PYTHIA 8 (tune Monash 2013) and PYTHIA 6 (tune Perugia 2011) quantitatively 17 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 0 5 10 15 20 25 30 35 40 45 50 η /d ch N d 0.9 1 1.1 1.2 1.3 1.4 〉 T p 〈 -integrated 0 S Ratio to = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.8, 0.15 ≤ \| η\| 0-10% (Jetty) 90-100% (Isotropic) PYTHIA 8 PYTHIA 8 (rr=0) PYTHIA 6 EPOS LHC Fig. 7 Summary and conclusions 9: Average pT of jet-like (circles) and isotropic (triangles) events normalized to that for the spherocity- integrated event class. The measurements are compared with different Monte Carlo generators. Statistical uncer- tainties (error bars) are negligible compared to systematic uncertainties (boxes around the data points). Fig. 9: Average pT of jet-like (circles) and isotropic (triangles) events normalized to that for the spherocity- integrated event class. The measurements are compared with different Monte Carlo generators. Statistical uncer- tainties (error bars) are negligible compared to systematic uncertainties (boxes around the data points). reproduce the behavior of data, while EPOS LHC overestimates the value of the exponent. Nevertheless, all models quantitative describe the pT integrated yields. The use of power-law exponents facilitates the study of the particle production at high pT for different collision systems. Finally, the measurement of the average transverse momentum as a function of event multiplicity at mid- pseudorapidity was presented. The results for the spherocity-integrated class (nearly identical to INEL> 0 pp collisions) at √s = 13 TeV are consistent with previous measurements at lower energies. The increase of the average pT with increasing multiplicity is well captured by PYTHIA 8 and EPOS LHC. In order to get a better insight into the particle production mechanisms, the spherocity-integrated sample was separated into different sub-classes characterized by the event structure in the transverse plane. Jet- like and isotropic events were selected based on the spherocity of the events. Isotropic events are well described by the three models which were considered in this work. Interestingly, PYTHIA 6 reproduces these event classes better than the INEL> 0 sample. For jet-like events, the average pT is overestimated by PYTHIA 6 and 8 models in the full multiplicity interval reported. However, EPOS LHC gives the best description of the jet-like event subsample. The results presented in this paper illustrate the difﬁculties for the models to describe different observ- ables once they are differentially analyzed as a function of several variables. The measurements are important to better understand the similarities between heavy-ion and small collision systems, as well as for Monte Carlo tuning purposes. Acknowledgements The ALICE Collaboration would like to thank all its engineers and technicians for their invaluable con- tributions to the construction of the experiment and the CERN accelerator teams for the outstanding performance of the LHC complex. The ALICE Collaboration gratefully acknowledges the resources and support provided by all Grid centres and the Worldwide LHC Computing Grid (WLCG) collaboration. The ALICE Collaboration acknowledges the following funding agencies for their support in building and running the ALICE detector: A. I. Alikhanyan National Science Laboratory (Yerevan Physics In- stitute) Foundation (ANSL), State Committee of Science and World Federation of Scientists (WFS), Armenia; Austrian Academy of Sciences, Austrian Science Fund (FWF): [M 2467-N36] and National- stiftung f¨ur Forschung, Technologie und Entwicklung, Austria; Ministry of Communications and High 18 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration η /d ch N d ) c (GeV/ 〉 T p 〈 10 20 30 40 50 0.4 0.6 0.8 1 = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.15 0-10% PYTHIA 6 PYTHIA 8 PYTHIA 8 (rr=0) EPOS LHC 10 20 30 40 50 class 0 S 10-20% 10 20 30 40 50 class 0 S 20-30% 10 20 30 40 50 0.4 0.6 0.8 1 class 0 S 30-40% 10 20 30 40 50 class 0 S 40-50% 10 20 30 40 50 class 0 S 50-60% 10 20 30 40 50 0.4 0.6 0.8 1 class 0 S 60-70% 10 20 30 40 50 class 0 S 70-80% 10 20 30 40 50 class 0 S 80-90% Fig. 10: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for nine spherocity classes are compared with Monte Carlo predictions. Statistical uncertainties (error bars) are negligible compared to systematic uncertainties (shaded area around the data points). Fig. 10: Average transverse momentum as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for nine spherocity classes are compared with Monte Carlo predictions. Statistical uncertainties (error bars) are negligible compared to systematic uncertainties (shaded area around the data points). Acknowledgements Technologies, National Nuclear Research Center, Azerbaijan; Conselho Nacional de Desenvolvimento Cient´ıﬁco e Tecnol´ogico (CNPq), Universidade Federal do Rio Grande do Sul (UFRGS), Financiadora de Estudos e Projetos (Finep) and Fundac¸˜ao de Amparo `a Pesquisa do Estado de S˜ao Paulo (FAPESP), Brazil; Ministry of Science & Technology of China (MSTC), National Natural Science Foundation of China (NSFC) and Ministry of Education of China (MOEC) , China; Croatian Science Foundation and Ministry of Science and Education, Croatia; Centro de Aplicaciones Tecnol´ogicas y Desarrollo Nuclear (CEADEN), Cubaenerg´ıa, Cuba; Ministry of Education, Youth and Sports of the Czech Republic, Czech Republic; The Danish Council for Independent Research — Natural Sciences, the Carlsberg Foundation and Danish National Research Foundation (DNRF), Denmark; Helsinki Institute of Physics (HIP), Fin- land; Commissariat `a l’Energie Atomique (CEA), Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3) and Centre National de la Recherche Scientiﬁque (CNRS) and R´egion des Pays de la Loire, France; Bundesministerium f¨ur Bildung und Forschung (BMBF) and GSI Helmholtzzentrum f¨ur Schwerionenforschung GmbH, Germany; General Secretariat for Research and Technology, Ministry of Education, Research and Religions, Greece; National Research, Development and Innovation Ofﬁce, Hungary; Department of Atomic Energy Government of India (DAE), Department of Science and Tech- nology, Government of India (DST), University Grants Commission, Government of India (UGC) and Council of Scientiﬁc and Industrial Research (CSIR), India; Indonesian Institute of Science, Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi and Istituto Nazionale di Fisica Nucleare (INFN), Italy; Institute for Innovative Science and Technology , Nagasaki Institute 19 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration η /d ch N d Data/Model 10 20 30 40 50 0.8 0.9 1 1.1 1.2 class 0 S 0-10% 10 20 30 40 50 = 13 TeV s ALICE pp c 10 GeV/ ≤ T p ≤ 0.15 class 0 S 10-20% PYTHIA 6 PYTHIA 8 PYTHIA 8 (rr=0) EPOS LHC 10 20 30 40 50 class 0 S 20-30% 10 20 30 40 50 0.8 0.9 1 1.1 1.2 class 0 S 30-40% 10 20 30 40 50 class 0 S 40-50% 10 20 30 40 50 class 0 S 50-60% 10 20 30 40 50 0.8 0.9 1 1.1 1.2 class 0 S 60-70% 10 20 30 40 50 class 0 S 70-80% 10 20 30 40 50 class 0 S 80-90% Fig. References [1] C. 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Acknowledgements 11: Data to model ratios as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for nine spherocity classes are shown. Systematic uncertainties are displayed as shaded areas around unity. Fig. 11: Data to model ratios as a function of event multiplicity in pp collisions at √s = 13 TeV. Results for nine spherocity classes are shown. Systematic uncertainties are displayed as shaded areas around unity. of Applied Science (IIST), Japan Society for the Promotion of Science (JSPS) KAKENHI and Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; Consejo Nacional de Ciencia (CONACYT) y Tecnolog´ıa, through Fondo de Cooperaci´on Internacional en Ciencia y Tec- nolog´ıa (FONCICYT) and Direcci´on General de Asuntos del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO), Netherlands; The Research Coun- cil of Norway, Norway; Commission on Science and Technology for Sustainable Development in the South (COMSATS), Pakistan; Pontiﬁcia Universidad Cat´olica del Per´u, Peru; Ministry of Science and Higher Education and National Science Centre, Poland; Korea Institute of Science and Technology In- formation and National Research Foundation of Korea (NRF), Republic of Korea; Ministry of Education and Scientiﬁc Research, Institute of Atomic Physics and Ministry of Research and Innovation and In- stitute of Atomic Physics, Romania; Joint Institute for Nuclear Research (JINR), Ministry of Education and Science of the Russian Federation, National Research Centre Kurchatov Institute, Russian Science Foundation and Russian Foundation for Basic Research, Russia; Ministry of Education, Science, Re- search and Sport of the Slovak Republic, Slovakia; National Research Foundation of South Africa, South Africa; Swedish Research Council (VR) and Knut & Alice Wallenberg Foundation (KAW), Sweden; Eu- ropean Organization for Nuclear Research, Switzerland; National Science and Technology Development Agency (NSDTA), Suranaree University of Technology (SUT) and Ofﬁce of the Higher Education Com- mission under NRU project of Thailand, Thailand; Turkish Atomic Energy Agency (TAEK), Turkey; National Academy of Sciences of Ukraine, Ukraine; Science and Technology Facilities Council (STFC), United Kingdom; National Science Foundation of the United States of America (NSF) and United States 20 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration References Rev. 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Skobeltsyn Institute of Nuclear, Physics, Moscow, Russia epartment of Applied Physics, Aligarh Muslim University, Aligarh, India y, y , y , , v Department of Applied Physics, Aligarh Muslim University, Aligarh, India iv Department of Applied Physics, Aligarh Muslim University, Aligarh, India v Institute of Theoretical Physics, University of Wroclaw, Poland Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Zherebchevskii112 , N. Zhigareva64 , D. Zhou6 , Y. Zhou88 , Z. Zhou22 , J. Zhu6 , Y. Zhu6 , A. Zichichi27 ,1 .B. Zimmermann34 , G. Zinovjev2 , N. Zurlo140 , R. Rogalev90 , E. Rogochaya75 , D. Rohr34 , D. R¨ohrich22 , P.S. Rokita142 , F. Ronchetti51 , E.D. Rosas70 , K. Roslon142 , P. Rosnet134 , A. Rossi29 , A. Rotondi139 , F. Roukoutakis83 , A. Roy49 , P. Roy108 , O.V. Rueda80 , R. Rui25 , B. Rumyantsev75 , A. Rustamov86 , E. 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Zhou88 , Z. Zhou22 , J. Zhu6 , Y. Zhu6 , A. Zichichi27 ,10 , M.B. Zimmermann34 , G. Zinovjev2 , N. Zurlo140 , Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 25 Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 7 Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 8 7 Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 8 8 Centro de Aplicaciones Tecnol´ogicas y Desarrollo Nuclear (CEADEN), Havana, Cuba g y 9 Centro de Investigaci´on y de Estudios Avanzados (CINVESTAV), Mexico City and M´erida, Mexico g y y 10 Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi’, Rome, Italy 11 Chicago State University, Chicago, Illinois, United States 12 China Institute of Atomic Energy, Beijing, China 13 Chonbuk National University, Jeonju, Republic of Korea 14 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovak 15 15 COMSATS University Islamabad, Islamabad, Pakistan 15 COMSATS University Islamabad, Islamabad, Pakistan 17 Department of Physics, Aligarh Muslim University, Aligarh, India 18 Department of Physics, Pusan National University, Pusan, Republic of Korea 18 Department of Physics, Pusan National University, Pusan, Republic o 19 Department of Physics, Sejong University, Seoul, Republic of Korea 19 Department of Physics, Sejong University, Seoul, Republic of Korea 20 Department of Physics, University of California, Berkeley, California, United States 21 20 Department of Physics, University of California, Berkeley, California, 21 Department of Physics, University of Oslo, Oslo, Norway 22 Department of Physics and Technology, University of Bergen, Bergen, Norway 22 Department of Physics and Technology, University of Bergen, Bergen, Norway 24 Dipartimento di Fisica dell’Universit`a and Sezione INFN, Cagliari, Italy 24 Dipartimento di Fisica dell’Universit`a and Sezione INFN, Cagliari, Italy 4 Dipartimento di Fisica dell’Universit`a and Sezione Fisica dell’Universit`a and Sezione INFN, Cagliari, Ita 25 Dipartimento di Fisica dell’Universit`a and Sezione INFN, Trieste, Italy 26 Dipartimento di Fisica dell’Universit`a and Sezione INFN, Turin, Italy 27 Dipartimento di Fisica e Astronomia dell’Universit`a and Sezione INFN, Bologna, Italy Dipartimento di Fisica e Astronomia dell’Universit`a 28 Dipartimento di Fisica e Astronomia dell’Universit`a and Sezione INFN, Catania, Italy 29 Dipartimento di Fisica e Astronomia dell’Universit`a and Sezione INFN, Padova, Italy 31 Dipartimento DISAT del Politecnico and Sezione INFN, Turin, Italy 32 Dipartimento di Scienze e Innovazione Tecnologica dell’Universit`a del Piemonte Orientale and INFN 32 Dipartimento di Scienze e Innovazione Tecnologica dell’Universit`a del Sezione di Torino, Alessandria, Italy Sezione di Torino, Alessandria, Italy 33 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy 34 33 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy 34 34 European Organization for Nuclear Research (CERN), Geneva, Switzerland 35 34 European Organization for Nuclear Research (CERN), Geneva, Switzerland 35 35 Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, University of Split, Split, Croatia 36 35 Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, University of Split, Split, Croatia 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37 l f l S i d h i l i i C h h i l i i i 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague Czech Republic p 38 Faculty of Science, P.J. ˇSaf´arik University, Koˇsice, Slovakia 39 38 Faculty of Science, P.J. ˇSaf´arik University, Koˇsice, Slovakia 39 38 Faculty of Science, P.J. Collaboration Institutes 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 Bogolyubov Institute for Theoretical Physics, National Academy of Sciences of Ukraine, Kiev, Ukraine 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 B l b I tit t f Th ti l Ph i N ti l A d f S i f Uk i Ki Uk i 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 Bogolyubov Institute for Theoretical Physics, National Academy of Sciences of Ukraine, Kiev, Ukraine 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), Kolkata, India 2 Bogolyubov Institute for Theoretical Physics, National Academy of Sciences of Ukraine, Kiev, Ukraine 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), Kolkata, India 4 Budker Institute for Nuclear Physics, Novosibirsk, Russia 5 5 California Polytechnic State University, San Luis Obispo, California, United States 6 6 Central China Normal University, Wuhan, China 26 Sezione di Torino, Alessandria, Italy ˇSaf´arik University, Koˇsice, Slovakia 39 39 Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universit¨at Frankfurt, Frankfurt, Germany 39 Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universit¨at Frankfurt, Frankfurt, Germany y 40 Gangneung-Wonju National University, Gangneung, Republic of Korea 41 41 Gauhati University, Department of Physics, Guwahati, India 41 Gauhati University, Department of Physics, Guwahati, India 41 Gauhati University, Department of Physics, Guwahati, India 42 Helmholtz-Institut f¨ur Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universit¨at Bonn, Bonn Germany Helmholtz-Institut f¨ur Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universit¨at Bonn, Bonn Germany 43 Helsinki Institute of Physics (HIP), Helsinki, Finland 44 43 Helsinki Institute of Physics (HIP), Helsinki, Finland 44 44 High Energy Physics Group, Universidad Aut´onoma de Puebla, Puebla, Mexico 44 High Energy Physics Group, Universidad Aut´onoma de Puebla, Puebla, Mexico 45 45 Hiroshima University, Hiroshima, Japan 6 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 48 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Roma 48 Indian Institute of Technology Bombay (IIT), Mumbai, India 48 Indian Institute of Technology Bombay (IIT), Mumbai, India 49 Indian Institute of Technology Indore, Indore, India 50 Indonesian Institute of Sciences, Jakarta, Indonesia 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 52 INFN, Sezione di Bari, Bari, Italy 53 INFN, Sezione di Bologna, Bologna, Italy 54 INFN, Sezione di Cagliari, Cagliari, Italy 55 INFN, Sezione di Catania, Catania, Italy 56 INFN, Sezione di Padova, Padova, Italy 57 INFN, Sezione di Roma, Rome, Italy 27 74 Johann-Wolfgang-Goethe Univers Mathematik, Frankfurt, Germany 5 74 Johann-Wolfgang-Goethe Universit¨at Frankfurt Institut f¨ur Informatik, Fachbereich Informatik und Mathematik Frankfurt Germany Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 58 INFN, Sezione di Torino, Turin, Italy 58 INFN, Sezione di Torino, Turin, Italy 59 INFN, Sezione di Trieste, Trieste, Italy 59 INFN, Sezione di Trieste, Trieste, Italy 60 Inha University, Incheon, Republic of Korea 60 Inha University, Incheon, Republic of Korea y p 61 Institut de Physique Nucl´eaire d’Orsay (IPNO), Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3/CNRS) Universit´e de Paris-Sud Universit´e Paris-Saclay Orsay France y p 61 Institut de Physique Nucl´eaire d’Orsay (IPNO), Institut National de Physique Nucl´eaire et de Physique des 61 Institut de Physique Nucleaire d’Orsay (IPNO), Institut National de Physique Nucleaire et Particules (IN2P3/CNRS), Universit´e de Paris-Sud, Universit´e Paris-Saclay, Orsay, France Particules (IN2P3/CNRS), Universit´e de Paris-Sud, Universit´e Paris-Saclay, Orsay, France 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, Netherlands 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, N 64 Institute for Theoretical and Experimental Physics, Moscow, Russia 64 Institute for Theoretical and Experimental Physics, Moscow, Russia 65 Institute of Experimental Physics, Slovak Academy of Sciences, Koˇsice, Slovakia 65 Institute of Experimental Physics, Slovak Academy of Sciences, Koˇsice, Slovakia 66 Institute of Physics, Homi Bhabha National Institute, Bhubaneswar, India y 67 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 67 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 68 Institute of Space Science (ISS), Bucharest, Romania 69 Institut f¨ur Kernphysik, Johann Wolfgang Goethe-Universit¨at Frankfurt, Frankfurt, Germany 69 Institut f¨ur Kernphysik, Johann Wolfgang Goethe-Universit¨at Frankfurt, Frankfurt, Germany 70 Instituto de Ciencias Nucleares, Universidad Nacional Aut´onoma de M´exico, Mexico City, Me 70 Instituto de Ciencias Nucleares, Universidad Nacional Aut´onoma de M´exico, Mexico City, Mexico 71 Instituto de F´ısica, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 71 Instituto de F´ısica, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 72 Instituto de F´ısica, Universidad Nacional Aut´onoma de M´exico, Mexico City, Mexico 72 Instituto de F´ısica, Universidad Nacional Aut´onoma de M´exico, Mexico City, Mexico 73 iThemba LABS, National Research Foundation, Somerset West, South Africa 74 73 iThemba LABS, National Research Foundation, Somerset West, South Africa 4 74 Johann-Wolfgang-Goethe Universit¨at Frankfurt Institut f¨ur Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany Mathematik, Frankfurt, Germany 75 Joint Institute for Nuclear Research (JINR), Dubna, Russia 76 75 Joint Institute for Nuclear Research (JINR), Dubna, Russia 76 76 Korea Institute of Science and Technology Information, Daejeon, Republic of Korea 77 76 Korea Institute of Science and Technology 77 77 KTO Karatay University, Konya, Turkey 78 78 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS-IN2P3, Grenoble, France 78 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS-IN2P3, Grenoble, France 79 Lawrence Berkeley National Laboratory, Berkeley, California, United States 80 79 Lawrence Berkeley National Laboratory, Berkeley, California, United States 80 Lund University Department of Physics, Division of Particle Physics, Lund, Sweden 80 Lund University Department of Physics, Division of Particle Physics, Lund, S 81 Nagasaki Institute of Applied Science, Nagasaki, Japan 81 Nagasaki Institute of Applied Science, Nagasaki, Japan 82 Nara Women’s University (NWU), Nara, Japan 83 National and Kapodistrian University of Athens, School of Science, Department of Physics , Athens, Greece 83 National and Kapodistrian University of Athens, School of Science, Department of Physics , Athens, Greece National Centre for Nuclear Research, Warsaw, Poland 85 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 85 National Institute of Science Education and Research, Homi Bhab 85 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 85 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India 86 N ti l N l R h C t B k A b ij 86 National Nuclear Research Center, Baku, Azerbaijan 87 National Research Centre Kurchatov Institute, Moscow, Russia 87 National Research Centre Kurchatov Institute, Moscow, Russia 88 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 88 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89 89 Nikhef, National institute for subatomic physics, Amsterdam, Netherlands 90 NRC Kurchatov Institute IHEP, Protvino, Russia 1 NRNU Moscow Engineering Physics Institute, Mo 92 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom ˇ 93 Nuclear Physics Institute of the Czech Academy of Sciences, ˇReˇz u Prahy, Czech Republic 94 4 Oak Ridge National Laboratory, Oak Ridge, Tenne 94 Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States 95 95 Ohio State University, Columbus, Ohio, United States 96 Petersburg Nuclear Physics Institute, Gatchina, Russia 97 Physics department, Faculty of science, University of Zagreb, Zagreb, Croatia 98 97 Physics department, Faculty of science, University of Zagreb, Zagreb, Croatia 98 Department, Panjab University, Chandigarh, India 99 Physics Department, University of Jammu, Jammu, India 100 Physics Department, University of Rajasthan, Jaipur, India 100 Physics Department, University of Rajasthan, Jaipur, India 101 Physikalisches Institut, Eberhard-Karls-Universit¨at T¨ubingen, T¨ubingen, Germany 101 Physikalisches Institut, Eberhard-Karls-Universit¨at T¨ubingen, T¨ubingen, Germany Physikalisches Institut, Eberhard-Karls-Universit¨at T 102 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 102 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 103 Physik Department, Technische Universit¨at M¨unchen, Munich, German 103 Physik Department, Technische Universit¨at M¨unchen, Munich, Germany 104 Politecnico di Bari, Bari, Italy Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Petersburg, Russia 113 Stefan Meyer Institut f¨ur Subatomare Physik (SMI), Vienna, Austria 113 Stefan Meyer Institut f¨ur Subatomare Physik (SMI), Vienna, Austria y y 114 SUBATECH, IMT Atlantique, Universit´e de Nantes, CNRS-IN2P3, Nantes, France 114 SUBATECH, IMT Atlantique, Universit´e de Nantes, CNRS-IN2P3, N 115 Suranaree University of Technology, Nakhon Ratchasima, Thailand 115 Suranaree University of Technology, Nakhon Ratchasima, Thailand 5 Suranaree University of Technology, Nakhon Ratch 116 Technical University of Koˇsice, Koˇsice, Slovakia 116 Technical University of Koˇsice, Koˇsice, Slovakia 117 Technische Universit¨at M¨unchen, Excellence Cluster ’Universe’, Munich, Germany 117 Technische Universit¨at M¨unchen, Excellence Cluster ’Universe’, Munich, Germany The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland y y y 119 The University of Texas at Austin, Austin, Texas, United States 119 The University of Texas at Austin, Austin, Texas, United States 119 The University of Texas at Austin, Austin, Texas, United S versity of Texas at Austin, Austin, Texas, United State 120 Universidad Aut´onoma de Sinaloa, Culiac´an, Mexico 120 Universidad Aut´onoma de Sinaloa, Culiac´an, Mexico 121 Universidade de S˜ao Paulo (USP), S˜ao Paulo, Brazil 121 Universidade de S˜ao Paulo (USP), S˜ao Paulo, Brazil 122 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 123 122 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 2 Universidade Estadual de Campinas (UNICAMP), 123 Universidade Federal do ABC, Santo Andre, Brazil 123 Universidade Federal do ABC, Santo Andre, Brazil 124 University College of Southeast Norway, Tonsberg, Norway 124 University College of Southeast Norway, Tonsberg, Norway 125 University of Cape Town, Cape Town, South Africa 126 126 University of Houston, Houston, Texas, United States 127 126 University of Houston, Houston, Texas, United 127 University of Jyv¨askyl¨a, Jyv¨askyl¨a, Finland 128 University of Liverpool, Liverpool, United Kingdom 128 University of Liverpool, Liverpool, United Kingdom 129 University of Science and Techonology of China, Hefei, China 130 129 University of Science and Techonology of China, Hefei, China 130 130 University of Tennessee, Knoxville, Tennessee, United States 130 University of Tennessee, Knoxville, Tennessee, United States 131 131 University of the Witwatersrand, Johannesburg, South Africa 131 University of the Witwatersrand, Johannesburg, South Africa 132 132 University of Tokyo, Tokyo, Japan 133 University of Tsukuba, Tsukuba, Japan 134 Universit´e Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 134 Universit´e Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 134 Universit´e Clermont Auvergne, CNRS/IN 135 Universit´e de Lyon, Universit´e Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France y y y y 136 Universit´e de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France, Strasbourg, France g, , , g, , g, 137 Universit´e Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, D´epartment de Physique Nucl´eaire (DPhN), Saclay, France g g g 137 Universit´e Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, D´epartment de Physique Nucl´eaire (DPhN), Saclay, France 137 Universit´e Paris-Saclay Centre d’Etudes de Saclay (CEA), IRFU, D´epartment de Physique Nucl´eaire Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration Particle production as a function of multiplicity and spherocity in pp collisions ALICE Collaboration 109 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 110 110 Secci´on F´ısica, Departamento de Ciencias, Pontiﬁcia Universidad Cat´olica del 110 Secci´on F´ısica, Departamento de Ciencias, Pontiﬁcia Universidad Cat´olica del Per´u, Lima, Peru 111 Shanghai Institute of Applied Physics, Shanghai, China 111 Shanghai Institute of Applied Physics, Shanghai, China g pp y g 112 St. Petersburg State University, St. Petersburg, Russia g pp y g 112 St. Petersburg State University, St. (DPhN), Saclay, France 138 Universit`a degli Studi di Foggia, Foggia, Italy 139 Universit`a degli Studi di Pavia, Pavia, Italy 139 Universit`a degli Studi di Pavia, Pavia, Italy 140 Universit`a di Brescia, Brescia, Italy 141 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, In 141 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, India 105 Research Division and ExtreMe Matter Institute EMMI Schwerionenforschung GmbH, Darmstadt, Germany 105 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum f¨ur 105 Research Division and ExtreMe Matter Institute EMM Schwerionenforschung GmbH, Darmstadt, Germany Research Division and ExtreMe Matter Institute EMM Schwerionenforschung GmbH, Darmstadt, Germany 106 Rudjer Boˇskovi´c Institute, Zagreb, Croatia 106 Rudjer Boˇskovi´c Institute, Zagreb, Croatia 107 Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 107 Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 108 Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, India 108 Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, India 28 141 Variable Energy Cyclotron Centre, H 142 Warsaw University of Technology, Warsaw, Poland 142 Warsaw University of Technology, Warsaw, Poland 143 Wayne State University, Detroit, Michigan, United States 143 Wayne State University, Detroit, Michigan, United States 144 Westf¨alische Wilhelms-Universit¨at M¨unster, Institut f¨ur Kernphysik, M¨unster, Germany 5 144 Westf¨alische Wilhelms-Universit¨at M¨unster, Institut f¨ur Kernphysik, M¨unster, German 146 Yale University, New Haven, Connecticut, United States 146 Yale University, New Haven, Connecticut, United States 147 147 Yonsei University, Seoul, Republic of Korea 29
https://openalex.org/W2342479827	https://europepmc.org/articles/pmc5253049?pdf=render	English	null	Dietetic Treatment of Diabetes	Medical clinics of North America/The Medical clinics of North America	1,960	cc-by	557	strictly , y tients, although no doubt to the apparent cure Dr. Shingleton Smith of Bristol, stated, as is of some. Dr. Shingleton Smith, of Bristol, stated, undoubtedly true, that patients often prefer a shorter life with a liberal diet to the hope of a longer one with a greatly restricted menu. With the exception of Dr. Duncan, of Glasgow, there seemed to be a general agree- ment with the lines laid down by Dr. Saundby in open- ing the discussion. Dr. Saundby pointed out the futility of persisting in withholding carbohydrates in the hope of removing the last trace of glycosuria. Carbohydrate food was not the only source of sugar. In severe diabetes sugar produc- tion and elimination continued, although no doubt much reduced in amount, even when the patient was placed on a flesh diet, a carbohydrate molecule being formed in the process of converting albumin into urea. Putting physiology on one side, however, clinical experience equally condemned the old routine method of treatment by " anti-diabetic" diet. Experiment must be tried in each case to find out how much carbo- hydrate the patient could take without injury, or rather what combination of proteid, fat, and carbohydrate gave the best result. To observe the progress of the case the patient should once a week get weighed, and collect and measure the whole of his urine for 2i hours, and a specimen of this urine, being of course an average, should be sent to the doctor for analysis. Of these observations that of the weight was really the most important, for if there was no loss of weight we had the best proof that the diet was right, even though there might be a moderate increase of sugar. Polyuria and great thirst were best checked by strict diet for a limited time, combined, if necessary, with the administration of a grain or two of extract of opium every night. -So far as the details of diet were con- cerned, the diet he gave might be described as one in which, after a fairly strict regimen for a time, which among other advantages gave a clue to the severity of g g y the case, a gradually increased amount of carbohydrate food was allowed so long as the distressing symptoms remained in abeyance and weight was not lost, but a long time must elapse before ordinary bread was taken, and often the patient had to do without it altogether. THE HOSPITAL. THE HOSPITAL. 440 Sept 25 1897 Sept 25 1897 Medical Progress a The Editor will be glad to receive offers of co-operation a should be addressed to The Editor, at the Office, 28 THE DIETETIC TREATMENT OF DIABETES. The discussion at Montreal on the Dietetic Treatment of Diabetes illustrates very forcibly tlie reaction which has taken place of late years in regard to the pre- scription of the strict anti-diabetic diet which was h i fi i i f nd Hospital Clinics d contributions f members f the f i All l tt t d contributions from members of the profession. All lettert & 29, Southampton Street, Strand, London, W.O.] strictly , y tients, although no doubt to the apparent cure Dr. Shingleton Smith of Bristol, stated, as is The starch contained in potatoes seemed to him to be much less injurious than that in cereals.
https://openalex.org/W4388910791	https://journals.uco.es/cco/article/download/14627/13037	es		La divina substancia como māsūra y muftaraqa. Un intento de reinterpretación de la terminología trinitaria de acuerdo con la enseñanza de al-Risāla fī l-thālūth al-muqaddas de Abū Rā’iṭa	Collectanea christiana orientalia	2,014	cc-by	10,193	The divine substance as māsūra and muftaraqa. An attempt of reinterpretation of the Trinitarian terminology in the light of the teaching of Abū Rā’iṭa’s al-Risāla fī l-thālūth al-muqaddas [La divina substancia como māsūra y muftaraqa. Un intento de reinterpretación de la terminología trinitaria de acuerdo con la enseñanza de al-Risāla fī l-thālūth al-muqaddas de Abū Rā’iṭa] Michal SADOWSKI Nicolaus Copernicus University, Toruń (Poland) sadowski.edu@gmail.com Resumen: Este artículo trata de la terminología trinitaria de Abū Rā’iṭa contenida en su al-Risāla fi l-thālūth al-muqaddas. Concretamente, se centra en una expresión que simboliza la unidad de la sustancia divina y la multiplicidad de hipóstasis, es decir “māsūra y muftaraqa”. Ofrecemos un intento de reinterpretación del significado de estos términos clave de acuerdo con la comprensión de la doctrina trinitaria de Abū Rā’iṭa. Abstract: The paper deals with Abū Rāi’ṭa’s Trinitarian terminology found in his al-Risāla fī l-thālūth al-muqaddas. In particular, it concentrates on an expression that epitomises the unity of the divine substance and multiplicity of hypostaseis, i.e. “māsūra and muftaraqa”. In the light of the Abū Rāi’ṭa’s understanding of the the Trinitarian doctrine, an attempt of reinterpretation of the meaning of these key-terms is presented. Palabras clave: Abū Rā’iṭa. Santísima Trinidad. Analogías. Terminología trinitaria. Key words: Abū Rā’iṭa. Holy Trinity. Analogies. Trinitarian terminology. Collectanea Christiana Orientalia 11 (2014), pp. 161-188; ISSN 1697–2104 162 Michal Sadowski Introduction The Jacobite Abū Rā’iṭa (+ after 830AD) is considered the first Arab Christian theologian to have presented the comprehensive description of the doctrine on the Trinity and Al-risāla fī l-thālūth al-muqaddas (On the Holy Trinity) is probably his most important work.1 Like the other Arab Christian texts, the pressing reason to write Al-risāla fī l-thālūth almuqaddas, as a matter of overriding importance, was to clarify the teachings of the “People of the South” )‫(اهل التمين‬, and to illustrate the doctrine of the “People of the Truth” )‫ (اهل احلق‬-i.e. the Jacobites-, as well as to explain the obscure aspects the teachings of the peoples (‫)اقاويل امم‬, presumably the Muslims. 2 or s li e that of Abū Rā’iṭa were mainly addressed to Christian congregations in their internal problems to preserve their own communities from conversion to Islam and, in that same context, to expose Christian doctrine and defend the faith against the accusations of polytheism.3 However, along with these external reasons that led the author 1 2 3 Georg GRAF, Die Schriften des Jacobiten Habîb ibn Hidma Abû Râ’ita. Edited and translated by G. Graf, col. «CSCO» 130 (Louvain: Imprimerie Orientaliste, 1951), pp. 126; Sandra Toenies KEATING, Defending the ‘people of truth’ in the early Islamic period. The Christian apologies of Abū Rāi’ṭah. Edited and translated by S. T. Keating (Leiden, Boston: Brill, 2006), pp. 164-215. Cf. Abū Rā’iṭa, Al-risāla fī l-thālūth al-muqaddas, in KEATING, Defending, p. 164. Selected apologetical works: ī tathlīth Allāh al- ā id, in Margaret Dunlop GIBSON (ed.), An Arabic Version of the Acts of the Apostles and the Seven Catholic Epistles from an Eighth or Ninth Century Ms. in the Convent of St. Catherine on Mount Sinai: with a Treatise on the Triune Nature of God, with translation from the same codex, col. «Studia Sinaitica» 7 (London: C. J. Clay and Son, 1889); THEODORE ABŪ QURRA, aymar yu a i u annahu lā yul amu l- a ārā an ya ūlū thalātha āliha idh ya ūlūna l- b ilāh a-l-Ibn ilāh a-Rū al- udus ilāh a-anna l- b a-l-Ibn a-Rū aludus ilāh a-la āna ull ā id minhum tāmm ‘alā idatihi, in Constantin BACHA (ed.), ayāmir Thā udūrus Abī urrah s uf arrān Beiruth aṭba at al-fawā’id, 1904), pp. 23–47; THEODORE ABŪ QURRA, aymar fī u ūd al- hāli a-l-dīn ala īm, in Louis CHEIKHO ed. , “ aymar li-Tāurus Ab urra f wu ūd al- hāli wa-ld n al- aw m»”, Mashriq 15 (1912), pp. 757–774, pp. 842–852; TIMOTHY, Almu ā arah al-dīniyya allatī arat bayna l- halīfat al- a dī a- imāthā us al- āthlī , The divine substance as māsūra and muftaraqa 163 to compose his writings, there is also other important information behind the text. Here, we will attempt to get insight into his Trinitarian theology in the context of its proper terminology. Consequently, this paper will focus neither on the list of the divine attributes nor on their provenience, since this has already been studied.4 4 in Robert CASPAR ed. , “Les versions arabes du dialogue entre le Catholicos Timothée I et le Calife Al- ahd`î IIe/IIIe siècle ohammed a suivi la voie des prophètes»,” Islamochristiana 3 (1977), pp. 107–175; IBRĀHĪ AL-ṬABARĀNĪ, u ādalah al-rāhib aliddīs Ibrāhīm al- abarānī ma‘a l-amīr ‘Abd al- ali ibn āli al Hāshimī, in Giacinto Bulūs MARCUZZO, Le dialogue d’Abraham de Tibériade avec ‘Abd al-Rahmân alHâsimî à Jérusalem vers 820: étude, édition critique et traduction annotée d’un texte théologique chrétien de la littérature arabe (Rome: Pontificia Universitas Lateranensis, 1986); EUTYCHIUS OF ALEXANDRIA, itāb al- urhān, in Pierre CACHIA (ed.), col. «Corpus Scriptorum Christianorum Orientalium» 192 (Louvain: Imprimerie Orientaliste, 1960); YAḤYĀ IBN ADĪ, a ālah yatabayyanu fīhā ghalaṭ Abī ūsuf ibn a‘ ūb ibn Is ā al- indī fī l-Radd ‘alā al- a ārā, in Augustin PÉRIER ed. , “Un traité de Yaḥyā ibn Ad , défense du dogme de la Trinité contre les ob ections d’al-Kind ”, Revue de l’Orient Chrétien 22 (1920-1921), pp. 3–21; IBN AL-ṬAYYIB, a ālah fī ltathlīth, in Gérard TROUPEAU ed. , “Le traité sur l’unité et la trinité de Abd Allāh ibn al-Ṭayyib”, Parole de l’Orient 2 (1971), pp. 71–89; Adel Theodore KHOURY, Apologétique Byzantine contre l’Islam (VIIIe-XIIIe s.) (Altenberge: Verlag für Christlich-Islamisches Schriftum, 1982), pp. 13-14; Richard BULLIET, Conversion to Islam in the medieval period: an essay in quantitative history (Cambridge: Harvard University Press, 1979), pp. 131; Philip JENKINS, The Lost History of Christianity (New York: HarperOne, 2008), p. 111; Arthur Stanley TRITTON, Muslim Theology (London: Luzac & Co., 1947), p. 89. Bibliography of Arab Christian literature and Christian Muslim relations: Georg GRAF, Geschichte der christlichen arabischen Literatur (Città del Vaticano: Biblioteca Apostolica Vaticana, 1944-1953), V vol.; Samir Khalil SAMIR, “Bibliographie du dialogue islamo-chrétien : Auteurs arabes chrétiens (XIe-XIIe siècles ”, Islamochristiana 2 (1976), pp. 201-249; Samir Khalil SAMIR, “Bibliographie du dialogue islamo-chrétien: Addenda et corrigenda aux auteurs arabes chrétiens des XIe et XIIe s.”, Islamochristiana 5 (1979), pp. 299-317; David THOMAS, Barbara ROGGEMA (eds.), Christian-Muslim Relations. A Bibliographical History (Leiden, Boston: Brill, 2009), vol. I; David THOMAS, Alex MALLETT (eds.), Christian-Muslim Relations. A Bibliographical History (Leiden; Boston: Brill, 2010-2011), vols. II & III. Sandra Toenies KEATING, “An Early List of the Ṣifāt Allāh in Abū Rā’iṭa al-Ta r t ’s “First Risāla On the Holy Trinity’”, Jerusalem Studies in Arabic and Islam 36 (2009), pp. 339-355. 164 Michal Sadowski Sandra Toenies Keating in her unpublished doctorate analyzed Abū Rāi’ṭa’s writings and theology from almost every possible angle. 5 However, an attentive reader would notice that this particular Trinitarian treatise (Thālūth) presents something more than a mere description of the Trinitarian dogma supported by a passing analogical references. For this reason I wish to focus my attention on a particular expression that is found in Abū Rāi’ṭa’s treatise on the Trinity, i.e. continuous and divided.6 With respect to these two terms I propose to examine them under the following aspects: 1) continuity - “homogeneity and process”; and 2 division “which does not divide”. At the end of the paper some remarks concerning the understanding of that terminology will be given. Since this two-fold way of speaking about God, in terms of continuity and division, was not created by Arabic speaking theologians, but it has its roots in the 4th century Trinitarian debate, the following section will deal briefly with the Cappadocian contribution to this issue. 1. The Cappadocian Fathers The discourse on the understanding of the divine unity and the trinity of the hypostaseis is a key-issue in the theological legacy of the Cappadocian Fathers. The Cappadocian distinction between hypostasis and substance is fundamental to their teaching on the continuity and distinction of the divine Being. Gregory of Nyssa places the Christian doctrine of God accurately between the Jewish monotheism and Greek polytheism, when he draws the 5 6 Sandra Toenies KEATING, Dialog between Muslims and Christians in the Early Ninth Century: The Example of abīb ibn idmah Abū Rā’iṭah al-Ta rītī’s Theology of the Trinity (The Catholic University of America, Washington D.C., 2001). See also: Salim DACCACHE, “Polemi ue logi ue et élaboration théologi ue chez Abû Rā’iṭa at-Ta r t ”, Annales de Philosophie 6 (1985), pp. 33-88; Sara Leila HUSSEINI, Early Christian Explanations of the Trinity in Arabic in the Context of Muslim Theology (University of Birmingham: Birmingham, 2011). The terms in italics are borrowed from S. Toenies KEATING translation. The divine substance as māsūra and muftaraqa 165 conclusion about the character of Christian doctrine, which is rooted in the Jewish conception of the unity of nature and the Hellenistic distinction of the persons.7 This principle was established most notably in his De differrentia essentiae et hypostaseos commonly nown as Basil’s Letter 38), written to clarify the Trinitarian teaching. In this work, we find an interesting passage that deals with the concept and its terminology.8 In the second paragraph of De differentia, Gregory deals with the proper understanding of the nouns (ὀνομάτων), which are predicated of plural and numerically various subjects. These general nouns are used to indicate a common nature (κοινὴν φύσιν) of things and are not confined to any particular element of the set. These individual elements, which are described by the identical definition of their essence or substance, are of the same common essence or substance (όμοούσιοι).9 Further, in Ad Ablabium quod non sint tres dei (On ‘ ot Three Gods’), we read that the oneness of nature, which is designated by a singular predicative noun, is an absolutely indivisible unit (καὶ ἀδιάτμητος ἀκριβῶς μονὰς), not capable of increase by addition or of diminution by subtraction, but in its essence being one and continually remaining one, inseparable even though it appear in plurality, continuous (συνεχὴς), complete (ὀλόκληρος), and not divided (οὐ συνδιαιρομένη) with the individuals who participate in it.10 Between the three divine Persons there is a certain indissoluble and continuous communion (συνεχή καὶ ἀδιάσπαστον κοινωνίαν). This guarantees that there is nothing inserted between the hypostaseis, nor is there anything else beyond the nature that separate it from itself. Gregory 7 8 9 10 Cf. GREGORY OF NYSSA, Oratio Catechetica Magna, III, PG 45, 17D. On the authorship of this work consult: Tomasz GRODECKI, “Autor i data powstania O rozróżnieniu między istotą a hipostazą (tzw. Listu 38 św. Bazylego iel iego)”, Vox Patrum 17 (1997), pp. 121-131. Cf. GREGORY OF NYSSA, De differentia essentiae et hypostaseos 2, PG 32, 325B-328A. Cf. GREGORY OF NYSSA, Ad Ablabium quod non sint tres dei, PG 45, 120B. English text after: Philip SCHAFF, A Select Library of the Nicene and Post-Nicene Fathers of the Christian Church (Grand Rapids, Michigan: William B. Eerdmans, 1979), vol. V, p. 332. 166 Michal Sadowski also refutes the existence of any “vacuum of interval, void of subsistence, which can make a break in the mutual harmony of the divine essence and solve the continuity (συεχὲς) by the inter ection of emptiness”.11 This emphasis on the indivisible, continuous state of the divine substance is followed by the parallel exposition concerning the distinction of the hypostaseis. To introduce the problem of hypostasis, Gregory speaks about the separation of certain circumscribed conceptions from the general idea. It is said that the particular elements of a set are characterized by the differentiating properties (ἰδιάζοντα τρέψῃ) that serve to distinguish one from another. 12 The distinctively apprehended hypostaseis are in mutual distinction (κεχωρισμένον τῶν ὐποστάσεων). 13 They are multiple but distinct from the others by the name, which belongs to each as its own and signifies the particular subject. Moreover, the three hypostaseis share in the common nature. Gregory explains that this distinction among the hypostaseis is caused by the particular attributes considered in each severally and, when they combined, is presented to us by means of number. These two characteristics of the Triune God are united in one inseparable junction through their operation (ἐνέργεια). No hypostasis, though individual and subsisting in the common nature, acts separately. None of them does anything that is not also being done by the two others. Every divine operation ad extra has its origin in the Father, proceeds through the Son, and is perfected and fulfilled in the Holy Spirit. Hence, the Trinity accomplishes each of its operation not by means of separate action according with the number of the hypostaseis, but in one motion and disposition, communicated from the Father and fulfilled in the Spirit.14 Unity and trinity are expressed not only conjunctively but also conversely to emphasize their mutual inseparability and how they work 11 12 13 14 GREGORY OF NYSSA, De differentia 4, PG 32, 332B. English text after: Philip SCHAFF, Fathers (Grand Rapids, Michigan: William B. Eerdmans, 1979), vol. VIII, p. 139. Cf. GREGORY OF NYSSA, De differentia 2, PG 32, 328A. Cf. GREGORY OF NYSSA, De differentia 4, PG 32, 332A. Cf. GREGORY OF NYSSA, De differentia 4, PG 32, 332A; GREGORY OF NYSSA, Ad Ablabium, PG 45, 125C-127B. The divine substance as māsūra and muftaraqa 167 simultaneously. This is made clear in the following passage of De differentia essentiae et hypostaseos: But the communion (κοινωνία) and the distinction (διάκρισις) 15 apprehended in Them are, in a certain sense, ineffable and inconceivable, the continuity (συνεχὲς) of nature being never rent asunder by the distinction of the hypostaseis (τῶν ὑποστάσεων διαφορᾶς), 16 nor the notes of proper distinction confounded in the community of essence. Marvel not then at my speaking of the same thing as being both conjoined and parted (συνημμὲνον καὶ διακεκριμένον), and thinking as it were darkly in a riddle, of a certain new and strange conjoined distinction (διάκρισίν τε συνημμένην) and distinct conjunction (διακεκριμένην συνάφειαν).17 This quotation sums up and confirms what was said above, namely that the parallel and, somehow, opposing properties of the divine Being as such, are intrinsically “convergent” and “coexisting”. Gregory of Nazianzus turns the expression on the continuity (conjunction) and distinction into another one, which deals explicitly with the “numeric” character of God, i.e. oneness and threeness, and conversely, threeness and oneness.18 An important re15 16 17 18 The term is rendered in English either by separation or distinction. In the Patristic sources it is used to distinguish the hypostaseis in Godhead. Cf. JOHN OF DAMASCUS, De fide orthodoxa I, 7, PG 94, 808A. In the field of Trinitarian theology the use of the term διαφορά is not allowed in reference to the divine substance, but it is commonly accepted with respect to the hypostaseis; “Κατὰ τοῦτο γὰρ καὶ θεὸτης μία·δηλονότι κατὰ τὸν τῆς οὐσίας λόγον τῆς ἐκότητος νοουμένης, ῶστε ἀριθμῷ μὲν τὴς διαφορὰν ὑπάρχειν, καὶ ταῖς ἰδιότησι ταῖς χαρακτηριζούσαις ἐκάτερον· ἐν δὲ τῷ λόγῳ τῆς θεότητος τὴν ἐνότητα θεωρεῖσθαι.” BASIL THE GREAT, Contra Eunomium I, 19, PG 29, 556B; cf. GREGORY OF NYSSA, Contra Eunomium VII, PG 45, 757B; GREGORY OF NYSSA, De differentia 5, PG 32, 336B. GREGORY OF NYSSA, De differentia 4, in PG 32, 332D-333A. English text: cf. SCHAFF, Fathers, vol. VIII, 139; GREGORY OF NYSSA, Epistula XXXV, 4n, in Anna M. SILVAS, Gregory of Nyssa: The Letters. Introduction, Translation and Commentary. Edited and translated by A.M. Silvas, Supplements to «Vigiliae Christiane» 83 (Leiden, Boston: Brill, 2007), p. 255. “Ἐκ μονάδος Τριάς ἐστι, καὶ ἐκ Τριάδος μονὰς αὖθις.” GREGORY OF NAZIANZUS, 168 Michal Sadowski mark with respect to the proper understanding of the terminology is also made. Gregory maintains that the divine hypostaseis are, in fact, divided without division and united in division (διαιρεῖται γὰρ ὰδιαιρέτως, συνάπτεται διῃρημένως).19 The Godhead (θεότης)20 is one in three and the three are one, while the proper understanding of this relation consists in not making the unity a confusion, nor the distinction a separation (οὔτε τὴν ἕνωσιν σύγχυσιν ἐργαζὸμενοι, οὔτε τὴν διαἱρεσιν, ἀλλοτρίωσιν). 21 To continue this thinking, the Three are neither so separated from one another as to be divided in nature, nor so contracted as to be circumscribed by a single person.22 2. The Unity of God The theological significance of the divine unity is the starting point of Abū Rā’iṭa’s study and exploration of the Trinity. In Christian theology the definition of the divine “unity” is always posited as God being one in the multiplicity of His hypostaseis, and this approach was already known to have its origin in Patristic times. To explain this, the Church Fathers applied the Aristotelean philosophical understanding of unity to their theological investigation. 23 Although the Trinitarian theology knows different 19 20 21 22 23 Carmina dogmatica I, PG 37, 413A. The term διαἱρεσις has a twofold meaning: disallowed in the Trinity, in the sense of division, and accepted in that of distinction by orthodoxy. Cf. ATHENAGORAS, Legatio pro Christianis 10, PG 6, 909B; GREGORY OF NAZIANZUS, Oratio XXXIX, 11, PG 36, 345D, 348Α. A term derived from the Gree θέα beholding . Cf. GREGORY OF NYSSA, Ad Ablabium, PG 45, 120D-121A. Cf. GREGORY OF NAZIANZUS, Oratio XXXIX, 11, PG 36, 345D, 348Α. Cf. GREGORY OF NAZIANZUS, Oratio XXXIV, 8, PG 36, 219A; GREGORY OF NAZIANZUS, Oratio XXXI (Theologica Quinta), 14, PG 36, 119A. Aristotle distinguished five types of unity 1 Unity by accident κατὰ συβεβηκός ; accidents which inhere in a subject may be called one together with the subject in which they inhere; 2 Unity by continuity κατὰ συνεχῆ): any number of objects may be considered one if combined to form a single collection; 3 Unity of substratum κατὰ The divine substance as māsūra and muftaraqa 169 models of unity, like: unity of substratum, unity by genus, and unity in species (definition),24 the Jacobite author was inclined to choose the latter of these, the model of unity in species.25 This model, as taught by Aristotle, unites the individuals that fall under one definition and are centered around a kind of a certain similarity (ὁμοιότης , such as, all water being the same everywhere, for it bears a kind of certain similarity. In this case the similarity is all the greater if water comes from the same source κρήνης .26 This statement is an important note we will refer and return to later in this paper. The unity in species is also discussed in the context of the unity in number, it is supported by an Alexandria-originated numerical theological “proof” concerning belief in the Trinity.27 This refers to the two groups of 24 25 26 27 ὑποκείμενον any number of substances are called one if they have a common underlying element; 4 Unity by genus κατὰ γένος an example is found between horse, human, and dog which all are animals; and 5 Unity in species εἶδος , or in definition λόγος two individuals of the same species are one, because either they have one definition, or they belong to the same species. ARISTOTLE, Metaphysics VI, 1015b, 16-35; 1016a, 1-17; 1016a, 17-24; 1016a, 24-32, 1016b, 31-32. Cf. Harry Austryn WOLFSON, The Philosophy of the Church Fathers (Cambridge, MA: Harvard University Press, 1956), p. 315. Cf. ABŪ RĀ’IṬA, Thālūth, pp. 172-174. On the further explanation of that choice read: Sandra Toenies KEATING, Dialog between Muslims and Christians in the Early Ninth Century: The Example of abīb ibn Ḫidmah Abū Rā’itah al-Ta rītī’s Theology of the Trinity (Washington: The Catholic University of America, 2001) (unpublished doctoral dissertation), pp. 382-385. Cf. ARISTOTLE, Topics I, 7, 103a, 19-23. Cf. ARISTOTLE, De coelo 268a. Philo maintained the number “three” to be a reflection of fulness and perfection, for it contains beginning, centre and the end. The Alexandrian tradition considered the number “three” to be perfect and holy from the theological perspective. According to their exegesis the number “three” was the symbol of divinity referred to sacrum, meanwhile its biblical use was aways identified with the Trinity. Cf. PHILO OF ALEXANDRIA, Quaestiones in Genesim II, 5. Translated by Ch. Mercier. Edited by Roger ARNALDEZ, Claude MONDESERT, Jean POUILLOUX, «Les oeuvres de Philon d’Alexandrie» 34-A (Paris: Cerf, 1979); PHILO OF ALEXANDRIA, Quaestiones in Genesim, III, 3. Translated by Ch. Mercier. Edited by Roger ARNALDEZ, Claude MONDESERT, Jean POUILLOUX, «Les oeuvres de Philon d’Alexandrie» 34-B (Paris: Cerf, 1984); PHILO OF ALEXANDRIA, Quaestiones in Exodum II, 100. Translated by F. Petit. 170 Michal Sadowski numbers, odd and even, when the perfect unity of species that belong to the set of numbers is enclosed in a number that contains both, odd and even numbers. Hence, this is the case of the number “three”, which encloses both odd and even. This numerical distinction found in God is then followed by another argument on the divine attributes that provides a crossing point between two theological reflections on the nature of God discussed in the treatise.28 However, it is worth noting, that Abū Rāi’ṭa did not focus his attention and explanations merely on the problem of the divine attributes, but rather he was intent on further reflection. He turns the language of attributes into a language of three categories that describe the inner life of Trinitarian relations. This issue requires further scrutiny and exploration. Referring to the divine hypostaseis Abū Rāi’ṭa claims that the divine attributes are “a perfect thing from something perfect”, and analyzes the problem in three different aspects. The “morphology” of the substance is classified in the following categories, as: a) divided and dissimilar ( ‫مفرتقة‬ ‫)متباينة‬. It is said, that in this case God is limited and isolated, having no continuity (‫ ;)ال اتصال‬b) continuous and connected (‫)متصةل مأسورة‬, having no dissimilarity (‫ ;)ال تباين‬and c) connected and divided (‫ )مأسورة مفرتقة‬at the same time (‫)مجيع ًا مع ًا‬.29 Each of these solutions to the problem consequently has different theological repercussions. If we take into consideration the model that sees the divine attributes as divided and dissimilar, the result is a polytheist model of separated gods. They differ and have no continuity that seems to constitute their equality and communion. The solution proposed 28 29 Edited by Roger ARNALDEZ, Claude MONDESERT, Jean POUILLOUX, «Les oeuvres de Philon d’Alexandrie» 33 (Paris: Cerf, 1978); CLEMENT OF ALEXANDRIA, Stromata VII, 40, 4, in SC 428, pp. 142-145. ore about the sybolism of the number “three” read in Mariusz SZRAM, Duchowy sens liczb w alegorycznej egzegezie aleksandryjskiej (II-V w.) (Lublin: RW KUL, 2001), pp. 209-217. For more about Abū Rā’ita’s doctrine on the divine attributes, read: Keating, “An Early List”, pp. 339-355. This oxymoronic expression is also used by Timothy I in his dialogue with al-Mahdi “...‫ ”والثالثة متّصةل ابنفصال منفصةل ابت ّصال‬. Cf. ABŪ RĀ’IṬA, Thālūth, 182; TIMOTHY, Almu ā arah, pp. 130-131. The divine substance as māsūra and muftaraqa 171 by number two sees the attributes as contrary to the solution number one. The attributes here are said to be continuous and connected, the thing that guarantees their cooperation, leeds to their unification and, consequently, makes their distinction and dissimilarity impossible. Therefore, in the light of orthodox Christian theology these two models must be rejected. The third model deals with a simultaneous connection and division in the divine substance, and once accepted, it is further developed. 3. Continuity - “homogeneity and process” The concept of continuity calls to mind a sense of unity and “homogeneity” of the divine substance (‫)جوهر‬. This factor is not to be understood either as a “linear,” “spacial” continuity, or as an Aristotelian unity by continuity κατὰ συνεχῆ). In the analogies presented in the Thālūth, Abū Rāi’ṭa makes an effort to visualize the complexity of the Trinitarian doctrine. Although he does not provide any definition of the term “substance” sensu stricto, nonetheless one can find some attempts to describe the very divine substance in his works. In his ī ithbāt one reads that the singularity of the substance is seen as being one in (‫)واحد يف‬: eternity (‫)الازلية‬, knowledge (‫)العمل‬, power )‫(القوة‬, honor (‫)اجملد‬, majesty (‫)العظمة‬, as well as being one in substantial attributes other than these (‫)وغريذكل من الصفات اجلوهرايت‬. In general, Abū Rāi’ṭa understands a substance of a thing and its quiddity (‫ )ماهية‬as something that embraces every component participating in that thing, and which is unchanging.30 The divine substance is said to be perfect (‫)اكمال‬, unmixed ( ‫مل‬ ‫)خيتلط به‬, simple (‫)بس يط‬, without density (‫)غري كثيف‬, spiritual (‫)روحاين‬, and incorporeal (‫)غري جسامين‬. 31 Speaking about the continuity of this substance, Abū Rāi’ṭa refutes any kind of its plurality (‫)اكثار‬.32 It is also said, that God 30 31 32 Cf. ABŪ RĀ’IṬA, Risāla fī ithbāt dīn al-na rāniyya a ithbāt al-thālūth al-muqaddas, in KEATING, Defending, pp. 106, 108. Cf. ABŪ RĀ’IṬA, Thālūth, p. 176; BASIL THE GREAT, Epistola 8, PG 32, 248C. Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 112; BASIL THE GREAT, Epistola 38, 2, PG 32, 325BC. 172 Michal Sadowski is in agreement (‫ )املتفق‬in all His affairs, harmonious (‫ )املتسق‬in all His states (‫)حاالت‬, immaterial in His substance (‫)ولطف جوهره‬, without difference in His power (‫)قوته‬, will (‫ )مشيته‬and operations (‫)افعاهل‬.33 Such a description of the divine substance calls for great scrutiny of the author’s idea of continuity, that he gives to describe the inner-Trinitarian life. The teaching on the Trinity is given by means of analogy, which was a common pedagogical method used in Christian theological debate at the time. In support of the arguments that he presented, three analogies are here described. These analogies, although limited, seem to transmit the author’s major ideas concerning his understanding of the inner-Trinitarian life. It is worth noting that Abū Rāi’ṭa acknowledges the limits that any argument per analogiam proposes. They may describe the very same things and relations, but in fact each of them highlights another aspect of the Trinitarian reality. The first example that is presented is the analogy of the three lamps, which is also used by other Arab Christian writers.34 Abū Rāi’ṭa uses this analogy in both of his treatises, the ī ithbāt and the Thālūth. It is used in reply to the following uestion is the manner of God’s unity (‫ )اتفاق‬different from the manner of His division (‫ ? افرتاق‬The “homogeneity” of the substance, as shown in this example, is demonstrated by the union of light (‫)واحد فالتفاقها مجيع ًا يف الضوية‬. The three lamps are one, with respect to the light they emit, although it is said that they constitute three “sources”, one for each flame.35 Abū Rāi’ṭa reasonably states that in the case of God, one must not speak about three sources but about one cause (‫ )عةل‬of the two other hypostaseis.36 Thus the “cause” becomes the center of the analogy of the three lamps. According to the definition of the unity in species, the similarity of the elements (species) is dependent on their source, namely, the cause. Conse uently, this static “homogeneity”, “sameness” of the 33 34 35 36 ABŪ RĀ’IṬA, Thālūth, p. 190. Cf. ABŪ QURRA, aymar yu a i u, p. 36. ABŪ RĀ’IṬA, ī ithbāt, pp. 106; ABŪ RĀ’IṬA, Thālūth, p. 186. Cf. ABŪ RĀ’IṬA, Thālūth, p. 184. The divine substance as māsūra and muftaraqa 173 divine substance appears to be based on the monarchial concept of God. The continuity of God, in this point, shifts from its seemingly “static” or “spatial” notion substantial oneness of light to its dynamic understanding as a “causative process”, which is characterized as being “without beginning and without time” ‫)بال بدى وال زمان‬. This is an important statement because it alludes to the character of the relation between the two hypostaseis and their cause (‫ عةل‬. Abū Rāi’ṭa confirms that this relation is substantial and natural (‫)اضافة جوهرية طباعية‬,37 and cannot be treated only from an individual dimension or perspective. The substantial and natural character of that relation assumes that it is a common determinant of the hypostaseis and their activity. The concept of continuity is further developed by the analogy of Adam, Abel and Eve. The analogy is present in the theological legacy both from the Church Fathers and the Arab Christian writers.38 The author proposes the analogy as a way of finding an answer on the question concerning the nature of their intransitive (unchanging) proper characteristics. The relation and similarity between Adam, Abel and Eve is said to be “something perfect from something perfect”. The perfection that describes each of them attests to their continuity i.e. “sameness” of substance that Eve and Abel share with Adam.39 Their “homogeneity” is made evident by an unbro en substantial relation (‫ )اضافة جوهرية‬that bounds Eve, Abel and Adam, and is conveyed by their common humanity (‫)الانسانية واحد‬.40 Furthermore, Abel and Eve are species whose unity is founded on a one, single cause. This idea of unifying monarchy is clearly elaborated here. Also the shift from the “static” or exclusively “substantial” understanding of the continuity to its dynamic dimension is in this case even more perceptible than in the previous analogy. The Jacobite author presents his view, with a detailed 37 38 39 40 Cf. ABŪ RĀ’IṬA, Thālūth, p. 186. ABŪ QURRA, aymar yu a i u, p. 36; ABŪ QURRA, aymar fī RĀ’IṬA, ī ithbāt, p. 114; ABŪ RĀ’IṬA, Thālūth, p. 188. Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 114; ABŪ RĀ’IṬA, Thālūth, p. 186. Cf. ABŪ RĀ’IṬA, Thālūth, pp. 184-186. u ūd, p. 224; ABŪ 174 Michal Sadowski and explicit statement, naming the particular relations that characterize the divine hypostaseis. The inner-Trinitarian relations are reflected by the proper characteristics (‫ )خاصة‬ascribed to each person of the analogy: Adam is the begetter and not the begotten (‫)وادل الودل‬, Abel is the begotten and not the begetter (‫ )ودل الوادل‬and Eve is the one who proceeds from Adam, neither begetter nor begotten (‫)خارجة من ادم ال وادل وال ودل‬.41 Unlike Adam, Abel and Eve, the divine hypostaseis are not limited either by time or by place. They are not divided either in power, will or in operation. The process of begetting and procession is atemporal. On the one hand, it is completed, but, on the other, it is eternally ongoing. Singularity is interchangeable with plurality, and the continuity is interconnected with division. The example of the Biblical triad of Adam, Abel and Eve emphasizes the role of common cause in the process of procession of the hypostaseis. This common source is not only the cornerstone of their communion but it is, moreover, the principle and guarantee of their distinction. The third analogy that was given to reflect the inner-Trinitarian life is that of the Sun.42 This is probably the most popular metaphor used by the Church Fathers and Arab Christians in their Trinitarian works. 43 In the Thālūth the analogy appears in a section concerning the temporal relation between the continuity and division of the divine substance. Using the image of the Sun (‫ )الشمس‬and its two properties, i.e. its light (‫ )ضوء‬and its 41 42 43 Cf. ABŪ RĀ’IṬA, Thālūth, p. 188. Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 112; ABŪ RĀ’IṬA, Thālūth, pp. 192-194. Cf. TEOGNOSTUS, Hypotyposeon, in PG 10, 240A; ATHANAIUS, In illud, omnia mihi traditia sunt, in PG 25, 216AB; ATHANAIUS, Orationes contra Arianos II, 41, in PG 26, 236A; ATHANASIUS, Orationes contra Arianos III, 4, in PG 26, 329A; Gregory of Nazianzus, Sermo 31, in PG 36, 162B; GREGORY OF NYSSA, Contra Eunomium 8, in PG 45, 773B; JACOB OF SERUGH, “Homélies contre les uifs”, in PO 38 1976 , p. 51, p. 53; ANASTASIUS, Explicatio fidei orthodoxae, in PG 89, 1404C; JEROME OF JERUSALEM, Dialogus de S. Trinitate inter Iudaeum et Christianum, in PG 40, 852C; JOHN OF DAMASCUS, De fide orthodoxa I, 8, in PG 94, 833A; ī tathlīth, p. 76; cf. TIMOTHY, Almu ā arah, p. 129; ABŪ QURRA, aymar yu a i u, pp. 40–41; AL-ṬABARĀNĪ, u ādalah, p. 371; EUTYCHIUS OF ALEXANDRIA, itāb al- urhān, pp. 33-34, n. 47. The divine substance as māsūra and muftaraqa 175 heat (‫)حرارة‬, the author intends to show the simultaneous nature of continuity and division; the Sun disc with its light and its heat. In the same way the divine Being may be characterized by a simultaneous continuity and division. The two substantial solar attributes, its light and its heat, proceed from their source, i.e. the solar itself. Their generation takes place within the disc and both, while dwelling in it, are also emitted by it. The generation of light and heat, their mutual indwelling, eternal and prior to time (‫)ازلي ًا ابزليته قدمي ًا بقدمه‬, may show not only the continuity and consubstantiality of the hypostaseis, but also attest the unity that is realized by their mutual indwelling, their reciprocal perichoresis.44 Their dwelling in one another is not only a static mode of being, but as in the image of the Sun, its light and its heat are continually emitted, so by parallel argument, the Father, who is the cause of the Son and the Spirit, is the principle of their mutual and continuous coinherence. This coinherence, in turn, supposes the existence of distinct subjects. 4. Division “which does not divide” The aspect of continuity presented previously, is inseparably bound to its complement, i.e. the notion of division. The division of the divine hypostaseis cannot be achieved by the existence of something “absolute”, for that would lead to a form of tritheism. The only way to distinguish the hypostaseis and to keep the unity of the substance inviolate is to distinguish them by means of relations. The relational character of the hypostaseis is also presented by means of analogy in the work of our author. Now we will examine how the division is explained and what arguments are used to show its inseparable link with the continuity. The well-known analogy of the three lamps, already presented in this 44 Cf. Jn 10:30.38, 14:9–10.20, 17:21; cf. JOHN OF DAMASCUS, De fide orthodoxa I, 8, in PG 94, 829A. 176 Michal Sadowski study, shows both the unity of the lights and their multiplicity. The division of the lights is said to consist in their self-subsistence (‫ )قامئ بعينه‬and endurance in their being (‫)اثبت بذاته‬. It is a proper characteristic that constitutes an identity of a being that does not pass away. Each of the lights is an individual, unchangeable being. He maintains also that each light is defined by its proper state of being (‫)قوام ذاته‬.45 Besides the numeric identity of the flames, no further details are provided on the nature of their division. Although the text does make use of the Trinitarian terminology, the proper state of being is not defined, and consequently the analogy does not explain fully what is meant. Manifestly more helpful guidelines are provided by the analogy of Adam, Abel and Eve. As in the case of the lights, the division of persons is expressed by the unchanging character of their properties (‫)خاصة‬. A certain distinctiveness of the analogy of Adam, Abel and Eve is that the text gives explicit names of these properties. Abū Rāi’ṭa lists here three properties that distinguish the persons from one another: the begetter (‫)وادل‬, the begotten (‫)ودل‬, and the one who proceeds (‫)خارجة‬.46 It is also said that the property of the first person is not the begotten (‫)الودل‬.47 This property is also extended to the third person to make it distinct from the second. The different ways of procession ascribed to Abel and Eve reflect the difference in procession of the divine persons. Eve’s procession is said to be “ad extra, external” ‫ خارجة‬to Adam, though she is “bone of his bone and flesh of his flesh”. 48 In conse uence, one cannot spea about Eve’s parental generation and she cannot be called Adam’s daughter. The parental relation 45 46 47 48 Cf. ABŪ RĀ’IṬA, Thālūth, p. 184. In the context of the divine hypostaseis Abū Rāi’ṭa uses here the following terms: fatherhood (‫)ابوة‬, sonship (‫)بنوة‬, procession (‫ الانمبثاق‬،‫)خروج‬. Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 114; ABŪ RĀ’IṬA, Thālūth, p. 188. The property of the Father is thus his unbegottenness (‫)ال هو مهنام‬. ABŪ RĀ’IṬA, ī ithbāt, p. 114. Cf. Gen. 2:23. The divine substance as māsūra and muftaraqa 177 is noted in the case of Abel, who is said to be begotten.49 This explanation reflects the theological insight into the inner-Trinitarian life. The relations are things that really exist in the divine Being, and do not differ from the divine substance. In consequence, the one substance which is said to be continuous, is equal to the three hypostaseis, whose distinction in based on the properties that are identical with the continuous substance. The names of the relations found in the analogy of Adam, Abel and Eve testify to the relational character of the persons, and signify their relational mode of being. Aristotle, in his teaching about the category of relation, says that the correlative beings come into existence simultaneously.50 Since the second (the Son) and third hypostasis (the Spirit) are related to the first hypostasis (the Father), as their cause, 51 they are co-eternal. With respect of the temporal dimension of these two features of the undivided substance, Abū Rāi’ṭa maintains that the continuity of the substance is not antecedent to the division of the hypostaseis. The continuity and division are related, interchangeable, inseparable and reciprocally indwelling notions that describe the divine Being. The last analogy to be analyzed is that of the Sun. The Sun and its three existent inseparable components (individuals) (‫)ذات ثلثة اشخاص‬52 is a reasonable object to picture the problem the simultaneity of continuity and division. The analogy discerns three distinct properties (‫)خواص‬: the Sun (visible solar disc, ‫)القرص‬, the heat (‫ )احلرارة‬and the light (‫)النور‬. The solar attributes are said to be unceasing (‫)مل تزل‬, continuously generated (‫)مل يزل وادل‬, existing atemporally (‫ )بال زمان‬and simultaneous (‫)شابق لوجود احدهام قبل غريه‬. The heat proceeds (‫ )منبثة‬from the Sun, but is carried by the light (‫)يف النور‬, which 49 50 51 52 An interesting account on this analogy wrote Theodor of Mopsuestia. THEODOR OF MOPSUESTIA, Controverse avec les Macédoniens, in PO 9 (1913), pp. 656-658. Also noted by Abū Rāi’ṭa (‫)من غري تقدمي وال اتخري‬. Cf. ARISTOTLE, Categories 7, 7b15; ABŪ RĀ’IṬA, ī ithbāt, p. 114. The author clearly states that the relationship of the Son, the Spirit to the Father has a continuous, unceasing character. This results from the fact that the Father is in eternal causative (‫ )عةل ازلية‬relation to the Son and the Spirit. ABŪ RĀ’IṬA, ī ithbāt, p. 114. Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 112. 178 Michal Sadowski is generated (‫ )املولود‬eternally. The division of these elements is based on the “differentiation of specific existence” ‫ )امتياز الوجود اخلاص للك واحد‬that belongs to each one.53 The property of each element of the triad is unch-anging, the solar disc is neither the light nor the heat, and the light is not the heat. The analogy of the Sun is limited and does make clear what the common substance of the three components is. Hence, their nature remains undefined. The Sun is the light and the heat, but it cannot be said that light and heat are the Sun. From the perspective of a contemporary reader, we might say, that the common principle for these three components of the analogy is radiation, which in the Sun assumes the form of a wide electromagnetic spectrum while, in the case of a visible light and sensible heat, it is only a section of this spectrum.54 As demonstrated in the three analogies, the concept of continuity is related to that of division of the divine hypostaseis. This inseparable bond shapes also the character of the division. This Trinitarian theological language must be precise, not only with respect to the terms that describe the common and particular categories of being but, first of all, it should be unambiguous with regard to such a sensitive and crucial issue as the relations. Therefore, in the context of what was said so far, the term division must not be used in the theological description of the Trinitarian life. Speaking about the division of the divine substance, or division between the three divine hypostaseis, introduces separation that leads to false conclusions and supports erroneous doctrine. Therefore in the next section we will examine theological vocabulary that Abū Rāi’ṭa uses when talking about the concepts of continuity and division. 53 54 Cf. ABŪ RĀ’IṬA, ī ithbāt, p. 112. To picture this, it is worth noting that the solar electromagnetic spectrum extends from the radio waves (300 GHz - 3 Hz) to the Gamma rays (more then 30 EHz), while the heat corresponds to the infrared radiation (430 THz - 300 GHz) and the light to the visible light frequencies (790 THz - 430 THz). The divine substance as māsūra and muftaraqa 179 5. Terminology The proper understanding of theological concepts relies on the nonambiguity of their terminology, especially in the field of Trinitarian theology. In respect to this issue, Latin theology is explicit in the terms it uses to describe the status of the hypostaseis. Each hypostaseis is said to be distinct (distinctio) from the other, never divided (divisio) nor separated (separatio).55 In case of Greek Trinitarian theological patrimony, the situation is not as clear. A reader who wants to know the Greek terms used to characterize the reciprocal status of hypostaseis has to be very careful. G.W.H. Lampe, renowned for his lexicon of Patristic Greek, lists two following terms used by the Church Fathers to render the meaning of distinctio διαίρεσις but as division denied within Trinity);56 and διάκρισις (meaning also: separation, division).57 This terminological principle is explicitly articulated by John of Damascus in his De fide orthodoxa.58 Abū Rāi’ṭa’s Trinitarian study is replete with terminology that is already developed and in use by the other Arabic speaking theologians at the time. Such terms as: substance, nature, being, hypostasis, individual, property, attribute, subsistence, generation, procession and so on, are found in almost every Trinitarian treatise of the time. 59 This changes when we take into 55 56 57 58 59 Cf. TERTULLIAN, Adversus Praxeam 12, in: PL 2, 168AB; AMBROSE, I De fide, 2 in: PL 16, 532BC; THOMAS AQUINAS, Summa theologiae I, q. 31, a. 2. On the use of Trinitarian terminology in English read: George Leonard PRESTIGE, God in Patristic Thought (London: S.P.C.K., 1952); Christopher A. BEELEY, Gregory of Nazianzus on the Trinity and the Knowledge of God (Oxford: Oxford University Press, 2008); Gilles EMERY, The Trinity. An Introduction to Catholic Doctrine on the Triune God (Washington: The Catholic University of America Press, 2011). Cf. G.W.H. LAMPE, A Patristic Greek Lexicon (Oxford, Clarendon Press: 1961), pp. 348-349. Cf. LAMPE, Lexicon, p. 354. Cf. JOHN OF DAMASCUS, De fide orthodoxa I, 14, in PG 94, 860B; JOHN OF DAMASCUS, De fide orthodoxa III, 5 in PG 94, 1000B; JOHN OF DAMASCUS, De fide orthodoxa IV, 18 in PG 94, 1181B. For more details on the Arabic Trinitarian vocabulary read: Rachid HADDAD, La Trinité 180 Michal Sadowski consideration terms that appear occasionally. Abū Rāi’ṭa’s exposition of the doctrine on the Trinity uses some terms that are correlated with the plurality of the hypostaseis and their mutual relations. The Trinitarian treatises of the Arabic speaking theologians, written between the 9th and the 10th century, make relevant remarks about Trinitarian language. In these texts we find some expressions that deal with the inner-Trinitarian relations, and provide foundations for further developpment of the proper understanding of Trinitarian dogma as well as its terminology. For instance, Timothy I (+ 823AD) refutes any separation between the hypostaseis (‫)مل تفرتق مهنام‬60 and difference (‫ )انفصال‬between God, His Word and His Spirit.61 To render the idea of multiplicity of the hypostaseis and the relations between them, he uses the m-y-z derivate words ّ ‫م‬, ‫)متيّي‬.62 An Arab-Orthodox Buṭrus al-Bayt Ra’s (Pseudo-Eutychius, (e.g. ‫متّية‬ 877-940 AD) describing the relation between the divine hypostaseis, explicitly denies their mutual separation (‫)ليس مبفرتق منه‬.63 The hypostaseis are ّ ) and conjoined without distinct without separation (‫ميّي بيهنا بال فرقة‬ 64 intermingling. Besides his re ection of “separation”, he spea s about the “distinction” not only by way of proper characteristic ‫ )خاصة‬but also by use of the m-y-z derivate terms.65 A similar remark concerning the terminology is found in Ibrāh m al-Ṭabarān ’s u ādalah al-rāhib al- iddīs Ibrāhīm alabarānī ma‘a l-amīr ‘Abd al- ali ibn āli al-Hāshimī. Discussing the doctrine of the Trinity, he notices that just as both heat and light come from the Sun without separation, in the same way God, His Spirit and His Word 60 61 62 63 64 65 divine chez les théologiens arabes (750-1050) (Paris: Beauchesne, 1985). TIMOTHY, Al-mu ā arah, 131. TIMOTHY, Al-mu ā arah, 129. TIMOTHY, Al-mu ā arat al-dīniyya allātī arat bayna l- halīfa l- ahdī a īmāthā us al- āthlī , in: Clint HACKENBURG, An Arabic-to-English Translation of the Religious Debate between the Nestorian Patriarch Timothy I and the ‘Abbāsid Caliph al-Mahdi (The Ohio State University, 2009), pp. 61-62. Cf. EUTYCHIUS OF ALEXANDRIA, itāb al- urhān, no. 31, p. 24, no. 32, p. 24. “ ‫ ولكهنّ م اهل واحد ليس يفرق بيهنم يشء‬،‫خباصته الّيت يه هل دون الآخر غريُ الآخر‬ ّ ‫”فلكّ واحد‬. EUTYCHIUS OF ALEXANDRIA, itāb al- urhān, no. 36, p. 28. Cf. EUTYCHIUS OF ALEXANDRIA, itāb al- urhān, no. 44, p. 32. The divine substance as māsūra and muftaraqa 181 are without division and separation (‫)بال تفريق وال فصل‬. 66 God’s ord and Spirit play a revealing role in our nowledge of God. Ibrāh m sees their separation from God as destructive for the deity as such, and at the same time he underlines the oneness and undivided character of the divine substance.67 In the previous section we dealt with the description of God proposed by the Jacobite theologian. However, I decided to follow the expressions used in the English edition of the treatise for the following reasons. First, to avoid the ambiguity that would rise after the use of more than a one term with respect to the “multiplicity”; second, to focus our attention on the importance of proper terminology in general; third, to signal the need of a deeper study of the context the key-terms that are used. For this purpose we notice that in spea ing about hypostaseis, Abū Rāi’ṭa uses three different words derived from the three roots: f-r-q, b-y-n and m-y-z. The terms based on two roots: f-r-q and b-y-n are used quite frequently (the f-r-q rooted words occur 29 times, the b-y-n rooted words occur 10 times), while the my-z derivates occur only 3 times. We may group the terms in respect of the context in which they are used. a) Relation between the divine attributes In most of the cases, the proper Trinitarian terminology is used in the description of the relations between the divine attributes and the divine substance,68 but there are also a few passages that deal with the relation 66 67 68 Cf. IBRĀHĪ AL-ṬABARĀNĪ, u ādalah, pp. 369-371. The expression “‫ ”لاكن هل ابتداء وانهتاء‬probably refers to 21 22. Cf. AL-ṬABARĀNĪ, u ādalah, p. 369. This is so in the following texts: on a rejection of the erroneous and a choice of the correct hypostasis-substance model #16 , the e uality of God’s substance and His hypostaseis (#18), the substance-hypostaseis simultaneity (#17, #24). Cf. ABŪ RĀ’IṬA, Thālūth, p. 183-191, 200. 182 Michal Sadowski between the components of the analogies.69 Below, the use of the f-r-q and and the b-y-n derivate terms, in their explicit reference to God or His attributes, will be presented. The first occurrence describes possible models of reciprocal relations between the divine attributes: And if this is the case, then it is necessary that [the attributes of life, knowledge and wisdom] be described either as divided (‫ )مفرتقة‬and dissimilar (‫ )متباينة‬having no continuity, or as continuous and connected, having no dissimilarity (‫)ال تباين‬, or as connected (‫ )ماسورة‬and divided (‫)مفرتقة‬ simultaneously. If they say that they are divided (‫ )مفرتقة‬without being continuous, then they are describing God as limited, because it is not possible that part of a single thing is divided (‫ )مفارق‬and separated (‫)مباين‬ form the other part, unless it is outside of its ousia, so that the two parts are isolated from each other.70 Abū Rāi’ṭa’s teaching on the nature of the relation between the divine substance and the divine attributes is important for a proper understanding of the terms he uses in the field of the Trinitarian theology. God is said not to be subject to any fragmentation, and His attributes are said to be originated from His substance.71 He is simple (‫)بس يط‬, without density ( ‫غري‬ ‫)كثيف‬, spiritual (‫)روحاين‬, and incorporeal (‫)غري جسامين‬.72 The three models of a possible substance-attribute configuration presented by the author, should be re-read in the context of his teaching on the divine Being. Since Abū Rāi’ṭa considers the divine attributes as the substantial ones, originated from God’s very substance ‫)من جوهره‬, certain requirements must be met, so that the principle of God simplicity remain inviolate. The first two models 69 70 71 72 Here, we may point out the following passages: relation between the soul, intellect and the faculty of speech (#25), the Sun, its light and its heat (#26-27), and the five bodily senses (#26). Cf. ABŪ RĀ’IṬA, Thālūth, pp. 193-195. ABŪ RĀ’IṬA, Thālūth, p. 183. Cf. ABŪ RĀ’IṬA, Thālūth, p. 182. Cf. ABŪ RĀ’IṬA, Thālūth, p. 176. The divine substance as māsūra and muftaraqa 183 do not suit Christian doctrine on God. The expressions used suggest that they are pairs of synonymous characteristics, rather than contrastive, antonymous juxtapositions. The divine attributes are said to be both ‫ماسورة‬ and ‫مفرتقة‬. The term ‫ ماسورة‬renders and idea of continuity, “homogeneity”, or “simplicity” of the divine substance. This should determine the understanding of the ‫ مفرتقة‬meaning that reflects the idea of multiplicity in God. Abū Rāi’ṭa defends the orthodoxy of the Christian faith’s position and his chosen terminology does not weaken the argument. Therefore, it seems that the latter term (‫ )مفرتقة‬should not negate the former. If one were to understand the ‫( مفرتقة‬and all the f-r-q derivate terms) as a division, one would introduce a fragmentation, a fraction, an atomization into the one divine substance. This would seem to be opposed to Abū Rāi’ṭa’s idea or intention. 73 The meaning that would perhaps better correspond to his theology is “difference”; 74 then God’s substance would be described as connected and different. The nature of this difference has been well elaborated by the author. But it would not be proper to speak about the divine hypostaseis as “different”, although we may accept that they are mar ed by a “difference,” which ma es them distinct. This so-called “difference” in them, is their way of procession. In fact, in the Trinity we distinguish two different processions: the generation of the Son and the procession of the Spirit. This difference further leads to another distinction in God, namely the properties: the fatherhood and the unbegotteness, the sonship and the procession (as presented by the analogy of Adam, Abel and Eve). 73 74 It is worth noting Lane’s indication that, in addition to “distinction”, the word may also represent the “union” or “connection” that strengthens even more its “unifying” dimension. Cf. Edward William LANE, An Arabic-English Lexicon (London: Williams and Norgate, 1893), Book I, Part 1, p. 286. Cf. Albert DE BIBERSTEIN KAZIMIRSKI, Dictionnaire arabe-français contenant toutes les racines de la langue arabe, leurs dérivés tant dans l’idiome vulgaire que dans l’idiome littéral, ainsi que les dialectes d’Alger et de Maroc (Paris: Maissoneuve et CIE Éditeurs, 1860), vol. II, p. 533; Edward William LANE, An Arabic-English Lexicon, Book I, Part 6, pp. 2383-2384. 184 Michal Sadowski In addition to what has been said so far, the cited passage contains another term that needs to be revised. Usually the dictionaries translate the b-y-n derivate nouns in a variety of ways, and they are often rendered in English by such words as “dissimilarity” or “separation”. However, the context in which Abū Rāi’ṭa uses them is specific. In accordance with the meaning of the f-r-q derivates, also the b-y-n derivates reflect the multiplicity in God in such a way that it does not harm His simplicity. A significant fragment that deals with the inner-Trinitarian relation reads: Now, does continuity precede division (‫ )افرتاق‬in the senses of the body, or does division (‫ )افرتاق‬anticipate continuity? For if the soul and the body and the senses are creatures, created things [which are] continuous and divided (‫ )مفارقة‬simultaneously without continuity anticipating division (‫)افرتاق‬, and division (‫[ )وافرتاقها‬preceding] continuity, then [this] is established as fact as we have described [it, namely] that God, may He be praised! is three hypostaseis bound through the coincidence of their ousia, and separated (‫ )متباينة‬through the state of existence of the being (‫ )حلال قوام ذات‬of each one of them, without their continuity preceding division (‫ )افرتاق‬and division (‫[ )وافرتاق‬preceding] continuity.75 The text in English interprets the ism al-fā’il ‫متباينة‬, referred to the divine hypostaseis, by separated. However, as stated above, the idea of separation is not proper in speaking about the divine hypostaseis. Furthermore, the hypostaseis are said to be ‫ متباينة‬through the state of their being (‫)حلال قوام ذات‬. The state of being, nown in Gree theology as the τρόπος ὑπάρξεως, is a property that constitutes a mode of divine Being. Therefore, the ‫حال قوام‬ should be understood as a mode of subsistence of the essence ( ‫ )ذات‬in each hypostasis. The state of being does not break either the unity of the substance or the inter-communicability of the hypostaseis, but it is rather the cause of their identity and individuality. Thus, the hypostaseis are said to be “distinct” and never separated. For that reason this text helps us to 75 ABŪ RĀ’IṬA, Thālūth, p. 195. The divine substance as māsūra and muftaraqa 185 understand the nuances of Abū Rāi’ṭa terminology. The third term that is found in the following passage describes the divine hypostaseis: For they are coincident, distinguished (‫)ممتّية‬, and different (‫)خمتلفة‬: coincident in their quiddity and their existence, and distinguished (‫ )وممتّية‬because of a distinguishing (‫ )ملّية‬characteristic of the substantial being (‫ )قوام ذات‬of each one of them, just as we have explained before in this passage. And [they] are different (‫ )وخمتلفة‬because of the difference (‫ )الختالف‬in property (‫ )خاصة‬of each one of them, although their ousia is not different because of the difference of their properties. [...] For Adam is the begetter and not the begotten, and Abel is begotten and not the begetter, and Eve is the one who proceeds, neither the begetter nor the begotten: [they have] different (‫)خمتلفة‬ properties belonging to distinguished (‫ )ممتّية‬hypostaseis, [and] coincident ousia.76 Abū Rāi’ṭa is also familiar with the m-y-z derivate terms. In the quoted passage, he maintains that the hypostaseis are distinguished (‫ )ممتّية‬by the characteristic of each one’s substantial being ‫)قوام ذات‬. This characteristinc is further rendered by a term ‫خاصة‬, a proper characteristic. Since it is the same factor as in the previous text that makes the hypostaseis different (i.e. ‫)قوام ذات‬, the relation between them should be described also by the same term, here “distinction”. It means that in this particular context the b-y-n and m-y-z derivate terms are synonymous. b) Relations in analogies The Trinitarian analogies belong to the second group of texts where Abū Rā’iṭa follows his theological terminology. The terms are not referred directly to the divine reality, but concern the components of the analogies. We 76 ABŪ RĀ’IṬA, Thālūth, p. 200. 186 Michal Sadowski may count among these passages the analogies where the f-r-q and the b-yn derivate terms are found. In these cases they characterize the relation between such things like: the Sun, its light and its heat;77 the soul, the intellect and the faculty of speech;78 and the five bodily senses.79 It is worth noting the analogy of the soul, the intellect and the faculty of speech, in which an interesting use of the ‫ مباينة‬term appears. Are they continuous or are they divided (‫)مفرتقة‬, or do they have both attributes, I mean continuity and division (‫ ?)افرتاق ًا‬Was the soul ever separate (‫ )مباينة‬from the intellect and the faculty of speech, or one of these two from the others, then joined [together] later? Or is it not the case that their continuity and division (‫[ )وتبايهنا‬occurred] together from their very beginning, [so that] one of them did not precede the other? Now, the thing is [in fact] as our description [explains] the continuity of the soul with its faculty of speech, and their division (‫)وافرتاقها‬.80 In this passage, Abū Rāi’ṭa poses a uestion “ as the soul ever ‫ مباينة‬from the intellect and the faculty of speech, or one of these two from the others, then oined [together] later?” This uestion highlights two points. First, although the analogy is an imperfect way of demonstrating things, its psychological model refers to the mutual indwelling and unity of the soul, the intellect and the faculty of speech, reflecting the Trinitarian perychoresis. Second, since the elements of the analogy were never anterior nor posterior to each other, their existence is parallel, simultaneous and atemporal. In consequence, they were never separated (‫)مباينة‬. Here, Abū Rāi’ṭa seems to be using the word ‫ مباينة‬in the sense of “separation” to point out what kind of relations are not to be ascribed to the divine hypostaseis. 81 77 78 79 80 81 ABŪ RĀ’IṬA, Thālūth, p. 193. ABŪ RĀ’IṬA, Thālūth, p. 193. ABŪ RĀ’IṬA, Thālūth, p. 193. ABŪ RĀ’IṬA, Thālūth, p. 193. A similar context of use of that term is found in the analogy on the Sun. Cf. ABŪ The divine substance as māsūra and muftaraqa 187 Moreover, in the same passage there is another place where the b-y-n derivate term is used “Or is it not the case that their continuity and division (‫[ )وتبايهنا‬occurred] together from their very beginning, [so that] one of them did not precede the other?” This statement is an explicit expression of an inseparable bound between the “one” and “many”. Its particularity is based on their “uninterrupted” coexistence. Continuity is not bro en by multiplicity and multiplicity by continuity. Therefore, in contrary to the previous use of that word, one cannot speak here about separation but rather about “distinction”. Conclusions Abū Rāi’ṭa’s demonstration of the Trinity in a form presented in the Thālūth is a study that, probably for the first time, appears in this form in Arab Christian works at the time. Its distinctive feature is the way it deals with the Trinitarian dogma. Abū Rāi’ṭa follows an interesting, vivid method of exposition, which, founded on the doctrine of the divine attributes, explains Christian perspective ―one could almost say― pictorially. How is it achieved? Our knowledge on God is realised through a combination of the two aspects (that which is common and proper, or continuous and distinct). The expression that epitomises the unity of the divine substance and multiplicity of hypostaseis is that of “continuity and difference”. It is repeated many times across the wor , and it creates a deep impression on the reader’s mind. The three analogies that were used in this paper also played an important role in the understanding of Abū Rāi’ṭa’s work. These analogies that are used, are not just mere examples, but were used in the process of developing and understanding of “continuity and difference”. The chosen analogies are not randomly pic ed but allow for a reflection on the inner-Trinitarian life from their true perspective, showing the reciprocal dependence of the “continuity and difference”. Such a RĀ’IṬA, ī ithbāt, p. 112. 188 Michal Sadowski fruitful exploration allows us to depart from conceiving the divine unity as a monolith, and at the same time, allows for an explicit negation of the strict numeral form of monotheism. The elaboration of the issue of “distinction” by means of the analogy of Adam, Abel, Eve introduces the reader to the reality of the inner-Trinitarian relations. Furthermore, it also highlights that the divine hypostaseis not only proceed from the common, one cause ―in reference to the unity of species― and attests to their substantial equality and individual identity, but it also suggests that they (hypostaseis) are turned to one another. Such a communion of Persons finds its climax in their reciprocal indwelling, as pictured by the analogy of the Sun. Speaking about the multiplicity of the hypostaseis along with their inalienable substantial unity and perfect similarity, leads inevitably to the development of terminology. As we can see in the Thālūth the Arabic abounds in the variety of words that may be used in to express the plurality of hypostaseis. However, because of this profusion, the terminology referring to multiplicity has to be read carefully. Unlike the other authors, Abū Rāi’ṭa may be accused of lacking clarity in the terminology he uses. Therefore, as it was shown, each root-derivate term should be, so to speak, deciphered in the very context of its use. The Trinitarian analogies applied to the exposition of the dogma are helpful to grasp the Abū Rāi’ṭa’s teaching. As presented above, they do not only serve to illustrate Abū Rāi’ṭa’s understanding of the the Trinitarian doctrine, but they help to get the right understanding of his terminology. Recibido / Received: 13/02/2012 Informado / Reported: 12/05/2014 Aceptado / Accepted: 19/06/2014
https://openalex.org/W2264687410	https://europepmc.org/articles/pmc4694703?pdf=render	English	null	Genome-Wide Association Study of Grain Appearance and Milling Quality in a Worldwide Collection of Indica Rice Germplasm	PloS one	2,015	cc-by	14,528	RESEARCH ARTICLE Abstract Citation: Qiu X, Pang Y, Yuan Z, Xing D, Xu J, Dingkuhn M, et al. (2015) Genome-Wide Association Study of Grain Appearance and Milling Quality in a Worldwide Collection of Indica Rice Germplasm. PLoS ONE 10(12): e0145577. doi:10.1371/journal. pone.0145577 Citation: Qiu X, Pang Y, Yuan Z, Xing D, Xu J, Dingkuhn M, et al. (2015) Genome-Wide Association Study of Grain Appearance and Milling Quality in a Worldwide Collection of Indica Rice Germplasm. PLoS ONE 10(12): e0145577. doi:10.1371/journal. pone.0145577 Grain appearance quality and milling quality are the main determinants of market value of rice. Breeding for improved grain quality is a major objective of rice breeding worldwide. Identification of genes/QTL controlling quality traits is the prerequisite for increasing breed- ing efficiency through marker-assisted selection. Here, we reported a genome-wide associ- ation study in indica rice to identify QTL associated with 10 appearance and milling quality related traits, including grain length, grain width, grain length to width ratio, grain thickness, thousand grain weight, degree of endosperm chalkiness, percentage of grains with chalki- ness, brown rice rate, milled rice rate and head milled rice rate. A diversity panel consisting of 272 indica accessions collected worldwide was evaluated in four locations including Hangzhou, Jingzhou, Sanya and Shenzhen representing indica rice production environ- ments in China and genotyped using genotyping-by-sequencing and Diversity Arrays Tech- nology based on next-generation sequencing technique called DArTseq™. A wide range of variation was observed for all traits in all environments. A total of 16 different association analysis models were compared to determine the best model for each trait-environment combination. Association mapping based on 18,824 high quality markers yielded 38 QTL for the 10 traits. Five of the detected QTL corresponded to known genes or fine mapped QTL. Among the 33 novel QTL identified, qDEC1.1 (qGLWR1.1), qBRR2.2 (qGL2.1), qTGW2.1 (qGL2.2), qGW11.1 (qMRR11.1) and qGL7.1 affected multiple traits with relatively large effects and/or were detected in multiple environments. The research provided an insight of the genetic architecture of rice grain quality and important information for mining genes/QTL with large effects within indica accessions for rice breeding. Genome-Wide Association Study of Grain Appearance and Milling Quality in a Worldwide Collection of Indica Rice Germplasm Xianjin Qiu1☯, Yunlong Pang2☯, Zhihua Yuan1, Danying Xing1, Jianlong Xu2,3, Michael Dingkuhn4,5, Zhikang Li2, Guoyou Ye4 Xianjin Qiu1☯, Yunlong Pang2☯, Zhihua Yuan1, Danying Xing1, Jianlong Xu2,3, Michael Dingkuhn4,5, Zhikang Li2, Guoyou Ye4 1 Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education/College of Agriculture, Yangtze University, Jingzhou 434025, China, 2 Institute of Crop Science/National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China, 3 Shenzhen Institute of Breeding & Innovation, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China, 4 International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines, 5 CIRAD, UMR AGAP, F-34398 Montpellier, France ☯These authors contributed equally to this work. * g.ye@irri.org (GY); xujlcaas@126.com (JX) ☯These authors contributed equally to this work. * g.ye@irri.org (GY); xujlcaas@126.com (JX) Introduction Competing Interests: The authors have declared that no competing interests exist. Rice (Oryza sativa L.), a staple cereal crop, feeds more than half of the world’s population. Improvement of rice yield and grain quality is the major objective of rice breeding worldwide. Grain quality primarily includes grain appearance, milling, eating and cooking and nutrition qualities. Grain appearance quality mainly includes grain shape and chalkiness. Grain shape, described by grain length (GL), grain width (GW), grain thickness (GT) and grain length to width ratio (GLWR), contributes to grain weight and yield and has a great impact on the mar- ket values of rice grain products. A short and round rice grain is generally preferred by con- sumers in Northern China, Korea and Japan, whereas consumers in Southern China, the USA, and South and Southeast Asian countries prefer a long and slender rice grain [1]. Chalkiness, according to its location within the endosperm, can be divided into white belly, white core and white back in rice grain [2]. Chalky grains, filled with loosely packed, round and large com- pound of starch granules, are more prone to breakage during milling and reduce head milled rice rate (HMRR). Furthermore, when chalky grains are cooked, cracks occur easily and reduce the palatability of the cooked rice [3]. Degree of endosperm chalkiness (DEC) and percentage of grains with chalkiness (PGWC) are commonly used to measure grain chalkiness. Milling quality determines the final yield and the broken kernel rate of the milled rice, which is of con- cern for consumers and farmers. Milling quality is measured by brown rice rate (BRR), milled rice rate (MRR) and HMRR. Brown rice is the de-hulled rice with the palea and lemma removed that can be used for cooking and eating. Milled rice is the result of brown rice after removing all of the bran, which consists of aleurone and pericarp, and germ or embryo. Head milled rice is kernel longer than or equal to 3/4 full length of a kernel. Among the above-men- tioned three milling quality parameters, HMRR is the most important and greatly affects mar- ket value. HMRR depends on varietal characteristics, production factors, and harvesting, drying and milling processes. Most of the rice quality determining traits are quantitatively inherited, controlled by multi- ple genes/QTL [2] and affected by growing environment [4]. GWAS of Rice Grain Quality Editor: Rattan Singh Yadav, Aberystwyth University, UNITED KINGDOM Editor: Rattan Singh Yadav, Aberystwyth University, UNITED KINGDOM UNITED KINGDOM Received: September 24, 2015 Accepted: December 4, 2015 Published: December 29, 2015 Copyright: © 2015 Qiu 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: © 2015 Qiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was granted by Global Rice Science Partnership (GRiSP), National 863 High Technology Research and Development Program from the Ministry of Science and Technology of China (2014AA10A601), Shenzhen Peacock Plan, the Natural Science Foundation of China (31461143014, 31261140369), and Hubei Collaborative Innovation Center for Grain Industry. 1 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Introduction As for other complex traits, QTL mapping using bi-parental populations derived from parental lines of contrast performance has been widely used to identify QTL for various rice quality traits. Many genes/QTL for rice grain appearance quality and milling quality traits have been reported in the last decades. The GW1-1 and GW1-2 [5], qGRL1.1 [6], GS2 [7], GW3 and GW6 [8], qGL-4b [9], qPGWC-7 [10] qGL-7 [11], qGRL7.1 [6], gw8.1 [12], gw9.1 [13], tgw11 [14] have been fine mapped. The GW2 [15], GS3 [16], qGL-3 [17], qSW5 [18], GS5 [19], Chalk5 [20], TGW6 [21], GW6a [22], SRS1 [23], GL7/GW7 [24, 25], GW8 [26] and CycT1;3 [27] have been cloned. The usefulness of some of the well characterized genes/QTL was proven in an indica population of diverse breeding lines [4]. However, QTL mapping using a single bi-parental population has a few important limita- tions including the need to create a population segregating for the target trait, the ability to assess only two alleles per locus, and the limited number of meiosis. Only a limited number of QTL can be identified, since only QTL for which the two parents differ can be detected. Given the limited scope of each study, mapping the same trait in different bi-parental populations may yield different QTL. More than two alleles are likely to segregate per locus, and the direc- tions of QTL effects may vary depending on the genetic background because of epistasis, pleiot- ropy and QTL- by- environment interaction (QEI) [28]. An alternative QTL mapping approach is to conduct association analysis in a large germ- plasm collection, known as association mapping. Association mapping is based on linkage dis- equilibrium (LD) or the non-independence of alleles in a population. Association mapping studies using sparse SSR markers has been proven to be successful in identifying marker-trait PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 2 / 25 GWAS of Rice Grain Quality associations (MTA) in rice [29–32]. With the development of high-throughput sequencing and SNP chip techniques, genome-wide association study (GWAS) using high density markers becomes more and more popular in rice genetics. The first GWAS in rice was conducted by Huang et al. [33] using 517 rice landraces genotyped by sallow sequencing. Among the QTL identified two and five were for GW and GL, respectively [33]. Applying the same method to a large panel of 950 worldwide rice accessions, Huang et al. Association mapping panel A total of 272 indica rice accessions from 31 countries or regions (S1 Table) were used in this study. More accessions came from China (39), Philippines (36), Madagascar (29), India (28), Senegal (24), Sri Lanka (24) and Bangladesh (15). For other countries or regions the number of accessions was fewer than eight. Introduction [34] identified two, four and two QTL for GW, GL and thousand grain weight (TGW), respectively. Based on genotyping 44,100 SNP across 413 diverse accessions from 82 countries, Zhao et al. [35] identified eight,10 and 15 QTL for GW, GL and GLWR, respectively. Begum et al. [36] identified seven, 10 and 11 QTL for GW, GL and GLWR, respectively, using advanced lines from IRRI’s irrigated breeding pro- gram genotyped with 71,710 SNP generated by Genotyping-by-sequencing (GBS). Similarly, using 533 O. sativa landrace and elite accessions genotyped with 4,358,600 SNP derived from GBS, Yang et al. [37] identified 10, 11, 19 and 21 QTL for GLWR, TGW, GW and GL, respec- tively. These studies clearly demonstrated that GWAS offers higher resolution for QTL map- ping and can be used as a powerful complementary strategy to the classical linkage mapping using bi-parental segregation populations. Many of the reported association studies in rice included grain traits, such as GL, GW, GLWR, GT and TGW. However, rice chalkiness and milling quality traits were less studied. The objective of this study is to identify markers associated with 10 grain appearance quality and milling quality traits using an indica collection of 272 accessions. The panel was pheno- typed in four locations representing major indica rice production environments in China. GBS and Diversity Arrays Technology (DArT) based on next-generation sequencing technique called DArTseq™was used to generate genome-wide markers. The association panel was char- acterized for population structure using three different methods and for LD pattern by estimat- ing basal LD and LD decay in the whole population and subpopulations. Sixteen association mapping models were tested to choose the best model for each of the trait-environment combi- nations. A number of known QTL and novel QTL were identified. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Phenotypic analysis Due to various reasons not all accessions had phenotypic data in all the four testing environ- ments. The final population size for each of the trait-environment combinations varied greatly (Table 1). Phenotypic analysis was conducted using linear mixed models to properly handle the unbalance data. For single-site analysis, accession (genotype) was regarded as fixed effect and replicate as random effect. The best linear unbiased estimates (BLUE) of accessions were obtained. For multi-site analysis, all effects including accession (genotype), environment and rep- licate within environment were regarded as random to estimate variance components. The best linear unbiased predictions (BLUP) for each of the genotype-environment combinations were predicted. All analyses were conducted using the PBTools package of R [38] developed by IRRI (bbi.irri.org). Phenotypic correlations were computed from the BLUE using the “rcorr” function implemented in the R package Hmisc [39]. Narrow-sense heritability (h2) based on genotypic means was computed using the estimated variance components as VG / (VG + VGEI /s +Ve /sr). Where, VG, VGEI, Ve are the variance of genotype, genotype by environment interaction (GEI) and residual error, respectively, s is the number of environments and r is the number of replicates. Phenotypic evaluation Field trials were conducted using a randomized complete block design with two replicates in four environments including Hangzhou (HZ), Jingzhou (JZ), Sanya (SY) and Shenzhen (SZ) in China. The sowing and transplanting dates varied for the testing environments to fit into the local planting season. In all environments, the plot size was three rows of 10 plants planted at a spacing of 20cm x 25cm. Local farmers’ management practices were followed. At maturity, eight plants in the middle row were harvested. The institutions including Yangtze University, Zhejiang Academy of Agricultural Sciences, Shenzhen Institute of Breeding & Innovation per- mitted the field trials were conducted in their experimental fields. The nature dried seeds stored in room temperature for three months were used to measure quality traits. GL (mm), GW (mm), GLWR, GT (mm), TGW (g), BRR (%), MRR (%) and HMRR (%) were measured according to the National Rice Grain Quality Assessment Standard of China (GB/T17891-1999). The grain chalkiness characteristics were measured using a 3 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality JMWT12 Rice Appearance Quality Detector (Dong Fu Jiu Heng, Beijing). PGWC (%) was the percentage of head milled grains with chalkiness. DEC (%) was calculated as the product of PGWC and chalkiness size (the area of chalkiness divided by the area of whole grain). All mea- surements were conducted for two independent samples. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Genotyping Genomic DNA was extracted for each accession from the leaf tissues of a single plant using the MATAB method described in Risterucci et al. [40] and then diluted to 100 ng/μl. Genotyping was conducted at Diversity Arrays Technology Pty Ltd. (DArT P/L), Australia, using a method of DArTseq™. The method involves genome complexity reduction using PstI/TaqI restriction enzymes followed by Illumina short-read sequencing. PstI specific adapters tagged with 96 dif- ferent barcodes to encode a plate of DNA samples were ligated to the restriction fragments. The resulting products were amplified and checked for quality. The 96 samples were pooled and run in a single lane on an Illumina Hiseq2000 instrument. The PstI adapters included a sequencing primer so that the tags generated were always read from the PstI sites. As detailed by Courtois et al. [41], the resulting sequences were filtered and split into their respective target datasets, and the barcode sequences were trimmed. The sequences were trimmed at 69 bp (5 Table 1. Popoation size for 10 traits in four environments. Trait HZ JZ SY SZ GL 220 131 216 228 GW 218 132 213 229 GLWR 218 131 215 229 GT 198 132 217 221 TGW 167 130 214 206 DEC 147 128 206 197 PGWC 147 131 213 200 BRR 136 130 201 206 MRR 136 130 213 205 HMRR 136 130 213 205 doi:10.1371/journal.pone.0145577.t001 Table 1. Popoation size for 10 traits in four environments. e 1. Popoation size for 10 traits in four environments. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 4 / 25 GWAS of Rice Grain Quality bp of the restriction site plus 64 bases with a minimum Q score of 10). A proprietary analytical pipeline developed by DArT P/L was used to produce DArT score tables and SNP tables. The remaining 69 bp sequences were aligned to the Os-Nipponbare-Reference-IRGSP-1.0 [42] pseudomolecule assembly using Bowtie v0.12 [43] with a maximum of three mismatches to recover the position of the restriction site for the DArT markers and the position of the poly- morphism(s) within the 69 bp sequences for the SNPs. For the DArT markers, the position given is that of the second base of the 6 base PstI restriction site (5'-C\|TGCAG-3') because the mutated base is unknown and can be any of the six. Imputation of missing genotypic data Markers for which the minor allele frequency (MAF) is below 2.5% were discarded before imputing missing data. Missing data were estimated using Beagle v4.0 [44]. Beagle uses a local- ized haplotype cluster model. It is a special class of directed acyclic graph which empirically models haplotype frequencies on a local scale and therefore adapts to local structure in the data. The model determines a hidden Markov model that can be used to find the most likely haplotype pair for each individual, given the genotype data for that individual and the graphical haplotype frequency model. The method works iteratively using an expectation maximization type approach. The imputed missing data, probabilities of missing genotypes and inferred hap- lotypes are calculated from the model that is fitted at the final iteration. Analysis of population structure Different statistical methods were employed to infer the number of subgroups in the panel. First, a model based Bayesian clustering analysis method implemented in STRUCTURE soft- ware version 2.3.4 [45] was used. The program was run with the following parameters: k, the number of groups in the panel varying from 1 to 9; 10 runs per k value; for each run, 10,000 burnin iterations followed by 10,000 MCMC (Markov Chain Monte Carlo) iterations. The optimal number of groups (k) was determined by Δk following the method described in Evanno et al. [46]. STRUCTURE analysis was conducted for different numbers of markers evenly distributed on the whole genome. Very similar results were obtained when the number of markers was more than 1000. Second, a multi-dimensional scaling and cluster analysis method implemented in Awclust package [47] was used. Third, Tdiscriminant analysis of prin- cipal components (DAPC) method implemented in adegenet package [48] was used. The “find.cluster” function was used to identify clusters (k) and the optimal k value was determined according to the Bayesian Information Criterion (BIC), then the “dapc” function was used to check the classification quality. Genotyping The same sequences were then aligned to the pseudomolecules using BLAST (e-value <1.0 e-20) to assess whether additional sequences could be positioned. The sequences that had only one hit on the pseudomolecules or had more than one hit but with a difference of at least 1.0 e-5 between the first and the second hits were retained for further analyses. When the marker position fell within a Michigan State Univer- sity-annotated gene (http://rice.plantbiology.msu.edu/), the feature was determined (intron, exon, 3' or 5' UTR), and the name and function of the gene were retrieved. Call rates were mea- sured for all markers, and markers with call rates below 80% were discarded. The allele fre- quency of the remaining markers was then calculated, and markers for which the minor allele had a frequency below 2.5% were also discarded. Linkage disequilibrium LD was measured by squared allele frequency correlations (r2) values between the pairs of markers using TASSEL 5.2.6 [49]. Only markers with MAF greater than 10% were used. Marker pairs were discretized into bins of 10 kb and the median r2 value was used as the esti- mate of r2 of a bin. The estimated r2 values were formulated as a function of physical distances between markers. A power law curve (y = axk) was fitted to determine the physical position (x) corresponding to a given r2 value (y) [52]. The following scheme was used to estimate the back- ground LD beyond which LD was assumed to be caused by genetic linkage: One marker per chromosome was randomly sampled and the maximum of all the pairwise r2 values was taken. The median of the maximum r2 values of 10,000 samples was taken as the basal r2 value. The physical distance at which r2 reached to the basal value was determined as the LD decay dis- tance of the population. All analysis was done for the whole population and the three subpopu- lations inferred by STRUCTURE. Kinship coefficient Three methods were used to calculate kinship coefficient matrix (K). (1) Pairwise_IBS: the dis- tance between two accessions is computed as the proportion of SNP which are different. The 5 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality IBS is computed by subtracting all values of the distance matrix from 2 and then scaling the resultant matrix so that the minimum value is 0 and the maximum value is 2. It is implemented in TASSEL 5.2.6 [49]. (2) The scaled_IBS: the IBS was scaled to have the mean diagonal ele- ment equal to 1+F, where F is the inbreeding coefficient of the current population. It is the default method of the TASSEL 5.2.6 [49]. (3) The VanRaden method: the realized relatedness between individuals is computed according to ZZ'/(2∑pi(1-pi)) where Z = W-P, W is the marker matrix, P contains the allele frequencies multiplied by 2, pi is the allele frequency of marker i, and the sum is over all loci [50]. It is the default method of GAPIT [51]. Association analysis All association models under the unified model for association mapping [53] can be described by considering how the two factors, the population structure (Q) and genetic relatedness between genotypes (K), are considered. We used four options for handling Q and four options for handling K to create 16 models. The four options for Q were: no Q, Q3 derived from STRUCTURE, Q7 derived from STRUCTURE and C7 derived from adegenet. The four options for K were: no K, K computed as pairwise_IBS (Kp scaled_IBS (Ks) and VanRaden method (Kv). All analyses were conducted using TASSEL 5.2.6 [49]. For models without K, known as general linear model (GLM) 1,000 permutations were used. For models with K, known as mixed liner model (MLM), the compressed mixed linear model approach [53, 54] and P3D [54] algorithms were adopted to reduce computing time. The best model for each trait-environment combination was chosen using the mean square difference (MSD) between observed and expected p-values of all marker loci, as suggested by Stich et al. [55]. MSD was a measure for the deviation of the observed p-values from the uniform distribution. Model with a smaller MSD is more appropriate. The critical p-value for declaring significant MTA was set to 0.0001. Basic statistics of markers A total of 22,266 polymorphic markers were detected in the panel including 9,340 SNP and 12,926 DArT markers. By removing markers with MAF less than 5%, 18,824 high quality markers (7,885 SNP and 10,939 DArT) were used in association analysis. The number of mark- ers per chromosome ranged from 891 on chromosome 10 to 2,361 on chromosome 1. The size of chromosome varied from 22.8Mb for chromosome 9 to 43.2 Mb for chromosome 1. The 6 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality whole genome size was 371.7 Mb and the average distance between neighboring markers (marker spacing) was 20.2 kb. The average marker spacing ranged from 16.3 kb for chromo- some 11 to 25.9 kb for chromosome 10 (S2 Table). About 70.4% of the neighboring markers distances were below the average value and 97.4% were less than 100 Kb. There were eight gaps devoid of markers larger than 500 kb on chromosomes 1 (D01_26116212—S01_26770440), 2 (D02_13852683—D02_15083642), 4 (D04_8765494—D04_9302397 and D04_16774867— D04_17319136), 6 (D06_18215124—D06_18803206), 7 (D07_11700243—S07_12785983 and S07_13836518—D07_14505070) and 11 (D11_11988263—S11_12639397). More than half of the markers (55.7%) had MAF lower than 0.20 (Fig 1). whole genome size was 371.7 Mb and the average distance between neighboring markers (marker spacing) was 20.2 kb. The average marker spacing ranged from 16.3 kb for chromo- some 11 to 25.9 kb for chromosome 10 (S2 Table). About 70.4% of the neighboring markers distances were below the average value and 97.4% were less than 100 Kb. There were eight gaps devoid of markers larger than 500 kb on chromosomes 1 (D01_26116212—S01_26770440), 2 (D02_13852683—D02_15083642), 4 (D04_8765494—D04_9302397 and D04_16774867— D04_17319136), 6 (D06_18215124—D06_18803206), 7 (D07_11700243—S07_12785983 and S07_13836518—D07_14505070) and 11 (D11_11988263—S11_12639397). More than half of the markers (55.7%) had MAF lower than 0.20 (Fig 1). Fig 1. Frequency of markers in different MAF classes. SNP with MAF < 0.05 were excluded. Fig 1. Frequency of markers in different MAF classes. SNP with MAF < 0.05 were excluded. doi:10.1371/journal.pone.0145577.g001 Fig 1. Frequency of markers in different MAF classes. SNP with MAF < 0.05 were excluded. doi:10.1371/journal.pone.0145577.g001 Fig 1. Frequency of markers in different MAF classes. SNP with MAF < 0.05 were excluded. doi:10.1371/journal.pone.0145577.g001 doi:10.1371/journal.pone.0145577.g001 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 7 / 25 GWAS of Rice Grain Quality Population structure According to the value of Δk, there were three groups in the current panel (S1 Fig). Group-I consisted of 88 accessions from 21 countries with 36 from China (36). Group-II had 96 acces- sions from 19 countries with 30 and 23 from Philippines and Senegal. Group-III had 88 acces- sions from 13 countries with more accessions from Madagascar (28), India (20) and Sri Lanka (19) (S2 Fig). Hierarchical cluster analysis using Awclust suggested that the best number of clusters was also three based on Gap stastics (S1 Fig). The classification of accessions into groups was simi- lar to that derived from STRUCTURE. Fifteen accessions of the group Q3-2 of STRUCTURE were assigned to the C3-3 of Awclust (S3 Fig). The “find.clusters” function of adegenet showed a clear decrease of BIC until k = 7 clusters, after which BIC increased (S1 Fig). In this case, the elbow in the curve also matched the small- est BIC, and clearly indicated that seven clusters should be retained. Roughly speaking, the three groups detected by STRUCTURE were divided into two, two and three clusters by ade- genet (S3 Fig). If we also chose seven groups for STRUCTURE analysis the Q3-1, Q3-2 and Q3-3 were further divided into two, two and four groups (S3 Fig). There were some differences in the assignments of accessions between k = 7 in STRUCTURE and adegent. The biggest dif- ference was that the C7-4 of adegenet was distributed over five of the seven groups of STRUC- TURE (S3 Fig). Phenotypic variation and trait correlation In general, the panel revealed a wide range variation for all the evaluated traits (Fig 2). Most of the traits appeared to be normally distributed, but some trait-environment combinations showed skewed distributions. For instance, the distributions of DEC in all environments, HMRR and PGWC in HZ and BRR in JZ were seriously skewed (Fig 2). Variance components derived from across-site analysis were given in Table 2. For appearance quality traits, the VG was much larger than VGEI, indicating that GEI was negligible. For BRR, MRR and HMRR, the VGEI was much larger than VG, suggesting that GEI was very important. However, the Ve for HMRR was very large, indicating phenotyping precision was low. The h2 for the appearance quality traits were high ranging from 0.82 for PGWC to 0.98 for GL and GLWR, while were low for the milling quality traits ranging from 0.19 for MRR to 0.27 for HMRR. The phenotypic correlations between traits were similar in the four testing environments. Low correlations were found between grain appearance quality traits and milling quality traits in all environments. GL showed a moderate and negative correlation with GW and a low and negative correlation with GT. Correlation between GW and GT was moderate and positive. TGW positively correlated with GL, GW and GT. High correlation was found between PGWC and DEC in all environments with coefficient ranging from 0.90 in SZ to 0.94 in HZ. The corre- lation between TGW and GT was moderate in all environments ranging from 0.46 in SY to 0.57 in HZ. Negative and moderate correlation was found between PGWC and HMRR. The correlation between PGWC and DED was strong and positive. The correlations between the three milling quality traits were positive ranging from low to moderate (Fig 3). Whole genome patterns of LD decay The decay of LD along physical distances was computed for both the whole population (272 accessions) and three subpopulations inferred by STRUCTURE k = 3. The determination coefficients (R2) of regression models were 0.87, 0.93, 0.88 and 0.95 for the whole population, Q3-1, Q3-2 and Q3-3, respectively (S3 Table). The maximum r2 was observed in the 0–10 kb marker interval for the whole population and subpopulations. The maximum r2 was lower in 8 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality Fig 2. Box plot of 10 rice grain appearance and milling quality traits in four environments. HZ: Hangzhou. JZ: Jingzhou. SY: Sanya. SZ: Shenzhen. GL: Grain Length. GW: Grain Width. GLWR: Grain Length and Width Ratio. GT: Grain Thickness. TGW: Thousand Grain Weight. BRR: Brown Rice Rate. MRR: Milled Rice Rate. HMRR: Head Milled Rice Rate. PGWC: Percentage of Grains With Chalkiness. DEC: Degree of Endosperm chalkiness. Fig 2. Box plot of 10 rice grain appearance and milling quality traits in four environments. HZ: Hangzhou. JZ: Jingzhou. SY: Sanya. SZ: Shenzhen. GL: Grain Length. GW: Grain Width. GLWR: Grain Length and Width Ratio. GT: Grain Thickness. TGW: Thousand Grain Weight. BRR: Brown Rice Rate. MRR: Milled Rice Rate. HMRR: Head Milled Rice Rate. PGWC: Percentage of Grains With Chalkiness. DEC: Degree of Endosperm chalkiness. Fig 2. Box plot of 10 rice grain appearance and milling quality traits in four environments. HZ: Hangzhou. JZ: Jingzhou. SY: Sanya. SZ: Shenzhen. GL: Grain Length. GW: Grain Width. GLWR: Grain Length and Width Ratio. GT: Grain Thickness. TGW: Thousand Grain Weight. BRR: Brown Rice Rate. MRR: Milled Rice Rate. HMRR: Head Milled Rice Rate. PGWC: Percentage of Grains With Chalkiness. DEC: Degree of Endosperm chalkiness. doi:10.1371/journal.pone.0145577.g002 doi:10.1371/journal.pone.0145577.g002 the whole population (0.65) than in the subpopulations. Q3-2 had the largest maximum r2 than Q3-1 (0.76) and Q3-3 (0.71). The basal r2 was 0.11, 0.16, 0.16 and 0.19 for the whole pop- ulation, Q3-1, Q3-2 and Q3-3, respectively (S3 Table). The LD decay distance was 150 kb for the whole population, which was longer (slower) than those for Q3-1 (110 kb) and Q3-3 (80 kb) but shorter (faster) than that for Q3-2 (240 kb) (Fig 4 and S3 Table). Association analysis Comparison of models. A total of 16 models for detecting associations were compared to choose the best model for each of the trait-environment combinations using MSD. The com- plete MSD results were given in S4 Table. Fig 5 gave the quantile-quantile plots for the 16 mod- els for GL measured in HZ to illustrate the relative differences between models. The best model varied with traits and environments (S4 Table). For all the trait-environment combinations except BRR in SY, the naïve model was always inferior to other models (S4 Table), indicating complex population structure and genetic relatedness between accessions present in our panel affected the detection of MTA. According to the average MSD value across all trait-environ- ment combinations, the Q models (Q3, Q7 and C7)that corrected for population structure only, were inferior to the K models (Kp, Ks and Kv) that corrected for the genetic relationships between accessions and the QK models (Q3Kp, Q3Ks, Q3Kv, Q7Kp, Q7Ks, Q7Kv, C7Kp, C7Ks and C7Kv) that corrected for both of population structure and genetic relationship (S4 Table). The QK model was the best model for most of the trait-environment combinations. The Q7 model was slightly better than the other two Q models. The Kp model was better than the Ks model and Kv model. The C7Kp model was better than the other QK models. For the models with the same Q but different K, the Q-Kp and Q-Ks models were similar and were slightly better than the Q-Kv model. The models with the same K but different Q had very sim- ilar MSD values. In the following sections only the MTA identified using the best model for each of the trait-environment combinations were presented and discussed. Table 2. Variance components and heritabilty estimated for 10 quality traits by across-site analysis. Trait VE VG VGEI Ve Rep(Env) h2 GL 0.00 0.62 0.00 0.08 0.01 0.98 GW 0.00 0.07 0.00 0.02 0.01 0.97 GLWR 0.00 0.32 0.00 0.05 0.01 0.98 GT 0.00 0.02 0.00 0.01 0.01 0.94 TGW 0.97 8.72 1.92 0.29 0.01 0.94 PGWC 0.62 86.04 59.81 27.7 1.72 0.82 DEC 0.00 465.84 277.44 138.17 6.72 0.84 BRR 0.08 0.57 5.57 0.97 0.31 0.27 MRR 3.01 2.75 44.98 4.71 0.48 0.19 HMRR 108.45 13.64 67.41 230.5 37.29 0.23 VE: environment variance; VG: genotype variance; VGEI: genotype- by- environment interaction variance; Ve: residual variance; Rep(Env): replication i ithi i t h2 h it bilit . doi:10.1371/journal.pone.0145577.t002 VE: environment variance; VG: genotype variance; VGEI: genotype- by- environment interaction variance; Ve: residual variance; Rep(Env): replication variance within environment; h2: narrow-sense heritability. GWAS of Rice Grain Quality G S o ce G a Qu PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 9 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 9 / 25 doi:10.1371/journal.pone.0145577.t002 Association analysis Variance components and heritabilty estimated for 10 quality traits by across-site analysis. Table 2. Variance components and heritabilty estimated for 10 quality traits by across-site analysis. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 10 / 25 GWAS of Rice Grain Quality Fig 3. Correlations between 10 gain quality traits measured in HZ, JZ, SY and SZ. The values given were correlation coefficients multiplied by 100. The values without glyphs indicated insignificant at the level of 0.05. Marker-trait associations. A total of 74 markers on all 12 chromosomes were found to be significantly associated with the 10 measured traits. By delineating significant markers with LD higher than 0.2 into a single QTL, the 74 markers were grouped into 38 QTL (Table 3). For GL, eight QTL were identified on chromosomes 2, 3, 5, 7, 9 and 11. qGL2.1 (D02_25608957) was detected in SY and explained 7.5% of the phenotypic variation (R2 = 7.5%). qGL2.2 (S02_305731433, S02_30664746 and S02_30664811) was detected in HZ, SY and SZ with R2 being 8.1%, 7.4% and 7.0%, respectively. qGL3.1 (S03_16663793, S03_16731182, S03_16748937, S03_16762099, S03_16858510, S03_16996600 and S03_17000111) was identified in HZ, SY and SZ with R2 being 8.0%, 10.2% and 10.5%, respectively. qGL5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in SY with a R2 of 7.5%. The qGL7.1 (S07_22019132, S07_22087092 and S07_22099651) was detected in HZ, SY and SZ and explained 7.8%, 7.7% and 6.4% of the phenotypic variance. qGL7.2 (S07_22844850) was detected in SY with a R2 of 7.5%. qGL9.1 (S09_10774239) was detected in JZ and explained 11.7% of the phenotypic variance. qGL11.1 (S011_2576141) was detected in JZ and SY with R2 being 12.0% and 9.6%, respectively (Table 3). Fig 3. Correlations between 10 gain quality traits measured in HZ, JZ, SY and SZ. The values given were correlation coefficients multiplied by 100. The values without glyphs indicated insignificant at the level of 0.05. doi:10.1371/journal.pone.0145577.g003 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 11 / 25 Fig 3. Correlations between 10 gain quality traits measured in HZ, JZ, SY and SZ. The values given were correlation coefficients multiplied by 100. The values without glyphs indicated insignificant at the level of 0.05. Fig 3. Correlations between 10 gain quality traits measured in HZ, JZ, SY and SZ. The values given were correlation coefficients multiplied by 100. The values without glyphs indicated insignificant at the level of 0.05. d i 10 1371/j l 0145577 003 doi:10.1371/journal.pone.0145577.g003 Marker-trait associations. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Association analysis A total of 74 markers on all 12 chromosomes were found to be significantly associated with the 10 measured traits. By delineating significant markers with LD higher than 0.2 into a single QTL, the 74 markers were grouped into 38 QTL (Table 3). For GL, eight QTL were identified on chromosomes 2, 3, 5, 7, 9 and 11. qGL2.1 (D02_25608957) was detected in SY and explained 7.5% of the phenotypic variation (R2 = 7.5%). qGL2.2 (S02_305731433, S02_30664746 and S02_30664811) was detected in HZ, SY and SZ with R2 being 8.1%, 7.4% and 7.0%, respectively. qGL3.1 (S03_16663793, S03_16731182, S03_16748937, S03_16762099, S03_16858510, S03_16996600 and S03_17000111) was identified in HZ, SY and SZ with R2 being 8.0%, 10.2% and 10.5%, respectively. qGL5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in SY with a R2 of 7.5%. The qGL7.1 (S07_22019132, S07_22087092 and S07_22099651) was detected in HZ, SY and SZ and explained 7.8%, 7.7% and 6.4% of the phenotypic variance. qGL7.2 (S07_22844850) was detected in SY with a R2 of 7.5%. qGL9.1 (S09_10774239) was detected in JZ and explained 11.7% of the phenotypic variance. qGL11.1 (S011_2576141) was detected in JZ and SY with R2 being 12.0% and 9.6%, respectively (Table 3). 11 / 25 GWAS of Rice Grain Quality Fig 4. LD decay plot in the whole population and three subpopulations inferred by STRUCTURE. Y axis is the average r2 value of each 10 kb region and X axis is physical distance between markers in unit of Mb. A power law curve (y = axk) was fitted to determine the physical position (x) corresponding to a given r2 value (y) For GW, five QTL on chromosomes 4, 5, 7, 10 and 11 were identified. qGW4.1 (S04_7167748) was detected in SZ with a R2 of 6.8%. qGW5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in JZ, SY and SZ with R2 being 16.2%, 10.0% and 12.8%, respec- tively. qGW7.1 (S07_19590107 and S07_19592078) was detected in JZ with a R2 of 13.6%. qGW10.1 (S010_14195109 and S010_14262980) was detected in SZ with a R2 of 6.6%. qGW11.1 (D011_7124485) was detected in SZ with a R2 of 7.0% (Table 3). For GLWR, six QTL were identified on chromosomes 1, 3, 5, 7 and 11. qGLWR1.1 (S01_5811755) was detected in HZ and SZ with R2 being 7.4% and 5.8%, respectively. qGLWR3.1 (D03_14077712) was detected in SZ with a R2 of 5.8%. Association analysis qGLWR3.2 (S03_16858510) was detected in SY and SZ with R2 being 6.3% and 6.4%, respectively. qGLWR5.1 Fig 4. LD decay plot in the whole population and three subpopulations inferred by STRUCTURE. Y axis is the average r2 value of each 10 kb region and X axis is physical distance between markers in unit of Mb. A power law curve (y = axk) was fitted to determine the physical position (x) corresponding to a given r2 value (y) doi:10.1371/journal.pone.0145577.g004 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 12 / 25 Fig 4. LD decay plot in the whole population and three subpopulations inferred by STRUCTURE. Y axis is the average r2 value of each 10 kb region and X axis is physical distance between markers in unit of Mb. A power law curve (y = axk) was fitted to determine the physical position (x) corresponding to a given r2 value (y) doi:10.1371/journal.pone.0145577.g004 doi:10.1371/journal.pone.0145577.g004 For GW, five QTL on chromosomes 4, 5, 7, 10 and 11 were identified. qGW4.1 (S04_7167748) was detected in SZ with a R2 of 6.8%. qGW5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in JZ, SY and SZ with R2 being 16.2%, 10.0% and 12.8%, respec- tively. qGW7.1 (S07_19590107 and S07_19592078) was detected in JZ with a R2 of 13.6%. qGW10.1 (S010_14195109 and S010_14262980) was detected in SZ with a R2 of 6.6%. qGW11.1 (D011_7124485) was detected in SZ with a R2 of 7.0% (Table 3). For GLWR, six QTL were identified on chromosomes 1, 3, 5, 7 and 11. qGLWR1.1 (S01_5811755) was detected in HZ and SZ with R2 being 7.4% and 5.8%, respectively. qGLWR3.1 (D03_14077712) was detected in SZ with a R2 of 5.8%. qGLWR3.2 (S03_16858510) was detected in SY and SZ with R2 being 6.3% and 6.4%, respectively. qGLWR5.1 For GW, five QTL on chromosomes 4, 5, 7, 10 and 11 were identified. qGW4.1 (S04_7167748) was detected in SZ with a R2 of 6.8%. qGW5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in JZ, SY and SZ with R2 being 16.2%, 10.0% and 12.8%, respec- tively. qGW7.1 (S07_19590107 and S07_19592078) was detected in JZ with a R2 of 13.6%. qGW10.1 (S010_14195109 and S010_14262980) was detected in SZ with a R2 of 6.6%. qGW11.1 (D011_7124485) was detected in SZ with a R2 of 7.0% (Table 3). For GLWR, six QTL were identified on chromosomes 1, 3, 5, 7 and 11. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Association analysis qGLWR1.1 (S01_5811755) was detected in HZ and SZ with R2 being 7.4% and 5.8%, respectively. qGLWR3.1 (D03_14077712) was detected in SZ with a R2 of 5.8%. qGLWR3.2 (S03_16858510) was detected in SY and SZ with R2 being 6.3% and 6.4%, respectively. qGLWR5.1 q ( _ ) % ( ) For GLWR, six QTL were identified on chromosomes 1, 3, 5, 7 and 11. qGLWR1.1 (S01_5811755) was detected in HZ and SZ with R2 being 7.4% and 5.8%, respectively. qGLWR3.1 (D03_14077712) was detected in SZ with a R2 of 5.8%. qGLWR3.2 (S03_16858510) was detected in SY and SZ with R2 being 6.3% and 6.4%, respectively. qGLWR5.1 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 12 / 25 GWAS of Rice Grain Quality Fig 5. Quantile-quantile plots of 16 models for GL measured in HZ. The horizontal and vertical axes are -log10 transformed expected p-values and observed p-values, respectively. Model with more uniformly distributed p-values is better. doi:10 1371/journal pone 0145577 g005 (S05_5368086, S05_5368151 and S05_5369527) was detected in HZ, JZ, SY and SZ with R2 being 7.4%, 14.1%, 11.4% and 7.6%, respectively. qGLWR7.1 (S07_25383179) was detected in SY with a R2 of 6.6%. qGLWR11.1 (S011_2576141) was detected in JZ and explained 6.6% of the phenotypic variance (Table 3). For GT, four QTL were found on chromosome 1, 4 and 5. qGT1.1 (D01_2405901) was detected in HZ with a R2 of 9.4%. qGT4.1 (S04_29442031 and S04_29495556) was detected in SZ with a R2 of 7.0%. qGT5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in HZ, JZ, SY and SZ with R2 being 13.1%, 8.6%, 8.5% and 15.8%, respectively. qGT5.2 (S05_18307877, S05_18314309 and S05_18321742) was detected in SZ with a R2 of 6.7% (Table 3). Fig 5. Quantile-quantile plots of 16 models for GL measured in HZ. The horizontal and vertical axes are -log10 transformed expected p-values and observed p-values, respectively. Model with more uniformly distributed p-values is better. doi:10.1371/journal.pone.0145577.g005 Fig 5. Quantile-quantile plots of 16 models for GL measured in HZ. The horizontal and vertical axes are -log10 transformed expected p-values and observed p-values, respectively. Model with more uniformly distributed p-values is better. doi:10.1371/journal.pone.0145577.g005 (S05_5368086, S05_5368151 and S05_5369527) was detected in HZ, JZ, SY and SZ with R2 being 7.4%, 14.1%, 11.4% and 7.6%, respectively. qGLWR7.1 (S07_25383179) was detected in SY with a R2 of 6.6%. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Association analysis qGLWR11.1 (S011_2576141) was detected in JZ and explained 6.6% of the phenotypic variance (Table 3). For GT, four QTL were found on chromosome 1, 4 and 5. qGT1.1 (D01_2405901) was detected in HZ with a R2 of 9.4%. qGT4.1 (S04_29442031 and S04_29495556) was detected in SZ with a R2 of 7.0%. qGT5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in HZ, JZ, SY and SZ with R2 being 13.1%, 8.6%, 8.5% and 15.8%, respectively. qGT5.2 (S05_18307877, S05_18314309 and S05_18321742) was detected in SZ with a R2 of 6.7% (Table 3). 13 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality Table 3. QTL identified for 10 traits in four environments. QTL Env Marker Allelesa MAF p Effectb R2 (%)c Known gene/QTL qGL2.1 SY D02_25608957 T/A 0.16 5.93E-05 0.43 7.5 qGL2.2 SY S02_30573143 T/A 0.36 7.03E-05 0.5 7.4 SZ S02_30573143 T/A 0.35 5.04E-05 0.43 7.0 HZ S02_30664746 G/T 0.30 2.63E-05 0.51 8.1 SZ S02_30664746 G/T 0.29 7.44E-05 0.44 6.7 HZ S02_30664811 C/T 0.30 2.63E-05 0.51 8.1 SZ S02_30664811 C/T 0.29 7.44E-05 0.44 6.7 qGL3.1 HZ S03_16663793 T/C 0.30 2.97E-05 0.79 8.0 GS3 SY S03_16663793 T/C 0.28 3.26E-06 0.98 10.2 SZ S03_16663793 T/C 0.29 2.19E-05 0.69 7.7 SZ S03_16731182 C/T 0.41 2.42E-05 -0.46 7.6 SZ S03_16748937 C/T 0.47 3.92E-06 0.57 9.2 SZ S03_16762099 A/G 0.47 1.13E-05 0.53 8.3 HZ S03_16858510 C/G 0.45 7.31E-05 0.61 7.2 SY S03_16858510 C/G 0.42 2.00E-05 0.69 8.5 SZ S03_16858510 C/G 0.43 8.03E-07 0.65 10.5 SZ S03_16996600 C/A 0.38 4.74E-05 0.48 7.0 SZ S03_17000111 C/T 0.39 1.80E-05 0.5 7.9 qGL5.1 SY S05_5368086 C/T 0.38 8.44E-05 -0.48 7.2 qSW5 SY S05_5368151 A/G 0.38 8.10E-05 -0.48 7.2 SY S05_5369527 A/T 0.37 5.71E-05 -0.49 7.5 qGL7.1 HZ S07_22019132 G/A 0.05 3.51E-05 -1.04 7.8 SY S07_22019132 G/A 0.05 4.85E-05 -1.11 7.7 SZ S07_22019132 G/A 0.05 9.95E-05 -0.81 6.4 SY S07_22087092 T/A 0.13 6.54E-05 -0.66 7.4 HZ S07_22099651 A/G 0.05 3.51E-05 -1.04 7.8 SY S07_22099651 A/G 0.05 4.85E-05 -1.11 7.7 SZ S07_22099651 A/G 0.05 9.95E-05 -0.81 6.4 qGL7.2 SY S07_22844850 C/T 0.18 6.30E-05 -0.63 7.5 qGRL7.1 qGL9.1 JZ S09_10774239 A/T 0.50 7.39E-05 0.53 11.7 qGL11.1 JZ S11_2576141 T/G 0.35 5.76E-05 -0.57 12.0 CycT1;3 SY S11_2576141 T/G 0.35 6.41E-06 -0.63 9.6 qGW4.1 SZ S04_7167748 C/T 0.42 5.91E-05 0.16 6.8 qGW5.1 SY S05_5368086 C/T 0.38 1.94E-05 0.19 8.7 qSW5 SZ S05_5368086 C/T 0.35 3.26E-07 0.21 11.2 JZ S05_5368151 A/G 0.39 1.06E-05 0.23 16.0 SY S05_5368151 A/G 0.37 8.43E-06 0.19 9.5 SZ S05_5368151 A/G 0.35 1.57E-07 0.22 11.9 JZ S05_5369527 A/T 0.38 9.45E-06 0.24 16.2 SY S05_5369527 A/T 0.37 5.09E-06 0.2 10.0 SZ S05_5369527 A/T 0.34 5.54E-08 0.22 12.8 qGW7.1 JZ S07_19590107 G/C 0.08 4.41E-05 0.29 13.6 JZ S07_19592078 T/A 0.08 4.41E-05 0.29 13.6 qGW10.1 SZ S10_14195109 A/T 0.05 7.54E-05 0.36 6.6 SZ S10_14262980 A/G 0.05 7.54E-05 0.36 6.6 qGW11.1 HZ D11_7124485 T/A 0.17 8.76E-05 0.12 7.0 qGLWR1.1 HZ S01_5811755 A/T 0.08 4.83E-05 0.52 7.4 (Continued) Table 3. QTL identified for 10 traits in four environments. (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 14 / 25 GWAS of Rice Grain Quality Table 3. (Continued) QTL Env Marker Allelesa MAF p Effectb R2 (%)c Known gene/QTL SZ S01_5811755 A/T 0.08 9.32E-05 0.48 5.8 qGLWR3.1 SZ D03_14077712 T/A 0.15 9.18E-05 -0.4 5.8 qGLWR3.2 SY S03_16858510 C/G 0.42 9.44E-05 0.38 6.3 GS3 SZ S03_16858510 C/G 0.43 4.06E-05 0.36 6.4 qGLWR5.1 JZ S05_5368086 C/T 0.40 4.03E-05 -0.46 12.9 qSW5 SY S05_5368086 C/T 0.39 1.47E-06 -0.35 9.7 SZ S05_5368086 C/T 0.35 3.09E-05 -0.31 6.6 JZ S05_5368151 A/G 0.39 2.30E-05 -0.48 13.8 SY S05_5368151 A/G 0.38 4.80E-07 -0.37 10.7 SZ S05_5368151 A/G 0.35 1.42E-05 -0.33 7.2 HZ S05_5369527 A/T 0.36 4.95E-05 -0.32 7.4 JZ S05_5369527 A/T 0.38 1.96E-05 -0.48 14.1 SY S05_5369527 A/T 0.38 1.97E-07 -0.38 11.4 SZ S05_5369527 A/T 0.34 9.05E-06 -0.34 7.6 qGLWR7.1 SY S07_25383179 T/C 0.07 5.93E-05 0.5 6.6 Srs1 qGLWR11.1 JZ S11_2576141 T/G 0.35 1.03E-05 -0.42 15.1 CycT1;3 qGT1.1 HZ D01_2405901 A/T 0.10 2.77E-05 0.14 9.4 qGT4.1 SZ S04_29442031 T/C 0.16 6.57E-05 -0.13 7.0 SZ S04_29495556 T/C 0.16 6.57E-05 -0.13 7.0 qGT5.1 HZ S05_5368086 C/T 0.33 2.37E-06 0.13 12.0 qSW5 SZ S05_5368086 C/T 0.35 1.13E-08 0.13 14.9 HZ S05_5368151 A/G 0.33 9.02E-07 0.14 13.1 JZ S05_5368151 A/G 0.39 8.98E-05 0.09 8.6 SZ S05_5368151 A/G 0.35 4.47E-09 0.14 15.8 HZ S05_5369527 A/T 0.32 8.77E-07 0.13 13.1 SY S05_5369527 A/T 0.37 1.70E-05 0.09 8.5 SZ S05_5369527 A/T 0.34 4.91E-09 0.14 15.7 qGT5.2 SZ S05_18307877 G/C 0.21 8.96E-05 -0.11 6.7 SZ S05_18314309 C/T 0.21 8.96E-05 -0.11 6.7 SZ S05_18321742 A/G 0.21 8.96E-05 -0.11 6.7 qTGW2.1 SZ S02_30688426 C/A 0.30 8.86E-05 1.78 7.7 qTGW5.1 HZ S05_4808850 T/A 0.48 6.59E-05 -2.06 10.1 HZ S05_4856967 A/T 0.47 6.88E-05 -2.07 10.0 HZ S05_4880400 A/G 0.47 6.88E-05 -2.07 10.0 HZ S05_4889259 C/T 0.47 6.88E-05 -2.07 10.0 qTGW5.2 SZ S05_5368086 C/T 0.33 5.38E-05 2.07 8.2 qSW5 SZ S05_5369527 A/T 0.32 2.66E-05 2.14 8.9 qTGW7.1 HZ S07_21628162 C/T 0.08 5.77E-05 -3.72 10.3 qTGW7.2 SZ S07_22684516 C/A 0.05 9.17E-05 -4.26 7.7 qGRL7.1 qPGWC5.1 SZ S05_5368086 C/T 0.32 5.16E-08 28.21 15.9 qSW5 SZ S05_5368151 A/G 0.32 2.97E-07 26.78 14.0 JZ S05_5369527 A/T 0.38 6.52E-05 24.33 10.9 SZ S05_5369527 A/T 0.31 1.72E-08 29.41 17.1 qPGWC6.1 SZ S06_6372444 A/C 0.09 5.76E-06 35.63 10.8 qDEC1.1 SZ S01_5811836 A/C 0.17 8.66E-05 11.18 7.3 SZ S01_5972775 T/G 0.23 7.96E-05 10.37 7.4 qDEC5.1 HZ S05_4180511 T/C 0.10 7.25E-05 11.03 10.3 (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 15 / 25 GWAS of Rice Grain Quality Table 3. (Continued) QTL Env Marker Allelesa MAF p Effectb R2 (%)c Known gene/QTL qDEC5.2 SY S05_5368086 C/T 0.37 7.27E-05 7.89 7.5 qSW5 SY S05_5368151 A/G 0.36 4.18E-05 8.2 8.0 SY S05_5369527 A/T 0.36 2.05E-05 8.59 8.7 SZ S05_5369527 A/T 0.29 3.23E-05 9.75 8.3 qDEC5.3 SZ S05_23647280 A/G 0.35 4.93E-05 -8.5 7.9 qDEC9.1 HZ S09_16262697 T/C 0.07 4.02E-05 -16.93 11.1 qBRR2.1 SZ D02_8060607 A/T 0.26 2.96E-05 -1.02 8.2 SZ D02_8075892 T/A 0.27 2.24E-05 -1.03 8.5 qBRR2.2 JZ D02_25652984 A/T 0.06 1.96E-05 -4.75 15.6 qBRR3.1 HZ S03_22356459 T/C 0.07 8.52E-05 3.84 10.6 qBRR4.1 HZ S04_32002470 C/T 0.09 3.59E-06 3.65 14.5 qBRR7.1 HZ S07_29497149 T/A 0.21 8.03E-05 2.31 10.7 qMRR1.1 HZ S01_41957924 G/A 0.07 4.44E-05 10.06 6.6 HZ S01_41957989 G/T 0.07 4.44E-05 10.06 6.6 HZ S01_41970208 T/A 0.07 4.44E-05 10.06 6.6 qMRR2.1 JZ D02_26022390 A/T 0.07 1.61E-06 -8.71 19.9 JZ D02_26022455 A/T 0.07 1.61E-06 -8.71 19.9 JZ D02_26163208 T/A 0.05 3.29E-07 -7.59 22.8 JZ D02_26265084 A/T 0.05 5.22E-06 -7.04 17.8 JZ D02_26312559 A/T 0.06 4.14E-05 -7.86 14.1 qMRR3.1 SZ S03_4636350 A/G 0.21 9.69E-05 -1.91 7.3 qMRR7.1 JZ S07_5075055 A/G 0.06 9.29E-05 -5.45 12.8 JZ S07_5081323 T/A 0.06 9.29E-05 -5.45 12.8 qMRR11.1 SY D11_6895700 T/A 0.06 4.05E-05 -12.33 7.8 qHMRR3.1 SZ D03_31372694 T/A 0.40 2.85E-05 12.34 9.0 SZ D03_31380514 A/T 0.48 1.67E-05 12.53 9.5 qHMRR4.1 HZ S04_28018950 T/C 0.05 3.92E-06 -26.22 14.9 qHMRR5.1 SZ S05_12426396 T/C 0.18 8.01E-05 15.42 7.9 qHMRR6.1 HZ D06_2712642 A/T 0.12 9.37E-05 -14.26 11.0 qHMRR8.1 SZ S08_2638229 G/A 0.12 3.16E-05 17.8 8.9 qHMRR12.1 SZ S12_26692818 C/T 0.36 1.79E-05 12.81 9.5 SZ S12_26695253 G/A 0.32 3.18E-05 12.65 8.9 aMajor/Minor allele aMajor/Minor allele. For TGW, five QTL were found on chromosome 2, 5 and 7. qTGW2.1 (S02_30688426) was detected in SZ with a R2 of 7.7%. qTGW5.1 (S05_4808850, S05_4856967, S05_4880400 and S05_4889259) was detected in HZ with a R2 of 10.1%. qTGW5.2 (S05_5368086 and S05_5369527) was detected in SZ with a R2 of 8.9%. qTGW7.1 (S07_21628162) was detected in HZ with a R2 of 10.3%. qTGW7.2 (S07_22684516) was detected in SZ with a R2 of 7.7% (Table 3). For PGWC, two QTL on chromosomes 5 and 6 were identified. qPGWC5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in JZ and SZ with R2 being 10.9% and 17.1%, respectively. qPGWC6.1 (S06_6372444) was detected in SZ with a R2 of 10.8% (Table 3). aMajor/Minor allele. bEffect: Allelic effect with respect to the minor allele. cR2: Phenotypic variation explained. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 doi:10.1371/journal.pone.0145577.t003 qHMRR5.1 (S05_12426396) was identified in SZ with a R2 of 7.9%. qHMRR6.1 (D06_2712642) was identi- fied in HZ with a R2 of 11.0%. qHMRR8.1 (S08_2638229) was identified in SZ with a R2 of 8.9%. qHMRR12.1 (S012_26692818 and S012_26695253) was identified in SZ with a R2 of 9.5% (Table 3). Six QTL were detected for HMRR. They were on chromosomes 3, 4, 5, 6, 8 and 12. qHMRR3.1 (D03_31372694 and D03_31380514) was identified in SZ with a R2 of 9.5%. qHMRR4.1 (S04_28018950) was identified in HZ with a R2 of 14.9%. qHMRR5.1 (S05_12426396) was identified in SZ with a R2 of 7.9%. qHMRR6.1 (D06_2712642) was identi- fied in HZ with a R2 of 11.0%. qHMRR8.1 (S08_2638229) was identified in SZ with a R2 of 8.9%. qHMRR12.1 (S012_26692818 and S012_26695253) was identified in SZ with a R2 of 9.5% (Table 3). q qHMRR4.1 (S04_28018950) was identified in HZ with a R2 of 14.9%. qHMRR5.1 Some of the QTL for different traits were in the same chromosomal regions and were regarded as the same QTL when the number of QTL was counted across traits. They were qDEC1.1 and qGLWR1.1 on chromosome 1; qBRR2.2 and qGL2.1 on chromosome 2; qGL2.2 and qTGW2.1 on chromosome 2; qGLWR3.2 and qGL3.1 on chromosome 3; qGL5.1, qGW5.1, qGLWR5.1, qGT5.1, qTGW5.2, qPGWC5.1 and qDEC5.2 on chromosome 5; qGL7.2 and qTGW7.2 on chromosome 7; qGW11.1 and qMRR11.1 on chromosome 11; and qGLWR11.1 and qGL11.1 on chromosome 11. doi:10.1371/journal.pone.0145577.t003 For TGW, five QTL were found on chromosome 2, 5 and 7. qTGW2.1 (S02_30688426) was detected in SZ with a R2 of 7.7%. qTGW5.1 (S05_4808850, S05_4856967, S05_4880400 and S05_4889259) was detected in HZ with a R2 of 10.1%. qTGW5.2 (S05_5368086 and S05_5369527) was detected in SZ with a R2 of 8.9%. qTGW7.1 (S07_21628162) was detected in HZ with a R2 of 10.3%. qTGW7.2 (S07_22684516) was detected in SZ with a R2 of 7.7% (Table 3). For PGWC, two QTL on chromosomes 5 and 6 were identified. qPGWC5.1 (S05_5368086, S05_5368151 and S05_5369527) was detected in JZ and SZ with R2 being 10.9% and 17.1%, respectively. qPGWC6.1 (S06_6372444) was detected in SZ with a R2 of 10.8% (Table 3). 16 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality For DEC, five QTL were identified on chromosomes 1, 5 and 9. qDEC1.1 (S01_5811836 and S01_5972775) was detected in SZ with a R2 of 7.4%. qDEC5.1 (S05_4180511) was detected in HZ with a R2 of 10.3%. qDEC5.2 (S05_5368086, S05_5368151 and S05_5369527) was detected in SY and SZ with R2 being 8.7% and 8.3%, respectively. qDEC5.3 (S05_23647280) was detected in SZ with a R2 of 7.9%. qDEC9.1 (S09_16262697) was detected in HZ with a R2 of 11.1% (Table 3). For BRR, five QTL located on chromosomes 2, 3, 4 and 7 were detected. qBRR2.1 (D02_8060607 and D02_8075892) was identified in SZ with a R2 of 8.5%. qBRR2.2 (D02_25652984) was identified in SZ with a R2 of 15.6%. qBRR3.1 (S03_22356459) was identi- fied in HZ with a R2 of 10.6%. qBRR4.1 (S04_32002470) was identified in HZ with a R2 of 14.5% (Table 3). For MRR, five QTL located on chromosomes 1, 2, 3, 7 and 11 were detected. qMRR1.1 (S01_41957924, S01_41957989 and S01_41970208) was identified in HZ with a R2 of 6.6%. qMRR2.1 (D02_26022390, D02_26022455, D02_26163208, D02_26265084 and D02_26312559) was identified in JZ with a R2 of 22.8%. qMRR3.1 (S03_4636350) was identi- fied in SZ with a R2 of 7.3%. qMRR7.1 (S07_5075055 and S07_5081323) was identified in JZ with a R2 of12.8%. qMRR11.1 (D11_6895700) was identified in SY with a R2 of 7.8% (Table 3). Six QTL were detected for HMRR. They were on chromosomes 3, 4, 5, 6, 8 and 12. qHMRR3.1 (D03_31372694 and D03_31380514) was identified in SZ with a R2 of 9.5%. qHMRR4.1 (S04_28018950) was identified in HZ with a R2 of 14.9%. LD pattern The basal r2 was 0.11 and estimated LD decay distance was 150 kb for the whole population (S3 Table). The LD decay distance ranged from 80 kb to 240 kb in three subpopulations. This was in agreement with previous finding that cultivated rice has a long range LD from close to 100 kb to over 200 kb [59, 60]. Compared with the whole population, LD dropped much faster in Q3-1 and Q3-3 but more slowly in Q3-2. The effect of population structure on LD pattern has been widely reported [52, 60–62]. Varying patterns of LD decay in different subpopulations were likely reflecting their breeding histories and the origins of accessions in subpopulations [63] and may influence the QTL mapping resolution of the panel. Given that our average marker density (one marker per 20.2 kb) was higher than the r2 decay, we expected to have rea- sonable resolution and power to identify common variants of large effect associated with traits of interest. Phenotypic variation and trait correlation Great variation was observed for all traits in all testing environments in the present study, sug- gesting that the association panel can be effectively used for GWAS of various quality traits. Grain appearance quality traits, particularly grain size and weight traits had high heritability and showed only small GEI (Table 2), suggesting that breeding for a wide range of growing environments could be feasible and effective. Grain chalkiness and milling quality traits had low heritability and large contribution of GEI and as a result will be more difficult to improve through conventional selection breeding. The relationship between grain quality traits in rice is complex. In the present study, positive correlations were observed between TGW and GL, GW and GT, which was consistent with previous studies [2, 26, 56]. The coefficients were moderate, which might be the result of nega- tive correlations between GL and GW and GT. The correlation between GL and GW were -0.54, -0.64, -0.50 and -0.55 in HZ, JZ, SY and SZ, respectively. However, a weak and positive (0.11 in SY) or negative (-0.11, -0.07 and -0.09 in JZ, HZ and SZ, respectively) correlation was observed between TGW and GLWR, which was different with the result of Xu et al. [57]. They 17 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality found TGW and GLWR had a moderate and positive correlation (0.44). Consistent with previ- ous reports, BRR was negatively correlated with GL but positively correlated with GW, even though the correlation coefficients were small. In the process of milling, the long and slender rice grain is easier to break than short and round one. Chalkiness is also an important factor influencing rice milling traits especially HMRR. Chalky grains are also more prone to breakage at chalky area. We found HMRR was negatively correlated with PGWC and DEC, which was consistent with the study of Zheng et al. [58]. Model comparison It is well-known that the results of association analysis using different models would be very different. However, there is no generally acceptable best model. It is likely that the best model differs among populations, traits and growing environments. The most appropriate model should be chosen by comparing different models. In the present study, MSD proposed by Stich et al. [55] was used to determine the best model for each of the 40 trait-environment combina- tions. The results showed that the naïve model and the Q models were almost always inferior to the K and QK models (S4 Table). The average MSD value across all trait-environment com- binations of the naïve model was much higher compared to those of the Q models, indicating introduction of population structure could tremendously reduce false positives. Q derived from different numbers of subpopulations (k) also leaded to different results. According to MSD, increasing k from three to seven resulted in better control of false positives (S4 Table). When population structure was detected by different methods, the resultant Q and as a result the association results were different. For instance, the Q7 model, which used the Bayesian clustering analysis method implemented in STRUCTURE, was better than the C7 model, which used the Q from the DAPC method (S4 Table). It was clear from our results that the three Q models were rarely the best model and the K and QK models were better (S4 Table). This was consistent with most of the previous studies that showed that GLM method (Q model) was inferior to MLM method (K and QK models). A recent example in rice was the study ofCourtois et al. [41]. They performed association study for 16 root depth and associated traits in a panel of japonica accessions and chose the best model by comparing the likelihoods of each model using the BIC [64]. Their results showed that the QK model was the best model for 13 measured traits, the K model was the best for the other 3 traits, and the Q model was always inferior to the other two models. For the models with the same Q but different K, the Q-Kp and Q-Ks models were similar and were slightly better than the Q-Kv model (S4 Table). PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 Model comparison It was observed that models with very similar MSD values had very similar association results, indicating that MSD is a simple but effective method for choosing the appropriate model(s) in GWAS. The effects of environment and QEI In the present study, the total number of identified QTL varied greatly across environments, indicating that testing environment had important influence on GWAS. Out of the 38 identi- fied QTL only one (qGLWR5.1 (qGT5.1)), four (qGL2.2, qGL3.1, qGL7.1 and qGW5.1) and four QTL (qGLWR1.1, qGL11.1, qPGWC5.1 (qDEC5.2) and qGLWR3.2) were detected in four, three and two environments, respectively (Table 3). Furthermore, even for qGLWR5.1, which was detected in all the four environments, the R2 varied from 7.4% to 14.1% across environ- ments. These results may be partially attributed to QEI. For BRR, MRR and HMRR that showed significant GEI, all QTL were detected in only one of the four environments (Table 3). However, the big differences in population size across environments may also be partially responsible for the observed differences between environments. For most of the traits the pop- ulation size was much larger in SY and SZ than in HZ and JZ (Table 1). Model comparison The models with the same K but dif- ferent Q had very similar MSD values, indicating that once the genetic relatedness between PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 18 / 25 GWAS of Rice Grain Quality genotypes was well accounted, the differences between methods for inferring Q became less important. Therefore, when both Q and K were considered simultaneously, K played a more important role than Q and different K should be tried to determine the best model. The best model differed for the same trait measured in different environments and different traits measured in the same environment (S4 Table). For traits with low heritability, the best model changed greatly across environments. For instance, BRR (h2 = 0.27), MRR (h2 = 0.19) and HMRR (h2 = 0.23), there was no common best model in the four environments. However, for traits with high heritability the same best model tended to be found across environments. For instance, Q7Kv was the best model for GL (h2 = 0.98) in HZ, JZ and SY and C7Kv was the best model for GW (h2 = 0.97) in HZ and SZ and Kv was the best model for GW in JZ and SY. In the present study, we selected the model with the smallest MSD value as the best model. It was observed that models with very similar MSD values had very similar association results, indicating that MSD is a simple but effective method for choosing the appropriate model(s) in GWAS. The best model differed for the same trait measured in different environments and different traits measured in the same environment (S4 Table). For traits with low heritability, the best model changed greatly across environments. For instance, BRR (h2 = 0.27), MRR (h2 = 0.19) and HMRR (h2 = 0.23), there was no common best model in the four environments. However, for traits with high heritability the same best model tended to be found across environments. For instance, Q7Kv was the best model for GL (h2 = 0.98) in HZ, JZ and SY and C7Kv was the best model for GW (h2 = 0.97) in HZ and SZ and Kv was the best model for GW in JZ and SY. In the present study, we selected the model with the smallest MSD value as the best model. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS is effective In the present study, five out of the 38 identified QTL were adjacent to previously known genes or fine mapped QTL, suggesting that association mapping was an effective way to map QTL or fine mapped QTL, suggesting that association mapping was an effective way to map QTL for rice grain quality traits. On chromosome 5, qGL5.1, qGW5.1, qGLWR5.1, qGT5.1, qTGW5.2, qPGWC5.1 and qDEC5.2 were located in genomic region (S05_5368086, S05_5368151 and S05_5369527) close to the cloned gene qSW5 affecting GW and TGW [18]. qSW5 was in 5362625bp - 5370506bp in the Os-Nipponbare-Reference- IRGSP-1.0 according to the complete cds sequence of qSW5 in indica. The qSW5 functioned as negative regulator of grain width and weight and a deletion in qSW5 resulted in a significant increase in sink size owing to an increase in cell number in the outer glume of the rice flower and finally increased the grain width and weight [18]. The qSW5 was also reported to regulate GL, GLWR and GT [65]. However, the effects of qSW5 on PGWC and DEC have not been reported before. Our results suggested that qSW5 could be used to increase GL and reduce DEC and PGWC simulta- neously. The qGL3.1 (S03_16663793, S03_16731182, S03_16748937, S03_16762099, S03_16858510, S03_16996600 and S03_17000111) and qGLWR3.2 (S03_16858510) associated with GL and GLWR, were close to GS3 (16729228–16735641) affecting grain shape. GS3 func- tions as a negative regulator for grain size which is a major QTL for GL and TGW and a minor QTL for GW and GT [16]. The qGL7.2 (S07_22844850) and qTGW7.2 (S07_22684516) were located in the region harboring the previously fine mapped QTL affecting GL, GW and GLWR, qGRL7.1 (22127.4Kb—24526.7Kb) [6]. The qGLWR7.1 (S07_25383179) was located in the pp gg g pp g y p for rice grain quality traits. On chromosome 5, qGL5.1, qGW5.1, qGLWR5.1, qGT5.1, g q y q q q q qTGW5.2, qPGWC5.1 and qDEC5.2 were located in genomic region (S05_5368086, PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 19 / 25 GWAS of Rice Grain Quality region of Srs1 (25381698–25389547) affecting grain size [23]. The srs1 results in small and round seed due to the reduction in both cell length and cell number in the longitudinal direc- tion, and the elongation of the cells in the lateral direction of the lemma. The qGL11.1 (S11_2576141) and qGLWR11.1 (S11_2576141) were close to CycT1;3 (2730143–2736649) whose down regulation results in a shorter grain. The number of QTL identified is small Compared to the previous GWAS studies in rice the number of QTLs detected was small in the present study. Many of the known genes affecting traits measured were not detected in the present study. The most likely reason is the small population size. Most of the association pan- els had more than 400 accessions while the present panel only had 230 or fewer accessions. The effect of population size on GWAS is well known. It is particularly important for detecting loci with low MAF to use a large panel. Because the panel size was small markers with MAF <0.05 were filleted out before association analysis. The trait-affecting genes were missed out if mark- ers in strong LD with them had low MAF in our panel. For example, the gene PGL2 (LOC_Os02g51320) was involved in controlling grain length and weight of rice through inter- action with a typical bHLH protein APG. Marker S02_31422564 very close to this gene (1.4 kb) was removed before association analysis since its MAF was only 0.02. The big differences between environments in population size could also explain why most of the QTL (53%) were detected in SZ. Nevertheless, loci with large effects were also detected for some of the traits with small population size. For instance, the population size of GW in JZ is only 132, but qGW5.1 was identified with a high R2 of 16.2%. Therefore, provided that the MAF is high enough, major loci would still be detected even if the population size was small. Another possi- ble reason is that the present panel consists of only indica accessions while most of the previous studies used panels with a few subspecicies/ecotypes. It is expected that genes that determine the major trait differences between subspecies/ecotypes could not be detected in our study but might be detectable in other studies. We also expected that more false positives were present when a panel with very diverse accessions was used. The use of a single subspecies allows con- trolling population structure at finer scale and as results reduces false positives. The critical sig- nificant threshold value used to declare significant association also influenced the analysis results. For instance, the qGW5.1 was found to be significant in JZ, SY and SZ but not in HZ. GWAS is effective Thirty-three of the identified QTL were located on chromosomal regions where no QTL for the measured traits have been reported and can be regarded as novel QTL. Five of the QTL including qDEC1.1 (qGLWR1.1), qBRR2.2 (qGL2.1), qTGW2.1 (qGL2.2), qGW11.1 (qMRR11.1) and qGL7.1 affected multiple traits with relatively large effects and/or were detected in multiple environments. PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 The number of QTL identified is small By inspecting the p values in HZ, it could be seen that the p values of S05_5368086, S05_5368151 and S05_5369527, were 0.00065, 0.00035 and 0.00014, respectively, which were only slightly higher than the applied critical significant threshold 10−4. It might be worth point- ing out that the number of markers used in the present study was smaller than or similar to the reported GWAS in rice. However, the marker density was sufficient for most of the genomic regions based on LD decay pattern. Furthermore, the eight gaps devoid of markers below criti- cal LD did not harbor any known genes affecting the studied traits. Therefore, the relatively low marker density was unlikely a major contributor to the small number of detected QTL. Nevertheless, the marker density might be too low in some chromosomal regions where LD decays more rapidly or breaks down due to recombination events [41]. For instance, the GW2 (LOC_Os02g14720) controlling grain width and weight, was in the marker interval of S02_8069581—S01_8181973. However, we could not identify any markers associated with GW 20 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality or TGW in this region, since LD was very low (about 0.02). Additional markers in such regions are clearly needed. or TGW in this region, since LD was very low (about 0.02). Additional markers in such regions are clearly needed. Supporting Information S1 Fig. Population structure analysis of 272 indica accessions. (a) Log probability change (Δk) as function of number of subpopulations (STRUCTURE). (b) Gap statistics as function of number of clusters (Awclust). (c) Value of BIC versus number of clusters (adegenet). (TIF) Conclusions Wide ranges of genetic variations were present in the indica association panel for all 10 mea- sured grain quality traits. The accessions could serve as sources of novel genes and alleles for improving rice appearance quality and milling quality traits. GWAS conducted using proper statistical models is a powerful method for elucidating the genetic basis of rice quality traits. Although it is generally true that MLM is better than the naïve model and GLM (Q) model for GWAS, the most appropriate model varies across traits and environments and must be chosen by comparing different models. MSD was found to be a simple but effective method for choos- ing the appropriate model in GWAS. Based on the selected best model for each of the trait- environment combinations, 38 QTL were identified for 10 rice grain appearance quality and milling quality traits including GL, GW, GLWR, GT, TGW, PGWC, DEC, BRR, MRR and HMRR. Eight genomic regions harbored QTL for more than one trait. Five QTL were found to concur with previously cloned genes or fine mapped QTL. Thirty-three were novel. Five of the novel QTL including qDEC1.1 (qGLWR1.1), qBRR2.2 (qGL2.1), qTGW2.1 (qGL2.2), qGW11.1 (qMRR11.1) and qGL7.1 affected multiple traits with relatively large effects and/or were detected in multiple environments. Our results provided an insight into the genetic architec- ture of rice grain appearance quality and milling quality traits. The novel QTL are important candidates for future functional characterization studies. References 1. Unnevehr L, Duff B, Juliano BO. 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Distributions of markers on chromosomes. (DOCX) S3 Table. Basic characteristics of LD pattern in the whole panel and three subpopulations. (DOCX) S4 Table. Comparison of 16 association models based the mean squared difference (MSD) between the observed and expected p values for 40 traits (10)-environment (4) combina- tions. (DOCX) 21 / 25 PLOS ONE \| DOI:10.1371/journal.pone.0145577 December 29, 2015 GWAS of Rice Grain Quality Acknowledgments This research was granted by Global Rice Science Partnership (GRiSP), National 863 High Technology Research and Development Program from the Ministry of Science and Technology of China (2014AA10A601), Shenzhen Peacock Plan, the Natural Science Foundation of China (31461143014, 31261140369), and Hubei Collaborative Innovation Center for Grain Industry. Author Contributions Conceived and designed the experiments: JX GY. Performed the experiments: XQ ZY. Ana- lyzed the data: YP GY. Contributed reagents/materials/analysis tools: MD DX ZL. 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https://openalex.org/W4235753729	https://www.researchsquare.com/article/rs-3813/latest.pdf	English	null	Genetic and clinical characterization of BRCA-associated Hereditary Breast and Ovarian Cancer in Navarra (Spain)	Research Square (Research Square)	2,019	cc-by	7,265	Genetic and clinical characterization of BRCA- associated Hereditary Breast and Ovarian Cancer in Navarra (Spain) Ainara Ruiz de Sabando (  aruizdes@navarra.es ) Ainara Ruiz de Sabando (  aruizdes@navarra.es ) Universidad Publica de Navarra https://orcid.org/0000-0003-3634-465X Research article Keywords: Hereditary breast and ovarian cancer (HBOC), BRCA1/2, Recurrent mutations, Demographics, Sporadic breast and ovarian cancer, Laterality and stage of tumors, Overall survival, Navarra Posted Date: October 18th, 2019 DOI: https://doi.org/10.21203/rs.2.13095/v3 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 27th, 2019. See the published version at https://doi.org/10.1186/s12885-019-6277-x. Page 1/22 Abstract Background Genetic testing for BRCA1/2 genes is widely used as a strategy to reduce incidence and morbidity of hereditary breast and ovarian cancer. The purpose of this study is to analyse the demographic and molecular characteristics of BRCA germline mutations in Navarra, Spain, and to investigate the clinical profile of hereditary and sporadic breast cancer (BC) and ovarian cancer (OC) in the Community. Methods The study includes 1246 individuals assessed for BRCA1/2 genetic testing in Navarra, during 2000-2016, and a cohort of BC (n=4384) and OC (n=561) from the population-based Navarra Cancer Registry. Distribution and molecular characteristics of BRCA1/2 mutations, as well as, comparative analysis of the clinical course, pathologic features and overall survival of patients in different risk groups were investigated. Results BRCA mutation detection rate was 16%, with higher proportion (63%) of BRCA2 families. Nineteen per cent of mutations were recurrent, one of which, BRCA2 c.6024dupG, showed high association to OC. BRCA carriers had double risk (95% CI=1.04-4.33) of developing multiple malignancies than low risk families and were diagnosed at a much earlier age (16.6 and 11.7 years difference for BC and OC, respectively) when compared to the general population. For BC, BRCA carriers showed a more advanced histological stage, higher risk of bilateral neoplasms (OR=4.3; 95% CI=1.3-11.4, for BRCA2 carriers) and worse overall survival rate at 5-, 10- and 15- years, than women with sporadic tumors. For OC, over 70% of patients of all risk groups showed advanced stages at diagnosis, with the highest 91% among BRCA1 carriers (91%). Furthermore, they also had higher probability of developing bilateral tumors (OR=7.8, 95%CI=1.7-55.7) than the general population. Five- year overall survival rate was worse among women with sporadic OC than in BRCA carriers, but it levelled out over the 15-year period. Conclusions This study describes the molecular features of BRCA1/2 mutations in Navarra and defines the clinical course and outcomes of BRCA associated tumors compared with those of sporadic origin. Long term assessment of mortality and survival will be required to evaluate the impact of BRCA genetic testing program on the health of target population in our community. Background About 32,800 breast (BC) and 3,300 ovarian cancers (OC) are diagnosed every year in Spain, accounting for one third of the cancers among women [1-3]. Although survival has substantially improved over the past few years due to screening and the improvement of treatments, BC is the leading cause of cancer death among Spanish women, with about 6,500 deaths in 2017, and OC is the fifth, with 2,000 [4]. Between 5-10% [5] of all BC and 13-15% of OC [6, 7] are hereditary and approximately 25% are associated with the Hereditary Breast/Ovarian cancer (HBOC) Syndrome, caused by abnormalities in the DNA repair genes BRCA1 and BRCA2 [8]. Inherited mutations in the BRCA1 and BRCA2 (BRCA1/2) cancer susceptibility genes convey high lifetime risks of BC and OC, in the range of 40-66% and 13-46%, respectively [9, 10], as well as, increased susceptibility for other malignancies, such as prostate and pancreatic cancers [11]. On the other hand, it has been hypothesized that BRCA-associated BC has a Page 2/22 different prognosis as compared to the sporadic counterpart [12]. However, clinical findings regarding the prognostic role of BRCA mutational status are still controversial. different prognosis as compared to the sporadic counterpart [12]. However, clinical findings regarding the prognostic role of BRCA mutational status are still controversial. Genetic testing for BRCA1/2 mutations allows us to stratify families and individuals by their risk or predisposition to developing cancer, so that, preventive measurements can be offered to decrease cancer mortality and morbidity. Characterization of the most prevalent BRCA1 and BRCA2 mutations and their geographical distribution can be useful in designing efficient mutational screening in a given population. Additionally, it is important to evaluate genetic testing and preventive programs for HBOC, carrying out intermediate processes of data analysis and comparison of outcome indicators. Previous studies have analysed the mutation spectrum of BRCA1/2 in different regions of Spain [13-19]. Navarra, with about 650,000 people in 2018, has historically been in exchange with the neighbouring Basque territories in the north and Aragon and Castile in the southeast; additionally, in the last decades, it has experienced migration from other Spanish communities and countries. Hereditary Breast/Ovarian Cancer study population From 2000 to 2016, a total of 1246 individuals of Navarra, corresponding to 751 families with BC and/or OC, were evaluated at the Hereditary Cancer Clinic of the Department of Medical Genetics of the Complejo Hospitalario de Navarra (CHN), the reference public center for the HBOC program in the Autonomous Community. Since 2000, families with suspected familial/hereditary cancers are referred by medical specialists to the CHN for evaluation, counselling and genetic testing, if considered appropriate. Relevant information including personal and familial history, age of cancer diagnosis, tumor type and geographic family origin, is routinely obtained. Families are classified as high-risk families, if they meet the criteria established by the Breast Cancer Committee of the CHN (Supplementary Figure 1), or as low-risk families, if not. High-risk families are informed about their hereditary cancer risk, advised according to their personal and familial circumstances and offered genetic analysis of BRCA1 and BRCA2 genes. Additionally, members of BRCA1/2 positive families that request genetic testing are studied and followed according to their individual risk status. All tested individuals provide a signed informed consent, following the appropriate genetic counselling. For this study, a disaggregated identification number was assigned to all participants to guarantee confidentiality and data protection. The study was approved by the Research Ethical Committee of Navarra. Background The aim of the present study is two-fold: a) to describe the molecular and demographic characteristics of families with HBOC syndrome in Navarra, Spain, b) to define the associated clinical phenotype and overall survival (OS) of individuals with BC and OC in different hereditary cancer risk families and the general population, in an attempt to provide indicators that may allow us to evaluate and improve the HBOC testing program in our community. Mutation analysis of BRCA1 and BRCA2 Genomic DNA was extracted from peripheral blood of index cases of high-risk families. Sanger sequencing of BRCA1 and BRCA2 genes was performed using BigDye® Terminator kits and read through 3500 Genetic Analyzer (Applied BioSystems). Mutational analysis was performed with SeqScape Software v3.0 (Thermo Fisher Scientific). All index patients were additionally tested for the presence of Large Genomic Rearrangements in BRCA1 and BRCA2 genes by Multiplex ligation-dependent probe amplification (MLPA). Specific probes for each exon of BRCA1 (SALSA MLPA P002 and P087 BRCA1 probemix, MRC-Holland) and BRCA2 genes (SALSA MLPA P045 BRCA2/CHEK2 probemix, MRC-Holland) were used. The fragments were measured by capillary electrophoresis using the 3500 Genetic Analyzer (Applied Biosystems) and analyzed with Coffalyzer (MRC-Holland). Origin of families Page 3/22 Geographical origin of families was assessed by enquiring about the place of birth of parents and grandparents of index individuals. Positive BRCA1/2 families were classified in one of the following geographic groups, based on the place of origin of their ancestors: a) Navarra, when the transmitting parent and/or grandparents were born in the Community of Navarra, b) neighbouring communities, when the transmitting parent and/or grandparents were from the surrounding communities of Navarra (Basque Country, Rioja, Soria, Zaragoza and Huesca), c) other Spanish communities, and d) other countries. The results were represented using the Geographic Information System QGIS version 3.0 Girona. Variant nomenclature & classification GenBank reference sequences of NM_007294.3 and NM_000059.3 were used for BRCA1 and BRCA2 analysis, respectively. Mutation nomenclature was described according to Human Genome Variation Society [20]. Pathogenicity of the mutations was assessed using the following databases: the Breast Cancer Information Core [21], the Universal Mutation Database [22] for BRCA1 and BRCA2, and ClinVar at NCBI [23]. For novel mutations the standards and guidelines of the American College of Medical Genetics and Genomics were followed to determine the possible disease-association [24]. In silico prediction was evaluated considering the impact of the amino acid changes as well as their conservation across species. These analyses were performed with the following bioinformatic tools: PROtein Variation Effect Analyzer (PROVEAN) [25], Polymorphism Phenotyping-2 (PolyPhen-2) [26]. Clinical and pathological features of BRCA and non-BRCA related breast and ovarian cancer. Navarra Cancer Registry Clinical and pathological features of BRCA and non-BRCA related breast and ovarian cancer. Navarra Cancer Registry Clinical and pathological features of BRCA and non-BRCA related breast and ovarian cancer. Navarra Individuals of Navarra are registered for health care and administrative purposes by a unique personal identification number in regional medical registries. This number was used to cross-link the information between the HBOC study and the Navarra Cancer Registry, a population-based cancer registry that records all tumor malignancies diagnosed since 1973 until 2012 for this study in Navarra residents. We used this Registry as a reliable source of clinical information on a total of 593 participants of the HBOC study that had developed, at least, one infiltrating cancer. For comparison with tumors of sporadic origin, we also analysed a cohort sample of 4384 cases with BC and 561 with OC diagnosed in the general population Page 4/22 Page 4/22 from 2000 to 2012. The following variables were obtained: gender, number of infiltrating tumors per patient, age at diagnosis of each cancer, age at death, laterality, site, histology and stage of neoplasms. Site and histology were defined according to the International Classification of Diseases for Oncology (ICD-O) [27].The extent of the disease was coded according to the Condensed TNM of the European Network of Cancer Registries [28]. from 2000 to 2012. The following variables were obtained: gender, number of infiltrating tumors per patient, age at diagnosis of each cancer, age at death, laterality, site, histology and stage of neoplasms. Site and histology were defined according to the International Classification of Diseases for Oncology (ICD-O) [27].The extent of the disease was coded according to the Condensed TNM of the European Network of Cancer Registries [28]. Statistics Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 20.0 and OpenEpi [29]. Socio-demographic and clinical variables were summarized using descriptive statistics, such as mean and standard deviation (SD) and frequencies and percentages. The associated risk of developing any tumor, of having bilateral tumor, and of having regional/advanced stage by HBOC group, compared with that of the general population (Cancer Registry), was estimated using odds ratios (OR), with 95% confidence intervals. The association between selected clinical–pathological data and specific groups of HBOC, all categorical variables, was determined by using Chi-Square or Fisher’s exact test, and Student’s T test, as appropriate. Student’s T test was used to compare independent continuous variables. Kaplan- Meier survival curves (proportion surviving) with 5- 10- and 15-year survival were plotted, and log-rank test was used to compare different groups. Cox (hazard ratios, HR) regression was also performed, adjusting for age at diagnosis. All tests were two-sided and p-value less than 0.05 was considered to be statistically significant. 4. BRCA25216_5218insAAA (exon 11) This insertion originates a nonsense mutation with a stop codon in position 1739. The index patient was a woman that developed BC at 28 years old, whose mother, carrier of the mutation, had OC at the age of 60. 1. BRCA14343delG (exon 13) This deletion, located in exon 13, results in a frame shift variant that originates a stop codon in position 1455. The index patient in this family was a male, diagnosed of PC at age 67 with family history of BC and OC on the mother’s side. BCs were diagnosed at ages 90 (mother) and 55 (maternal aunt) and OC at age 42 (maternal aunt). 2. BRCA1 exon 5-7 duplication This variant was found in a 54 year old woman diagnosed with invasive ductal carcinoma. Her mother died of BC at 46. Two additional family members (a maternal cousin and her daughter), carriers of the same mutation, suffered from BC at ages 33 and 34, respectively. HBOC study (2000-2016) Among the 751 evaluated families, residents of Navarra, with clinical and family history that suggested HBOC, 233 were classified as low-risk families, with no further analysis, and 518 others as high-risk families, for which mutational analysis was performed in the index affected member (Table 1). The number of cases included in the study is represented in Figure 1, showing a remarkable increase of BRCA1/2 testing and diagnosis over time. From the high-risk group, 84 unrelated families (16.2%), 228 individuals, were found carrying pathogenic mutations: 31 (37%) in BRCA1 and 53 (63%) in BRCA2. Of them, 39% of the transmitting parents or grandparents were originally from Navarra (33 families), 18% came from the neighbouring communities (15 families) and the remaining families (42%) had ancestors from other communities (34 families) or countries (2 families) (Figure 2). Frequency of mutations in BRCA2 gene was higher than in BRCA1 in all geographic groups, and more so in families of local ancestors, in which two thirds (67%) of them were BRCA2 positive. BRCA1 families (n=31) carried a total of 23 different mutations (Supplementary Table 1); two were recurrent mutations and 21 were unique mutations. The most frequent mutation was c.5123C>A, present Page 5/22 Page 5/22 Page 5/22 in eight apparently unrelated families, followed by c.211A>G, identified in two families, representing a 26%, and 7%, respectively, of the total families with BRCA1 mutations. in eight apparently unrelated families, followed by c.211A>G, identified in two families, representing a 26%, and 7%, respectively, of the total families with BRCA1 mutations. Among BRCA2 positive families (n=53), 29 different mutations were observed (Supplementary Table 2). Eight were recurrent mutations and 21 unique mutations. Variant c.2806_2809delAAAC was highly represented (11 unrelated families). The five most frequent pathogenic variants accounted for 50% of BRCA2 mutations. Four novel, unreported, pathogenic mutations, responsible for early-onset B Four novel, unreported, pathogenic mutations, responsible for early-onset BC and OC were identified. 3. BRCA24132_4133delAC (exon 11) This alteration triggers a frame shift variant resulting in a stop codon in position 1380. The index patient, who inherited the mutation from her father, was diagnosed with invasive ductal carcinoma at 40 years old and a few months after she also developed cervix cancer. Two of her paternal aunts died of BC. Clinical data from the Navarra Cancer Registry Of all the 1246 individuals in the HBOC study, 593 were included in the Navarra Cancer Registry with a total of 693 tumor entries. Twenty four (4%) patients were males and 9 (37.5%) of them carried a BRCA deleterious mutation. The frequency of multiple tumors was significantly higher among BRCA1/2 carriers (21.6%) than in individuals of the low-risk families (11.5%), with an OR of 2.11 (95% CI=1.04 to 4.33, p=0.038) (Table 2). The difference did not reach statistical significance when compared with the high risk BRCA-negative Page 6/22 Page 6/22 group (HR-BRCA-negative). BRCA1 and BRCA2 mutations were associated with a different range of tumors. Cancer affecting breast, ovary, skin (SC), endometrium (EC), pancreas (PC) and prostate (PrC) accounted for 91.2% of all tumors registered in BRCA1/2 patients and 93.7% among HR-BRCA-negative and low risk cases. BRCA1 carriers had a higher frequency of OC (24.1%), SC (11.1%) and PC (3.7%) than individuals in the other three groups (9.3%, 5.3% and 0%, respectively). BC (70.7%) and PrC (2.7%), however, were overrepresented in BRCA2 carriers compared to BRCA1-positives (55.6% and 0%, respectively). Among men, there were 3 BRCA1 mutation carriers who developed PC (1) and SC (2), and 6 BRCA2 positive cases, presenting with a wide variety of tumors: breast (4), prostate gland (2), lung (1), colon (1), fossa piriform (1) and thyroid gland (1). Three of the 9 BRCA1/2 male mutation carriers, developed multiple tumors, all associated with BRCA2. To further study severity of the disease we analysed laterality, stage of the tumor, age at diagnosis and overall survival (OS) in patients of the HBOC study in comparison with a cohort sample of BC (n=4384) and OC (n=561) in the general population. As shown in Table 3, all four risk groups of the HBOC study showed a higher incidence of bilateral BC compared to the general population, although only in the group of BRCA2 positives the difference reached statistical significance (7.6% vs 1.8%; p=0.021, OR=4.3; 95% CI=1.3 to 11.4). For OC, BRCA1 carriers had a much higher frequency of bilateral tumors (77.8%) than individuals in the BRCA2 positive (33.3%), HR-BRCA negative (30.8%), low-risk HBOC (0%) and the general population (30.9%; p=0.007, OR=7.8; 95% CI=1.7 to 55.7). The stage at diagnose of BC tumors was significantly more advanced (p=0.043) in BRCA1/2 patients than in sporadic tumors. Clinical data from the Navarra Cancer Registry Over 55% of tumors were at regional/advanced stage among BRCA carriers compared to 40.4% and 43% in the low risk HBOC and the general population, respectively. The same trend was observed for OC among BRCA1 carriers, although numbers were too small to reach statistical significance. Mean age at diagnose of BC and OC was lower in all four groups of the HBOC study than in the general population (Table 3, Figure 3). BRCA1/2 carriers were diagnosed of BC 16.6 years earlier (44.2 vs 60.8 years), while for OC the time difference was 11.7 years (53.9 vs 65.6). Survival over the course of 15 years was also studied. For BC, 5- 10- and 15-year OS did not show significant differences among risk groups (Table 4). More than 64% of BRCA carriers lived 15 or more years after diagnosis, a percentage similar to that observed in sporadic BC. However, mean age at death differed between groups. BRCA1/2 mutation carriers died 20 years earlier than BC patients from the general population, indicating that age at diagnosis could be a co-variant of prognosis (HR=1.08; 95% CI =1.07 to 1.08; p<0.0001). Further analysis, adjusting by age at diagnosis, showed that BRCA1 and BRCA2 carriers had a significant worse prognosis than individuals of the other groups (Figure 4A), with HR of 3.2 (95% CI = 1.5 to 6.7; p=0.002) and 2.6 (95%CI = 1.5 to 4.7; p=0.001) respectively, when compared with BC of sporadic origin. Page 7/22 Page 7/22 For OC, the general population showed worst 5-year OS rate (41%), becoming, however, similar in all risk groups at 15 years after diagnosis, with OS rates of 20% in BRCA1 mutation carriers and 28% in the general population (Table 4, Figure 4B). Mean age at death differed notoriously, but sample size was not sufficient to use the modelling approach as for BC analysis. Nevertheless, when we analysed OS only in patients diagnosed before the age of 65 years, no significant differences were observed among risk groups (data not shown). Discussion We present a demographic, clinical and molecular study of families with HBOC in Navarra (Spain), through the analysis of retrospective data collected by the Department of Medical of the CHN, and the Cancer Registry for the Autonomous Community. Mutations in BRCA1 and BRCA2 genes were identified in 16% of families (26% of individuals) included in the high-risk HBOC sample. This rate is comparable with other studies [14, 18, 19] and similarly shows a decreasing trend over time, moving from 30% in 2000-2005, to 17% during (2006-2010) and 14% from 2011 to 2016. This is, most likely, due to the referral of the most severely affected families in the first years of cancer genetic services, and the use of less strict inclusion criteria in the later period, given the decrease in cost of the molecular analysis. It is noteworthy to highlight, however, the remarkable growth in the number of families and individuals assessed along the study period. Two thirds of the high-risk families were diagnosed in the last 6 years of the study, accounting for more than half of the families with pathogenic variants in BRCA1 and BRCA2. Navarra is a small community in the North of Spain that has experienced important demographic changes since the 1950 decade, due to migration from other Spanish communities or countries. This mixed population could explain, at least in part, the similarities in BRCA1/2 mutation distribution and spectrum with other areas of Spain [17, 19]. As previously reported national wide, with the exception of Galicia and Asturias [13, 14], deleterious mutations in BRCA2 were more frequent than in BRCA1, particularly among families of local ancestors in which 2/3 of BRCA associated families carried a BRCA2 deleterious variant. Nineteen per cent (10/52) of pathogenic variants were recurrent and were responsible of 50% of HBOC families (Supplementary Tables 1 and 2). Many common mutations in Navarra are also frequent in other Spanish populations [14-18]. More so, many of the recurrent mutations in Navarra coincide with the most frequent BRCA1/2 variants in Spain, as published in a recent international study [30]. Navarra is a small community in the North of Spain that has experienced important demographic changes since the 1950 decade, due to migration from other Spanish communities or countries. This mixed population could explain, at least in part, the similarities in BRCA1/2 mutation distribution and spectrum with other areas of Spain [17, 19]. Discussion As previously reported national wide, with the exception of Galicia and Asturias [13, 14], deleterious mutations in BRCA2 were more frequent than in BRCA1, In addition to these national similarities, BRCA1/2 mutations in Navarra present some unique features. Recurrent deleterious variants show geographic clusters in more isolated regions. Our most frequent mutation, BRCA2 c.2806_2809delAAAC, is mainly distributed in the north-western mountainous lands, while the also frequent BRCA2 c.6024dupG is present in the south-western riverbanks. Interestingly, BRCA2 c.6024dupG and c.7558C>T, are two recurrent mutations not reported previously in Spain. BRCA2 c.7558C>T, also found (non-recurrently) in Sweden and Honduras [30], was detected in 8 carriers of three Page 8/22 Page 8/22 families and was associated to 6 BC events. BRCA2 c.6024dupG was found in 16 carriers of 5 apparently unrelated families. This mutation, although never recurrently, has been reported mostly in Latin American countries [31], which may suggest a founder effect in the south-west of Navarra. This duplication has showed the highest correlation with OC in our study population: 4 out of the 7 BRCA2 positive OC identified in the HBOC study were associated to this mutation. In total, six mutation carriers developed ten different neoplasms, affecting ovary (4), breast (2), skin (1), endometrium (1), prostate (1) and lung (1). Three patients suffered multiple cancers. The information about the relationship between specific mutations in BRCA1/2 and the clinical expression is limited; however, there is evidence that mutations in the central part of BRCA2 (around exon 11), defined as the Ovarian Cancer Cluster Region, are associated with a higher OC risk and lower BC risk [32, 33]. Similarly, a Breast Cancer Cluster Region has been described at the end of BRCA2 (c.7394-c.8904) [33]. Our data from mutations BRCA2 c.6024dupG (exon 11) and c.7558C>T, with high association to OC and BC respectively, support these evidences. Many studies have investigated the prognosis role of BRCA germ line mutations with inconsistent results. Some early studies suggested that BRCA associated BC and OC had a better prognosis than sporadic malignancies [34, 35], perhaps due to increased sensitivity to chemotherapy. BRCA1/2 function as tumor suppressor genes and their proteins play an important role in repairing damaged DNA [16]. Deficiency of BRCA1/2 proteins results in carcinomas with a diminished capacity to repair DNA and, presumably, decreased ability to repair DNA breaks caused by chemotherapy. BRCA1/2 mutation carriers seem to have an improved response to platinum-based chemotherapy [36]. Discussion However, we observed that over a 15-year period, the apparent protective effect in BRCA is no longer present. Similar results have been described by other authors [40] suggesting that for BRCA1/2 positive women with OC, the short-term survival advantage does not lead to a long-term OS better prognosis. Finally, it is interesting to mention that, as it is well known, most high-risk families for HBOC do not carry a BRCA gene mutation [8]. However, they tend to share with them some of the phenotypic characteristics, such as, lower age at diagnosis and higher frequency of bilateral occurrence than cases in the low risk group and the general populations, which indicates that additional genetics and non-genetic factors remain to be identified. Finally, it is interesting to mention that, as it is well known, most high-risk families for HBOC do not carry a BRCA gene mutation [8]. However, they tend to share with them some of the phenotypic characteristics, such as, lower age at diagnosis and higher frequency of bilateral occurrence than cases in the low risk group and the general populations, which indicates that additional genetics and non-genetic factors remain to be identified. This study has some strengths and limitations. It has an unbiased design with practically complete ascertainment of BRCA carriers in the studied population, as the Department of Medical Genetics is the reference center for genetic testing in Navarra. Additionally, the study sample comprises a large population-based cohort of BC and OC with reliable clinical and outcomes data on BRCA and non-BRCA carriers. However, the small number of BRCA families and cancer events in carriers may have limited the precision estimates of main effects and reduced statistical power of the study. In summary, this retrospective study reviews HBOC due to mutations in BRCA1/2 genes and describes the associated clinical and molecular features in Navarra. Additionally, it presents the state of the art of clinical practice for HBOC in our community, providing information on intermediate process and results indicators that will allow us to evaluate long-term efficiency of the cancer genetic testing program and its impact on public health. Conclusions The spectrum of BRCA deleterious mutations in Navarra is similar to other Spanish regions, with some unique features. The possible founder effect of BRCA2 c.6024dupG and its high association with OC must be taking into account when performing genetic testing. Overall, severe phenotypic features such as early diagnosis, high tumor grading, occurrence of multiple malignancies and bilateral location, are more frequent among BRCA carriers than in other cancer risk groups, conferring a poorer prognosis than those of sporadic origin. For BC, BRCA carriers have a worse OS rate at 5-, 10- and 15 years than cases from the general population. This study provides intermediate indicators that will help to further evaluate long-term prognosis of patients with HBOC and improve health care in our community. Discussion However, whether the improved response translates into survival benefits still remains unclear. More recent studies indicate that BRCA mutations do not have a protective effect [37]. In our population, women with BC and BRCA mutations had worse prognosis than those with sporadic cancer, diagnosed at the same age, with HR of 3.2 for BRCA1 and 2.6 for BRCA2. Goodwin et al [38] suggested that prognosis was best predicted by the characteristic of the cancer developed by each individual rather than by their carrier status. Evaluating the effects of tumor histology and treatment-related variables was not the purpose of this study. However, we found that age at diagnosis, laterality (in BRCA2 carriers), tumor stage and presence of multiple neoplasms were significantly associated with BRCA mutations. Interestingly, BC in HR-BRCA-negative and low-risk patients also presented at an earlier age than in the general population, but there were no significant differences with respect to the stage and laterality of the disease. On the other hand, their OS adjusted by age at diagnosis showed no differences with that of the general population, but it was significantly higher than in BRCA1/2 carriers. These results may indicate that BRCA carriers present with more aggressive or severe tumor characteristics, and, consequently, a worse prognosis than sporadic BC. We cannot rule out, however, that it might be the result of an increased disease burden in BRCA positive patients, as they are also prone to suffer from other malignancies, in addition to BC. In OC, diagnosis occurred 11-12 years earlier in all HBOC risk groups than in the general population, although only BRCA1-associated tumors were mostly bilateral and more advanced in tumor development. With respect to OS, sporadic OC seem to have a worse 5-year prognosis than BRCA associated Page 9/22 Page 9/22 carcinomas. Other studies also support the hypothesis of a protective OS effect of a BRCA1/2 mutation carrier versus a non-carrier [39]. However, we observed that over a 15-year period, the apparent protective effect in BRCA is no longer present. Similar results have been described by other authors [40] suggesting that for BRCA1/2 positive women with OC, the short-term survival advantage does not lead to a long-term OS better prognosis. carcinomas. Other studies also support the hypothesis of a protective OS effect of a BRCA1/2 mutation carrier versus a non-carrier [39]. Abbreviations BC Breast Cancer BC Breast Cancer CDS Coding DNA Sequence Page 10/22 CHN Complejo Hospitalario de Navarra CI Confidence Interval EC Endometrium Cancer HBOC Hereditary Breast and Ovarian Cancer HR Hazard Ratio KM Kaplan-Meier LC Lung Cancer OC Ovarian Cancer OR Odds Ratio OS Overall Survival PC Pancreatic Cancer PrC Prostate Cancer SC Skin Cancer SD Standard Deviation TNM Tumor, Node and Metastases CHN Complejo Hospitalario de Navarra CI Confidence Interval EC Endometrium Cancer HBOC Hereditary Breast and Ovarian Cancer HR Hazard Ratio KM Kaplan-Meier LC Lung Cancer OC Ovarian Cancer OR Odds Ratio OS Overall Survival PC Pancreatic Cancer PrC Prostate Cancer SC Skin Cancer SD Standard Deviation TNM Tumor, Node and Metastases Consent for publication. Not Applicable. Ethics approval and consent to participate. The study was approved by the “Comité de Ética de la Investigación con medicamentos (CEIm)” of “Gobierno de Navarra”. The project number is “Ptyo2019/42”. All tested individuals provided a signed informed consent, following the appropriate genetic counselling. Availability of data and material. The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Page 11/22 Funding. Asociación Navarra de Cáncer de Mama (SARAY). The grant supported a full time researcher for the completion of the study. Authors' contributions. ARS organized the databases, performed the data analysis and wrote the manuscript. FGA and SM performed the mutational analysis and MLPA studies. EUL and EA managed the databases and extracted the information. AAS, LMG and MRA revised clinical data. MRA supervised the study and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. Acknowledgements. We would like to thank Asociación SARAY, as well as patients, families and referring physicians for their support and valuable contribution to the study. We would also like to acknowledge Berta Ibañez, from the Methodology Unit of Navarrabiomed and Sandra Eraso, from the Navarra Statistics Institute, for their assistance in the biostatistical analysis of the clinical and anatomo-pathological parameters and the use of QGIS Geographic Information System, respectively. Page 11/22 Page 11/22 Competing interests. The authors declare that they have no competing interests. References 1. Asociación Española Contra el Cáncer [https://www.aecc.es/es]. Accessed 30 September 2019. 1. Asociación Española Contra el Cáncer [https://www.aecc.es/es]. Access 2. Sociedad Española de Oncología Médica [https://seom.org/]. Accessed 30 September 2019. 3. Red Española de Registros de Cáncer [http://redecan.org/es/index.cfm]. Accessed 30 September 2. Sociedad Española de Oncología Médica [https://seom.org/]. Accesse 2. Sociedad Española de Oncología Médica [https://seom.org/]. Accessed 30 September 2019. 3. 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Rebbeck TR: Association of Type and Location of BRCA1 and BRCA2 Mutations With Risk of Breast and Ovarian Cancer. JAMA 2015, 313(13):1347-1361. 34. Verhoog LC: Survival and tumour characteristics of breast-cancer patients with germline mutations of BRCA1. Lancet 1998, 351(9099):316-321. 35. Cortesi L: Favourable ten-year overall survival in a Caucasian population with high probability of hereditary breast cancer. BMC Cancer 2010, 10:90. 36. Kriege M: Sensitivity to first-line chemotherapy for metastatic breast cancer in BRCA1 and BRCA2 mutation carriers. J Clin Oncol 2009, 27(23):3764-3771. 37. Schmidt MK: Breast Cancer Survival of BRCA1/BRCA2 Mutation Carriers in a Hospital-Based Cohort of Young Women. Journal of the National Cancer Institute 2017, 109(9). 38. Goodwin PJ: Breast Cancer Prognosis in BRCA1 and BRCA2 Mutation Carriers: An International Prospective Breast Cancer Family Registry Population-Based Cohort Study J Clin Oncol 2012, 30(1):19-26. 39. Huang Y-W: Association of BRCA1/2 mutations with ovarian cancer prognosis. Systematic Review and Meta-Analysis 2018, 97(2):e9380. 39. Huang Y-W: Association of BRCA1/2 mutations with ovarian cancer prognosis. Systematic Review and Meta-Analysis 2018, 97(2):e9380. 40. McLaughlin JR: Long-Term Ovarian Cancer Survival Associated With Mutation in BRCA1 or BRCA2. Journal of the National Cancer Institute 2013, 105(2):141-148. References Breast Cancer Res Treat 2007, 106(2):255-256. 16. Duran M: Mutational Analysis of BRCA2 in Spanish Breast Cancer Patients from Castilla-Leon: Identification of Four Novel Truncating Mutations. Human Mutation 2003, 21(4):448. 16. Duran M: Mutational Analysis of BRCA2 in Spanish Breast Cancer Patients from Castilla-Leon: Identification of Four Novel Truncating Mutations. Human Mutation 2003, 21(4):448. 17. Diez O: Heterogeneous prevalence of recurrent BRCA1 and BRCA2 mutations in Spain according to the geographical area: implications for genetic testing. Familial Cancer 2010, 9:187-191. 17. Diez O: Heterogeneous prevalence of recurrent BRCA1 and BRCA2 mutations in Spain according to the geographical area: implications for genetic testing. Familial Cancer 2010, 9:187-191. 18. Barrios XG: Molecular characterization and clinical interpretation of BRCA1/BRCA2 variants in families from Murcia (south-eastern Spain) with hereditary breast and ovarian cancer: clinical– pathological features in BRCA carriers and non-carriers. Familial Cancer 2017, 16:477-489. 19. Pajares B: Hereditary breast and ovarian cancer in Andalusian families: a genetic population study. BMC Cancer 2018, 18(1):647. 20. Human Genome Variation Society [https://www.hgvs.org/]. Accessed 30 September 2019. 20. Human Genome Variation Society [https://www.hgvs.org/]. Accessed 30 September 2019. 21. Breast Cancer Information Core [https://research.nhgri.nih.gov/bic/]. Accessed 30 September 2019. 21. Breast Cancer Information Core [https://research.nhgri.nih.gov/bic/]. Acc 22. Universal Mutation Database [http://www.umd.be/]. Accessed 30 Septem 23. ClinVar [https://www.ncbi.nlm.nih.gov/clinvar/]. Accessed 30 September 2019. 23. ClinVar [https://www.ncbi.nlm.nih.gov/clinvar/]. Accessed 30 September 2019. 24. Richards S: Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in medicine 2015, 17(5):405-424. 24. Richards S: Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in medicine 2015, 17(5):405-424. 25. PROtein Variation Effect ANalyzer (PROVEAN) [http://provean.jcvi.org/index.php]. Accessed 30 September 2019. 25. PROtein Variation Effect ANalyzer (PROVEAN) [http://provean.jcvi.org/index.php]. Accessed 30 September 2019. 26. Polymorphism Phenotyping-2 (PolyPhen-2) [http://genetics.bwh.harvard.edu/pph2/. Accessed 30 September 2019. 26. Polymorphism Phenotyping-2 (PolyPhen-2) [http://genetics.bwh.harvard.edu/pph2/. Accessed 30 September 2019. 27. World Health Organization, ICD-O‎ – 3rd edition, 1st revision, 3rd ed. [https://www.who.int/iris/handle/10665/96612]. Accessed 30 September 2019. Page 13/22 Page 13/22 28. Condensed TNM of the European Network of Cancer Registries [https://www.encr.eu/sites/default/files/pdf/extentofdisease.pdf 28. Condensed TNM of the European Network of Cancer Registries [https://www.encr.eu/sites/default/files/pdf/extentofdisease.pdf]. Accessed 30 September 2019. 29. OpenEpi [https://www.openepi.com/]. Accessed 30 September 2019. 30. Table 3: Clinical characteristics of breast and ovarian cancer cases included in the study. Tables Page 14/22 Table 1: Total number of subjects and families included in the Hereditary Breast/Ovarian Cancer (HBOC) study. Total HBOC Study High risk BRCA1+ n (%) High risk BRCA2+ n (%) High risk BRCA- n (%) Low risk n (%) No. Families 751 31 (4.1%) 53 (7.1%) 434 (57.8%) 233 (31%) No. Individuals 1246 94 (7.5%) 135 (10.8%) 648 (52%) 369 (29.6%) Table 1: Total number of subjects and families included in the Hereditary Breast/Ovarian Cancer (HBOC) Table 1: Total number of subjects and families included in the Hereditary Breast/Ovarian Cancer (HBOC) study. Table 1: Total number of subjects and families included in the Hereditary Breast/Ovarian Cancer (HBOC) study. Table 2. Number and type of malignancies among individuals of the Hereditary Breast/Ovary Cancer (HBOC) study. (HBOC) study. Total HBOC study High risk BRCA1+ n (%) High risk BRCA2+ n (%) High risk BRCA- n (%) Low risk n (%) AVARRA CANCER REGISTRY Patients with one or more tumors Single tumor 506 (85.3%) 33 (78.6%) 47 (78.3%) 303 (86.1%) 123 (88.5%) Multiple tumors (a) 87 (14.7%) 9 (21.4%) 13 (21.7%) 49 (13.9%) 16 (11.5%) Total 593 42 60 352 139 (a) BRCA1/2 vs Low Risk: OR=2.11 95%CI=1.04 to 4.33, p-value=0.038 Tumors by affected tissue BREAST 531 (76.6%) 30 (55.6%) 53 (70.7%) 327 (80.5%) 120 (76.4%) OVARY 55 (7.9%) 13 (24.1%) 7 (9.3%) 27 (6.6%) 8 (5.1%) SKIN 46 (6.6%) 6 (11.1%) 4 (5.3%) 24 (5.9%) 12 (7.6%) ENDOMETRIUM 13 (1.9%) 1 (1.8%) 2 (2.7%) 7 (1.7%) 3 (1.9%) PANCREAS 5 (0.7%) 2 (3.7%) 0 0 3 (1.9%) COLON 5 (0.7%) 0 1 (1.3%) 4 (1%) 0 PROSTATE GLAND 3 (0.4%) 0 2 (2.7%) 0 1 (0.6%) OTHERS 35 (5.1%) 2 (3.7%) 6 (8%) 17 (4.2%) 10 (6.4%) TOTAL 693 54 75 406 157 Table 3: Clinical characteristics of breast and ovarian cancer cases included in the study. Tables Page 15/22 HBOC study Cancer Registry p-value High risk BRCA1+ n (%) High risk BRCA2+ n (%) High risk BRCA- n (%) Low risk n (%) BREAST CANCER Laterality Right 11 (37.9%) 22 (41.5%) 153 (48.1%) 51 (44%) 2003 (47.4%) Left 17 (58.6%) 27 (50.9%) 155 (48.7%) 61 (52.6%) 2142 (50.7%) Bilateral 1 (3.5%) 4 (7.6%) 10 (3.2%) 4 (3.5%) 78 (1.8%) 0.021 (a) Total 29 53 318 116 4223 (a) BRCA2+ vs Cancer Registry: OR=4.3; 95% CI=1.3 to 11.4 Stage Localized 10 (40%) 24 (47.1%) 154 (57.2%) 56 (59.6%) 2396 (57%) Regional 13 (52%) 24 (47.1%) 105 (39%) 36 (38.3%) 1618 (38.5%) Advanced 2 (8%) 3 (5.9%) 10 (3.7%) 2 (2.1%) 189 (4.5%) 0.043 (b) Total 25 51 269 94 4203 (b) BRCA1/2 vs Cancer Registry Age at diagnosis in years Mean (standard deviation) 44.3 (8.98) 44.12 (10.17) 45.72 (10.34) 50.14 (11.44) 60.79 (15.47) Number 30 53 327 120 4384 p-value <0.001 <0.001 <0.001 <0.001 Reference OVARIAN CANCER Laterality Right 1 (11.1%) 0 6 (46.2%) 3 (60%) 100 (37.2%) Left 1 (11.1%) 2 (66.7%) 3 (23.1%) 2 (40%) 86 (32%) Bilateral 7 (77.8%) 1 (33.3%) 4 (30.8%) 0 83 (30.9%) 0.007 (c) Total 9 3 13 5 269 (c) BRCA1+ vs Cancer Registry: OR=7.8; 95% CI=1.7 to 55.7 Stage Localized 1 (9.1%) 1 (25%) 6 (25%) 2 (28.6%) 28 (23%) Advanced 10 (90.9%) 3 (75%) 18 (75%) 5 (71.4%) 94 (77%) 0.43 (d) (d) BRCA1/2 vs Cancer Registry Age at diagnosis in years Mean (standard deviation) 54.12 (12.03) 53.33 (14.68) 53.29 (10.86) 54.55 (13.08) 65.63 (15.43) Number 13 7 27 8 561 p-value 0.007 0.078 <0.001 0.057 Reference (d) BRCA1/2 vs Cancer Registry Table 4: Overall survival rates for breast and ovarian cancer. Tables Number Mean age at death Overall Survival Log Rank test Age adjusted 5-year 10- year 15- year Hazard Ratio (95%CI) p- value ancer + 30 58.79 ± 13.96 0.863 0.823 0.640 p<0.001 3.19(1.51, 6.74) 0.002 + 53 53.4 ± 11.06 0.882 0.786 0.703 2.63(1.48, 4.67) 0.001 sk - 327 50.73 ± 11.87 0.948 0.899 0.840 1.1(0.79, 1.54) 0.578 sk 120 55.41 ± 11.49 0.967 0.936 0.874 0.61(0.33, 1.14) 0.122 er ry 4384 77.12 ± 15.83 0.818 0.724 0.651 Reference Cancer + 13 62.68 ±13.43 0.615 0.538 0.202 p<0.001 − + 7 53.93 0.857 0.857 0.857 sk - 27 64.1 ± 10.05 0.889 0.572 0.525 sk 8 64.46 ± 12.27 0.875 0.500 0.333 er ry 561 72.85 ± 12.37 0.408 0.321 0.276 Table 4: Overall survival rates for breast and ovarian cancer. Figures Figures Supplementary Files Supplementary Table 1: BRCA1 mutations identified in this study. Germ line BRCA1 pathogenic mutations, molecular change, frequencies, geographical origin of the families (NC= neighbouring communities; OSC= Other Spanish communities, OC= other communities) and associated tumors. Page 17/22 Supplementary Table 2: BRCA2 mutations identified in this study. Germ line BRCA2 pathogenic mutations, molecular change, frequencies, geographical origin of the families (NC= neighbouring communities; OSC= Other Spanish communities, OC= other communities) and associated tumors. Supplementary Figure 1: Inclusion criteria of the Hereditary Breast/Ovarian Cancer study. Figures Figure 1 Supplementary Figure 1: Inclusion criteria of the Hereditary Breast/Ovarian Cancer study. Figure 1 Number of families included in the Hereditary Breast/Ovarian Cancer study (2000-2016). Number of families included in the Hereditary Breast/Ovarian Cancer study (2000-2016). Number of families included in the Hereditary Breast/Ovarian Cancer study (2000-2016). Page 18/22 Page 18/22 Figure 2 Geographical origin of the Hereditary Breast and Ovarian Cancer families in Navarra. Page 19/22 Page 19/22 Page 19/22 Figure 3 Mean age at diagnosis of breast and ovarian cancer cases included in the study. Mean age at diagnosis of breast and ovarian cancer cases included in the study. Mean age at diagnosis of breast and ovarian cancer cases included in the study. Page 20/22 4 Figure 4 Overall survival curves for breast and ovarian cancer. A) Cox regression adjusted by age at diagnosis for breast cancer; B) Kaplan-Meier curves for ovarian cancer. Overall survival curves for breast and ovarian cancer. A) Cox regression adjusted by age at diagnosis for breast cancer; B) Kaplan-Meier curves for ovarian cancer. SupplementaryTable1.doc Supplementary Files Page 21/22 Page 21/22 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTable1.doc SupplementaryTable2.doc Page 22/22
https://openalex.org/W1976555942	https://open.fau.de/bitstreams/8efd860a-071d-43c5-ab1e-9239eec4a1d3/download	English	null	Alouatta Trichromatic Color Vision: Cone Spectra and Physiological Responses Studied with Microspectrophotometry and Single Unit Retinal Electrophysiology	PloS one	2,014	cc-by	10,512	Abstract The howler monkeys (Alouatta sp.) are the only New World primates to exhibit routine trichromacy. Both males and females have three cone photopigments. However, in contrast to Old World monkeys, Alouatta has a locus control region upstream of each opsin gene on the X-chromosome and this might influence the retinal organization underlying its color vision. Post- mortem microspectrophotometry (MSP) was performed on the retinae of two male Alouatta to obtain rod and cone spectral sensitivities. The MSP data were consistent with only a single opsin being expressed in each cone and electrophysiological data were consistent with this primate expressing full trichromacy. To study the physiological organization of the retina underlying Alouatta trichromacy, we recorded from retinal ganglion cells of the same animals used for MSP measurements with a variety of achromatic and chromatic stimulus protocols. We found MC cells and PC cells in the Alouatta retina with similar properties to those previously found in the retina of other trichromatic primates. MC cells showed strong phasic responses to luminance changes and little response to chromatic pulses. PC cells showed strong tonic response to chromatic changes and small tonic response to luminance changes. Responses to other stimulus protocols (flicker photometry; changing the relative phase of red and green modulated lights; temporal modulation transfer functions) were also similar to those recorded in other trichromatic primates. MC cells also showed a pronounced frequency double response to chromatic modulation, and with luminance modulation response saturation accompanied by a phase advance between 10–20 Hz, characteristic of a contrast gain mechanism. This indicates a very similar retinal organization to Old- World monkeys. Cone-specific opsin expression in the presence of a locus control region for each opsin may call into question the hypothesis that this region exclusively controls opsin expression. Editor: Daniel Osorio, University of Sussex, United Kingdom Received May 13, 2014; Accepted October 27, 2014; Published November 18, 2014 Received May 13, 2014; Accepted October 27, 2014; Published November 18, 2014 Copyright: 2014 Silveira 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: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are included within the Supporting Information files. Alouatta Trichromatic Color Vision: Cone Spectra and Physiological Responses Studied with Microspectrophotometry and Single Unit Retinal Electrophysiology Luiz Carlos L. Silveira1,2, Ce´zar A. Saito1, Manoel da Silva Filho1, Jan Kremers3, Barry B. Lee5,6 1 Instituto de Cieˆncias Biolo´gicas, Universidade Federal do Para´, Bele´m, Para´, Brazil, 2 Nu´cleo de Medicina Tropical, Universidade Federal do Para´, Bele´m, Para´, Brazil, 3 Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany, 4 Division of Visual Science, Institute of Ophthalmology, University College London, London, England, United Kingdom, 5 State College of Optometry, State University of New York, New York, New York, United States of America, 6 Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany Abstract Funding: This research was supported by the following grants: CNPq-PRONEX / FAPESPA / UFPA / FADESP #2268; CNPq-PRONEX / FAPESPA #316799/2009; FINEP IBN Net #1723. LCLS is a CNPq research fellow. JK was supported by the Hertie Foundation. The authors would like to thank Dr. Jose´ Augusto Pereira Carneiro Muniz of the Centro Nacional de Primatas (Ananindeua, Para´, Brazil) for provision of animals. 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. Email: luiz@ufpa.br * Email: luiz@ufpa.br ation: Silveira LCL, Saito CA, da Silva Filho M, Kremers J, Bowmaker JK, et al. (2014) Alouatta Trichromatic Color Vision: Cone Spe sponses Studied with Microspectrophotometry and Single Unit Retinal Electrophysiology. PLoS ONE 9(11): e113321. doi:10.1371/journ November 2014 \| Volume 9 \| Issue 11 \| e113321 aito CA, da Silva Filho M, Kremers J, Bowmaker JK, et al. (2014) Alouatta Trichromatic Color Vision: Cone Spectra and Physiologica Microspectrophotometry and Single Unit Retinal Electrophysiology. PLoS ONE 9(11): e113321. doi:10.1371/journal.pone.0113321 Introduction vision can be attained. This has been demonstrated using different behavioral methods [6], [7] and physiologically recordings of opponent cone responses from retinal ganglion cells and lateral geniculate nucleus (LGN) relay neurons [8–11]. In catarrhine primates (Old-World monkeys, apes, and humans), the genes for the middle- (M) and long-wavelength (L) sensitive opsins form a tandem array on the X chromosome and, together with the short-wavelength (S) cone opsin, coded on chromosome 7, form the basis for trichromacy [1–3]. In diurnal platyrrhine primates (New-World monkeys), usually only one longer wavelength opsin gene is found on the X chromosome but, depending of the species, two or more alleles are present [4], [5]. This means that all males are dichromats but if a female possesses two different alleles on her X chromosome pair, trichromatic color A major exception is the Alouatta. Electroretinographic studies in this platyrrhine species showed that in males and females both M- and L-opsins are present [12], and there is evidence in males and females for behavioral trichromacy [13]. The Alouatta L- and M-photopigments have spectral absorption maxima at about 560 and 530 nm, close to those of catarrhines [12], but there may be some inter-individual variability [14]. The genes coding these November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 1 Alouatta Color Vision opsins are present in a tandem array on the X chromosome, as in catarrhines, but with a different structure [4], [14], [15]. regimen and drinking water at libitum. Animal housing conditions, feeding regimen, and health were supervised by the CENP veterinarian staff. The animals were kept in environmental enriched cages as recommended by CENP primatologists. In catarrhines, a single locus control region (LCR) is situated upstream of the tandem gene array, and this is thought to control which gene is expressed [4], [15]. In the Alouatta, a LCR is present upstream of each opsin gene, suggesting that the opsin gene and upstream mechanisms are both duplicated [4], [15]. As a result, Alouatta trichromacy may have a different etiology compared to catarrhines [4], [15]. g y p g Each animal room measured 2.50 m x 4.00 m x 2.25 m, 10 m2 of total area. Each room pair housed 1 male and 5–6 female Alouatta. Houses and rooms were cleaned daily. They were washed with jets of water containing 10% hypochlorite. Introduction Animals were moved from one of the paired rooms to the other during cleaning. Clean water was provided for primates in 500 ml animal drinking bottles, two bottles per room. Bottles were refilled 3 times per day. Primate diet consisted of the following items: a) food pellets provided in plates, ad libitum; b) fruits, vegetables, once a day; cooked eggs, twice a week; diluted milk, twice a week. In addition, Alouatta were provided, daily with Embauba leaves which are part of the natural diet of these Amazonian primates. All animals housed in the CENP received continuous veterinary care which followed the following protocol: inspection in the first hour every day to observe each animal and to inspect the feeding plates and drinking bottles; thereafter regular inspection until the evening. There are also facilities for primate anesthesia and surgery. The facilities allow primate full veterinary assistance, including X-ray, ultrasound, and complete blood, urine, and stool clinical laboratory examination and testing. Each room is enriched with trunks, branches, and ropes to facilitate primate exercising and escape during disputes and force display. Platforms are available for resting and access for feeding. Anatomically, the retinae of diurnal platyrrhines are very similar to those of catarrhines [16–19]. Physiologically, early studies performed, for example, on the squirrel monkey, demonstrated the differences in color opponency in LGN between males and females [20]. Later, physiology performed in further platyrrhine species (Cebus sp., Saimiri sp., and Callithrix sp.) confirmed color opponency in the retinal and LGN cells of trichromatic females, and suggested a pattern that is broadly similar to that of catarrhines, with low achromatic and high chromatic contrast sensitivity of PC cells, and higher contrast sensitivity of MC cells; dichromatic male platyrrhines also showed a low contrast sensitivity of PC cells, indicating an adherence to a primate pattern [8], [9], [11], [21–26]. The retinal anatomy of the Alouatta shows primate character- istics in addition to some unique features. Franco and colleagues estimated the cone density for both retinas of one individual and found that they had an extremely high cone density of about 429,000 cones/mm2 and 357,000 cones/mm2, in the foveola [17]. This density is higher than in any other primate described so far, including humans, for which the highest reported individual value was 324,000 cones/mm2 [27]. Microspectrophotometry However, physiological characteristics of the Alouatta retina remain unknown. If the LCR controls which opsin is expressed, the dual LCRs in the Alouatta could provide a potential for mixed expression of two opsins in the same cone; this seems unlikely in face of the behavioral data, but the mechanisms for expression control in Alouatta is unknown. Physiologically, the characteristics of ganglion cells of the male Alouatta might be expected to resemble those of catarrhines. We here address these issues. Alouatta specimens are rarely available for physiological experi- ments. This brief note contains limited data from two male animals suggesting that pigment expression is indeed cone specific and that ganglion cells closely resemble the catarrhine pattern. Preliminary reports of these experiments have appeared elsewhere [29], [30]. Two eyes, one eye of each animal, were lightly fixed in 2% glutaraldehyde and subsequently examined. All procedures were carried out under dim red light. The eyes were enucleated, hemisected, and the anterior portion removed. One or two small pieces of retina, about 1 mm square, were taken from the fovea and parafoveal regions each, placed on a coverslip and mounted in saline containing 10% dextran, before being squashed with a second coverslip, which was sealed with wax. Although the tissue was in a relatively poor state of preservation, a small number of cone and rod outer segments were identified. g Microspectrophotometric recordings from these cells were performed in the conventional manner using a Liebman dual- beam microspectrophotometer [2], [6], [31]. Spectra were recorded at 2 nm intervals from 750 to 350 nm and from 351 to 749 nm on the return scan. The outward and return scans were averaged. A baseline spectrum was measured for each cell, with both beams in an unoccupied area close to the cell, and this was subtracted from the intracellular scan to derive the final spectrum. Two baseline scans were recorded for each cell and averaged. All cells were fully bleached with white light and post bleach spectra recorded. Records that either had very low absorbance or were clearly distorted were discarded. The wavelength of maximum absorbance (lmax) of both the absorbance spectra and difference spectra were determined by a standard computer program that best fits a visual pigment template to the right hand limb of the spectra [2], [6]. Introduction On the other hand, the retinal ganglion cell distribution in the Alouatta is similar to that observed in other diurnal platyrrhines and catarrhines [28]. On the day of experiment, the animal were sedated with an intramuscular injection of ketamine (ca 20 mg kg21) and then transferred to the Biological Sciences Institute of the Federal University of Para. Ethical statement All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996), and were approved by the Ethical Research Committee for Animal Experiments of the Institute of Biological Sciences, Federal University of Para´ (#MED004/2008). November 2014 \| Volume 9 \| Issue 11 \| e113321 Animals Two male Alouatta caraya, were obtained from the breeding colony of the Centro Nacional de Primatas – CENP (Ananindeua, Para´, Brazil) (Appendix S1). The animals were bred and kept in the CENP facilities until the day the experiment started. There, they were kept in housing conditions permitting social interaction with their conspecifics and submitted to an appropriate feeding A standardized computer program was used to estimate the wavelength of maximum absorbance (lmax) as fully detailed in Mollon and colleagues [6]. In summary, each of 20 relative absorbance values on the long-wave limb of the absorbance spectrum (corresponding to a 40 nm segment of the record and to November 2014 \| Volume 9 \| Issue 11 \| e113321 November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 2 Alouatta Color Vision [35]. Mean chromaticity was 595 nm, retinal illuminance levels was ca 2000 Td, but because of the small Alouatta eye the retinal flux per Troland is about 2 times that in humans. absorbance in the range approximately 45–90% of the maximum for the cell) was then referred to a standard template curve to obtain an estimate of the lmax: this operation amounts to finding the spectral location of the standard curve that gives the percentage absorbance value under consideration. The 20 individual estimates were then averaged to give the values given in the paper. The segment of the right hand limb of the spectrum was used because it includes the steepest part of the photopig- ment’s absorbance spectrum and so small changes in the wavelength correspond to large changes in absorbance, and secondly, the short-wave region in microspectrophotometric measurements is the most variable owing to factors including wavelength dependent scattering and the potential presence of photoproducts. For each cell, we recorded responses to four different stimulus series to extend cell classification and investigate the retinal mechanisms for Alouatta trichromacy. 1) Responses to 400 ms luminance and chromatic pulses were measured to assess the time course of responses; luminance pulses were incremental or decremental; chromatic pulses were redward or greenward. The cells’ spectral sensitivity and cone opponency were assessed by measuring responses to sinusoids in two different protocols. 2) In a modified HFP protocol, the relative modulation depths of a 638 nm red diode and a 554 nm green diode were varied while keeping mean chromaticity and mean luminance constants [36]. Electrophysiology 4) MC cells and PC cells modulation transfer functions (MTFs) were obtained by recording cell responses to sinusoidal stimuli at twelve temporal frequencies and multiple contrast levels. Lumi- nance modulation, with the red and green LEDs in phase, was employed for all cells. Responses of color opponent cells to chromatic modulation, with the LEDs out of phase, were also measured. For luminance modulation, luminance contrast was calculated as (Lmax 2 Lmin)/(Lmax+Lmin). For chromatic contrast, cone contrast was calculated in a similar manner, but using cone absorptions. Animals were initially anaesthetized with an intramuscular injection of ketamine (ca 20 mg kg21). Later, anesthesia was maintained by intravenous infusion of sufentanyl (0.5–4 mg kg21 h21). EEG and ECG were continuously monitored to ensure adequate depth of anesthesia and analgesia. Muscular paralysis was achieved by infusion of 5 mg kg21 h21 of gallamine triethiodide, and the animals were respired with O2 to which ca 1–2% CO2 had been added. End-tidal PCO2 was kept between 4 and 5% and body temperature maintained within the normal limits. The eye was prepared in a similar way to the eye of the macaque and Cebus, and recording of ganglion cell activity was performed as in those species [9], [33]. Briefly, the conjunctiva was sewn to a ring for stabilization and a cannula inserted behind the limbus. A tungsten-in-glass microelectrode was passed through the cannnula, and on touching the retinal surface the electrode was permitted to penetrate into the ganglion cell layer and the activity of a single neuron isolated. A two-dimensional gimbal system, rotating about the point of entry to the globe, permitted us to target different retinal regions. Following completion of record- ings, animals were killed with an overdose of barbiturate, Thionembutal (Abbott, Abbott Park, Illinois, USA), 35 mg/kg or higher. Death was assessed by cessation of ECG activity. For all sinusoidal modulation conditions, about 6 s of activity was averaged for each condition, and first and second harmonic amplitudes and phases were extracted. Animals Non-opponent cells show a null or response minimum at a particular 638/554 nm ratio while red-green opponent cells show a vigorous response at or close to that same ratio [9], [33], [36], [37]. Two to four (4.88–39 Hz) temporal frequencies were measured. Because the absorbance spectra of visual pigments become narrower towards shorter wavelengths, an empirical template has to be used to estimate the lmax. The template used in this study, and in all previous microspectrophotometric measurements from this lab recorded from both Old and New World monkeys, was based on the classical Dartnall nomogram expressed in units of lJ, an empirical relationship that can be used over the limited spectral range of primate rods and L and M cones. More recent empirical templates, such as those by Govardovskii and colleagues [32]), that can be used over the full spectral range, show, over the limited spectral range used here, little if any variation from the template employed in this study. 3) The cells’ spectral sensitivity and cone opponency were also investigated using a phase protocol [38], [39]. The relative phase of the luminance modulation of the 638 nm and 554 nm diodes was varied, with constant modulation amplitude. A phase of 6180 deg corresponds to chromatic modulation and of 0 deg to luminance modulation. For the phase protocols, MC cells were studied at 20 or 50% modulation contrast while PC cells were studied at 50 or 100% modulation contrast. Six temporal frequencies (1.22–39 Hz) were measured. November 2014 \| Volume 9 \| Issue 11 \| e113321 Microspectrophotometry Plots in (A), (B), and (D) show, respectively, rod, middle-wave sensitive cone, and long-wave sensitive cone pre-bleach (open symbols) and post-bleach (filled symbols) spectra. Histogram in (C) shows spectral distribution of lmax of individual rods and cones obtained from absorbance spectra. Figure 1. Absorbance spectra of visual pigments from Alouatta caraya. Plots in (A), (B), and (D) show, respectively, rod, middle-wave sensitive cone, and long-wave sensitive cone pre-bleach (open symbols) and post-bleach (filled symbols) spectra. Histogram in (C) shows spectral distribution of lmax of individual rods and cones obtained from absorbance spectra. doi:10.1371/journal.pone.0113321.g001 support for these classifications was provided by the quantitative analyses as described below. possibility of a limited degree of co-expression within each cone class. No published microspectrophotometric data from monkeys or humans has ever explicitly tested for co-expression. It would require very careful and somewhat difficult partial bleaching experiments due to the small spectral separation in these animals. We then measured cell responses to 400 ms step changes in luminance (increment or decrement) and color (redward or greenward pulses) at various contrast levels. Figure 2 shows the responses of an MC off-centre cell (A) and a +M-L PC off-centre cell (B). The MC cell showed strong phasic responses to luminance changes and little response to chromatic pulses. The PC cell showed strong tonic response to the green pulse, was strongly inhibited by the red pulse, and showed small tonic responses to luminance changes. These responses are very similar to those of macaque ganglion cells [33], [36], [37]. Microspectrophotometry Ten cone outer segments were analyzed and these fell into two clear populations, eight with lmax close to 530 nm (529.565.5 nm) and two with lmax around 557 nm (557.564.9 nm). All these outer segments were fully bleached after exposure to white light. No S cones were identified. Seven rod outer segments gave a mean lmax of 499.861.8 nm, but bleaching with white light revealed, in addition to the typical ‘retinal’ peak at about 370 nm, a stable photoproduct absorbing around 465 nm, presumably metarhodopsin III, which is some- times seen in lightly fixed tissue. The absorbance of the cone outer segments was low, 0.009 and 0.004 for the MWS and LWS cones, respectively, whereas the rods, which were better preserved, had a transverse absorbance of 0.029. A summary of the microspectro- photometric results is presented in Figure 1. These data are consistent with selective expression of the M and L opsins in different cones. Visual stimuli were presented using a Maxwellian view system with red and green diodes (LEDs) as light sources [8], [34]. The system could be rotated about the pupil in order to center the stimulus on a cell’s receptive field. The temporal waveforms for the LEDs were generated by a computer through 12-bit digital-to- analog converters. The LEDs were driven by a frequency- modulated pulse train which provided a highly linear relationship between driving voltage and light output. The emission spectra of the LEDs were measured with a spectroradiometer (Model pro- 703/PC, Photo Research, Burbank, CA, USA). The dominant wavelengths of the LEDs were 638 and 554 nm. The mean luminances of LEDs were set using heterochromatic flicker photometry (HFP) matches by an observer whose spectral luminosity function matched the 2 deg Judd’s spectral sensitivity Although the data are limited, the records fall into two clear spectral groups of L and M cones, but as with all primate microspectrophotometric data, we cannot explicitly exclude the November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 3 Alouatta Color Vision Figure 1. Absorbance spectra of visual pigments from Alouatta caraya. Plots in (A), (B), and (D) show, respectively, rod, middle-wave sensitive cone, and long-wave sensitive cone pre-bleach (open symbols) and post-bleach (filled symbols) spectra. Histogram in (C) shows spectral distribution of lmax of individual rods and cones obtained from absorbance spectra. doi:10.1371/journal.pone.0113321.g001 Figure 1. Absorbance spectra of visual pigments from Alouatta caraya. November 2014 \| Volume 9 \| Issue 11 \| e113321 Electrophysiology We recorded from 34 ganglion cells from the retinae of the two animals (Dataset S1). Cell receptive fields were plotted on a tangent screen and were located within 10 deg of the fovea. Initial cell classification was performed with flashed spots of different colors and gave similar results to those previously obtained from recordings from the retinae of macaques and Cebus [9], [33], [37], [40]. Cells that were classified as PC cells at this stage could be clearly identified by their vigorous response, either excitatory or inhibitory, to green and red lights. KC cells with excitatory S-cone input strongly responded to short wavelength light. Cells that were classified as MC cells could be identified by their good response to low achromatic contrast. Using these criteria, we identified 6 PC cells, 1 S-cone On cell, and 17 MC cells. The remaining 10 cells of our sample remained unclassified mostly because these cells were not recorded for the necessary time for clear classification. Further Figures 3 and 4 shows Alouatta MC and PC cell responses to heterochromatically modulating stimuli. As described in the Methods section, an amplitude protocol based on HFP (Figure 3) and a phase protocol (Figure 4) were used to characterize the spectral sensitivity and color-opponency of ganglion cells. Alouatta MC cells’ responses to the HFP protocol displayed a minimum when the contrast ratio of the counterphase-modulated red to green lights was changed. A ratio of unity would suggest a spectral sensitivity similar to the human photopic luminosity function (Vl). As an example, Figure 3A shows responses of an November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 4 Alouatta Color Vision ll to 9.8 Hz stimuli; response amplitude and phase are against the relative modulation of the red and green LEDs. presence of a substantial second-harmonic component with a amplitude that peaked near a contrast ratio of unity (Figure 3 2. Ganglion cell responses to luminance and color step changes. Responses to 50% contrast, 400 ms step changes in luminan ent or decrement) and color (redward or greenward pulses) of an MC off-centre cell (A) and a +M-L PC off-centre cell (B). The MC cell show esponses to luminance changes and little responses to chromatic pulses. The PC cell showed strong tonic responses to the greenward puls ngly inhibited by the redward pulse, and showed small tonic responses to luminance changes. Electrophysiology Histograms are averages of 20 sweeps; 4 m 371/journal.pone.0113321.g002 ouatta Co o s o e 2. Ganglion cell responses to luminance and color step changes. Responses to 50% contrast, 400 ms step changes in luminanc ment or decrement) and color (redward or greenward pulses) of an MC off-centre cell (A) and a +M-L PC off-centre cell (B). The MC cell showe responses to luminance changes and little responses to chromatic pulses. The PC cell showed strong tonic responses to the greenward puls rongly inhibited by the redward pulse, and showed small tonic responses to luminance changes. Histograms are averages of 20 sweeps; 4 ms .1371/journal.pone.0113321.g002 Figure 2. Ganglion cell responses to luminance and color step changes. Responses to 50% contrast, 400 ms step changes in luminance (increment or decrement) and color (redward or greenward pulses) of an MC off-centre cell (A) and a +M-L PC off-centre cell (B). The MC cell showed phasic responses to luminance changes and little responses to chromatic pulses. The PC cell showed strong tonic responses to the greenward pulse, was strongly inhibited by the redward pulse, and showed small tonic responses to luminance changes. Histograms are averages of 20 sweeps; 4 ms/ bin. doi:10.1371/journal.pone.0113321.g002 doi:10.1371/journal.pone.0113321.g002 MC cell to 9.8 Hz stimuli; response amplitude and phase are plotted against the relative modulation of the red and green LEDs. There is a minimal response of the MC cell first harmonic (filled diamonds) amplitude (upper panel), coinciding with a sudden shift in phase of about 180 deg (middle panel), at a relative amplitude of the red and green LEDs of about 2. Alouatta MC cells also showed a pronounced frequency-doubled response, expressed by the presence of a substantial second-harmonic component with an amplitude that peaked near a contrast ratio of unity (Figure 3A, upper and middle panels, empty squares) whilst phase remained stable. The histograms below the plots illustrate actual responses to two cycles of modulation (Figure 3A, lower panel). Arrows indicate the R/G values for the histograms. The frequency-doubled response is clear in the central histogram. November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 5 Alouatta Color Vision Figure 3. Ganglion cell responses to heterochromatic flicker photometry stimuli. (A) MC and (B) PC cell responses to heterochromatic stimuli. HFP protocol: The relative modulation depths of counterphase modulated 554 and 638 nm LEDs were manipulated. Electrophysiology In the HFP protocol, Alouatta MC cell responses displayed a minimum, coinciding with a sudden shift in phase, when the modulation ratio of the red and green lights was slightly larger than unity. The amplitudes of the second harmonic component peaked around a contrast ratio of unity, where it was substantially larger than the amplitude of the first harmonic component, whilst its phase remained stable. The resultant frequency doubled response can be observed in the corresponding histogram. Alouatta PC cells showed vigorous response and no phase shift for nearly all stimulus conditions. doi:10.1371/journal.pone.0113321.g003 response shaping (Figure 3B, upper and middle panels, empty squares). Again, the histograms set below the plots illustrate actual responses to two cycles of modulation (Figure 3B, lower panel). On average, response minima of MC cells from the two animals tested was close to a luminance ratio between the red and green diodes of one (mean: 1.08, s.d.: 0.26, n = 16) but the two animals differed significantly in mean null values (means 1.25 (n = 9), 0.86 (n = 7), p,0.02, t test). It is established that HFP luminance nulls of humans is related to relative L/M cone numerosity in the retina [41], [42], but the mechanisms controlling relative numerosity are poorly understood; with the different genetic foundation in the Alouatta this problem is compounded. In conclusion, the responses of Alouatta MC cells and PC cells to the HFP protocol were similar to those of their counterparts found in the retinas of macaques and trichromatic Cebus [9], [33], [36], [37]. In the phase protocol, rather than changing the relative amplitude of modulated lights, their relative phase is varied. This protocol can determine relative weights and temporal properties of cone mechanisms in ganglion cell responses [39]. At 10 Hz and above, macaque MC cells respond to the phase protocol with a first-harmonic maximum when the green and red lights are in phase; their responses reach a minimum when the green and red stimuli are modulated in counterphase. At lower temporal frequencies, the phase of minimum response moves away from zero as in human psychophysical results [38], [39], [43]. At higher frequencies, a similar pattern was found in Alouatta MC cells, as shown in Figure 4A. Response amplitude and phase have been plotted as a function of the relative phase of the two LEDs. Electrophysiology MC and PC cells were stimulated at 9.8 Hz and 1.22 Hz, respectively. Stimulus size was, 4 deg mean retinal illuminance was 2000 Td. Each data point was obtained from the Fourier analysis on averaged responses in episodes each lasting 6 s. Top and middle panels: response amplitude and phase as a function of the red/ green amplitude ratio, respectively. Filled diamonds and empty squares represent the parameters for response first and second harmonics, respectively. Bottom panels: the histograms illustrate actual averaged responses to two modulation cycles, the arrows point at the employed red/ green ratios for the histograms, and the red and green curves represent the temporal luminance modulation of the red and green lights for each stimulus condition. In the HFP protocol, Alouatta MC cell responses displayed a minimum, coinciding with a sudden shift in phase, when the modulation ratio of the red and green lights was slightly larger than unity. The amplitudes of the second harmonic component peaked around a contrast ratio of unity, where it was substantially larger than the amplitude of the first harmonic component, whilst its phase remained stable. The resultant frequency doubled response can be observed in the corresponding histogram. Alouatta PC cells showed vigorous response and no phase shift for nearly all stimulus conditions. doi:10.1371/journal.pone.0113321.g003 Figure 3. Ganglion cell responses to heterochromatic flicker photometry stimuli. (A) MC and (B) PC cell responses to heterochromatic stimuli. HFP protocol: The relative modulation depths of counterphase modulated 554 and 638 nm LEDs were manipulated. MC and PC cells were stimulated at 9.8 Hz and 1.22 Hz, respectively. Stimulus size was, 4 deg mean retinal illuminance was 2000 Td. Each data point was obtained from the Fourier analysis on averaged responses in episodes each lasting 6 s. Top and middle panels: response amplitude and phase as a function of the red/ green amplitude ratio, respectively. Filled diamonds and empty squares represent the parameters for response first and second harmonics, respectively. Bottom panels: the histograms illustrate actual averaged responses to two modulation cycles, the arrows point at the employed red/ green ratios for the histograms, and the red and green curves represent the temporal luminance modulation of the red and green lights for each stimulus condition. November 2014 \| Volume 9 \| Issue 11 \| e113321 Electrophysiology MC and PC cells were stimulated at 9.8 Hz and 1.22 Hz, respectively. Stimulus size was 4 deg, mean retinal illuminance was 2000 Td. Response amplitudes (top panels) and phases (middle panels) are shown as a function of the phase difference between the luminance modulation in the red and green LEDs. Filled diamonds and empty squares represent the parameters for response first and second harmonics, respectively, extracted by Fourier analysis. Bottom panels: the histograms illustrate actual responses to two cycles of modulation, the arrows indicate the red/green phase difference for the histograms, and the red and green curves represent how the phase of the red and green lights changed for each stimulus condition. Luminance modulation corresponds to a relative phase of 0 deg, chromatic modulation to a relative phase of 6180 deg. MC cells responded to the phase protocol with a maximal response first harmonic amplitude when the green and red lights were modulated in phase and their response phase changed continuously with the phase difference between the modulation in the two LEDs. PC cells displayed a minimal response when the green and red lights were modulated in phase and their response phases changed abruptly in the region of minimal response. doi:10.1371/journal.pone.0113321.g004 Alouatta, a highly significant difference (p,0.001, t test). This would suggest a species difference. is a response minimum near +/2180 deg. The response phase changes gradually with a more rapid transition near +/2180 deg. The second-harmonic response is seen to be very vigorous at this phase. In the macaque [39], second-harmonic response compo- nents were sometimes seen but were seldom large enough to disturb analysis of first-harmonic components. In the sample of Alouatta cells, frequency-doubled components were very promi- nent and made analysis of first-harmonic components noisy, especially at lower temporal frequencies. To validate this observation, we compared the ratio of luminance 1st harmonic to chromatic 2nd harmonic responses for samples of cells from the two species, using very similar stimuli (50% modulation contrast, 4.88 Hz, same LED peak wavelengths and mean luminance; macaque data were taken from previous work [34]. Ratios were 2.65 (s.d. 1.42, n = 13) for macaque and 0.758 (sd. 0.31, n = 13) for is a response minimum near +/2180 deg. The response phase changes gradually with a more rapid transition near +/2180 deg. The second-harmonic response is seen to be very vigorous at this phase. Electrophysiology There The frequency-doubled response to chromatic modulation of MC cells in the Alouatta was very obvious as in the example in Figure 3. As in the macaque [36], it was not present at higher temporal frequencies (39 Hz), with some cell-to-cell variability. This is taken up below. Alouatta PC cells showed vigorous responses to nearly all heterochromatic stimuli; the first harmonic component to 1.22 Hz stimuli (Figure 3B filled diamonds) displayed a broad peak at ratios around unity (upper panel). The phases were similar for all ratios (middle panel). The second harmonic-response follows the first harmonic amplitude but is always smaller and thus is related to November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 6 Alouatta Color Vision Figure 4. Ganglion cell response to stimulus phase changes. (A) MC and (B) PC cell responses to heterochromatic stimuli. Phase protocol. The relative phases of the 554 and 638 nm LEDs were modulated with fixed modulation depths. MC and PC cells were stimulated at 9.8 Hz and 1.22 Hz, respectively. Stimulus size was 4 deg, mean retinal illuminance was 2000 Td. Response amplitudes (top panels) and phases (middle panels) are shown as a function of the phase difference between the luminance modulation in the red and green LEDs. Filled diamonds and empty squares represent the parameters for response first and second harmonics, respectively, extracted by Fourier analysis. Bottom panels: the histograms illustrate actual responses to two cycles of modulation, the arrows indicate the red/green phase difference for the histograms, and the red and green curves represent how the phase of the red and green lights changed for each stimulus condition. Luminance modulation corresponds to a relative phase of 0 deg, chromatic modulation to a relative phase of 6180 deg. MC cells responded to the phase protocol with a maximal response first harmonic amplitude when the green and red lights were modulated in phase and their response phase changed continuously with the phase difference between the modulation in the two LEDs. PC cells displayed a minimal response when the green and red lights were modulated in phase and their response phases changed abruptly in the region of minimal response. doi:10.1371/journal.pone.0113321.g004 Figure 4. Ganglion cell response to stimulus phase changes. (A) MC and (B) PC cell responses to heterochromatic stimuli. Phase protocol. The relative phases of the 554 and 638 nm LEDs were modulated with fixed modulation depths. November 2014 \| Volume 9 \| Issue 11 \| e113321 Electrophysiology In the macaque [39], second-harmonic response compo- nents were sometimes seen but were seldom large enough to disturb analysis of first-harmonic components. In the sample of Alouatta cells, frequency-doubled components were very promi- nent and made analysis of first-harmonic components noisy, especially at lower temporal frequencies. To validate this observation, we compared the ratio of luminance 1st harmonic to chromatic 2nd harmonic responses for samples of cells from the two species, using very similar stimuli (50% modulation contrast, 4.88 Hz, same LED peak wavelengths and mean luminance; macaque data were taken from previous work [34]. Ratios were 2.65 (s.d. 1.42, n = 13) for macaque and 0.758 (sd. 0.31, n = 13) for PC cells responded to the phase protocol with a minimum when the green and red lights were in phase; their responses increased with increasing phase difference between the green and red stimuli and were maximal when the two modulated in counterphase (Figure 4B, upper panel). In addition, the cell response showed an abrupt change in phase in the region of minimum response (Figure 4B, middle panel). These results suggest responses of Alouatta MC and PC cells were similar to those of the corresponding ganglion cells found in the retinas of macaques [39] and trichromatic Cebus [9], although differences in detail are likely to be present. There appeared to be more variability in the opponent weighting for PC cells than in similar macaque data, but our cell sample was not large. November 2014 \| Volume 9 \| Issue 11 \| e113321 November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 7 Alouatta Color Vision To explore further the comparison between Alouatta ganglion cell responses on the one hand and those of ganglion cells in macaques and in trichromatic female platyrrhines on the other, we employed sinusoidal luminance and chromatic modulation at different temporal frequencies and contrasts. Figure 5A–B illus- trates response amplitudes and phases of Alouatta MC (Figure 5A) and PC cells (Figure 5B) as a function of contrast; luminance modulation was used for the MC cell and chromatic modulation for the PC cell. Data representing response amplitude as a function of contrast were fitted with Naka-Rushton functions [44]. Electrophysiology MC cell vigorously responded to low levels of luminance contrast at frequencies of 5–40 Hz, but responses rapidly saturated accom- panied by response phase advancement, especially at intermediate temporal frequencies (9.8 Hz), as seen in the phase plot. PC cells were most sensitive to chromatic contrast (Figure 5B), but responses did not exhibit saturation or phase advance when contrast was increased. Figure 5C illustrates the averaged tempo- ral MTFs that were obtained from Alouatta MC cells (n = 7) and PC cells (n = 4). For both cell classes, we obtained luminance temporal MTFs and for PC cells we also obtained red-green temporal MTFs. The MTFs show the contrast gain as a function of temporal frequency, contrast gain being the initial slope (at zero contrast) of the Naka-Rushton functions fitted to the amplitude- versus-contrast data, cone contrast being used for the red-green temporal MTFs. MC cells were much more sensitive than PC cells to temporal luminance modulation across the temporal frequency range studied and responded vigorously to high temporal frequencies. On the other hand, PC cells were most sensitive to red-green contrast, especially at low and intermediate temporal frequencies. and responded to the HFP protocol with an amplitude peak near equal luminance. In addition, two specific features found in macaque ganglion cells were also encountered. MC cells responded to equal-luminance red-green modulation with twice the stimulation frequency [36]; this effect was marked in Alouatta MC cells. Also, MC cells showed response saturation and phase advancement with increasing contrast whereas PC cells did not. These latter features of MC cells are characteristic of a contrast gain control mechanisms [45], [46], and found in MC cells but not in PC cells of catarrhines [47], [48] and platyrrhines [8], [9]. Many properties are shared by the MC and PC cells of Alouatta, macaques [33], [37], and trichromatic female Callithrix [8] and Cebus [9]. The MC and PC cells of dichromatic male or female Callithrix [8] and Cebus [9] also exhibit the similar responses to luminance-modulated stimuli as in their trichromatic conspecifics. In conclusion, although a relatively small number of cells were measured, they displayed response properties that closely match those of other trichromatic species and individuals strongly suggesting that the processing in the retina of Alouatta is very similar. Electrophysiology Alouatta PC cells comprised the same subclasses found in other trichromats: some were excited by red light and inhibited by green light, while others were excited by green light and inhibited by red light. These responses reflected the excitatory or inhibitory inputs they received from M cones (530 nm absorption peak) or L cones (558 nm absorption peak). No study has yet morphologically identified Alouatta bipolar cells, but we suggest from the responses obtained from PC cells that single-cone midget bipolar cells similar to those that have been found in catarrhines [49] and platyrrhines [50], [51] will be present in the Alouatta retina. As complete trichromacy seems to have evolved relatively recently in Alouatta (with a different origin compared to catarrhines [4], [15]), it is likely that the receptoral mechanisms were able to take over pre-existent post-receptoral retinal wiring, already utilized in females of the species, and to use them to send trichromatic signals to higher visual centers. Physiological substrate for Alouatta trichromacy This work provides direct physiological data on the functional organization of the Alouatta retina for comparison with data in other species. The comparison may shed light on basic principles of primate retinal organization and on possible specific adaptations to trichromatic vision in different primate species. We therefore conclude that the functional organization of the retina is very similar in all anthropoid primates and was probably present before the split between catarrhines and platyrrhines 40 million years ago (MYA). There may well be differences in detail, so that the frequency-doubled MC response to chromatic modulation was very obvious in Alouatta, compared to earlier recordings in trichromatic Callithrix and Cebus. Another feature of trichromatic color vision is cone specificity in the surrounds of PC- cells of macaques [52], [53]. This is present to some degree in the Callithrix [54] but the situation in Alouatta is unknown. We stress that quantitative differences between retinae of different primate species are likely. We have found microspectrophotometric signatures of two populations of middle-to-long wavelength sensitive cones bearing separate expressions of photopigments with absorption peaks close to 530 nm and 558 nm absorption peaks. The presence of these two cone types is the receptoral prerequisite for a red-green color opponent pathway in the primate retina [1], [2], [5]. Cone-specific expression of M and L opsins is not unexpected from ERG and behavioral data [12], but it remains unclear how this occurs when each opsin gene has its own LCR. In addition, we have found post-receptoral red-green color opponent mechanisms with very similar properties of those previously found in macaques [33], [37], trichromatic female Callithrix [8] and trichromatic female Cebus [9]. Although the cell sample was limited due to the small number of animals available, the general features found in the macaque were apparent. Discussion Physiological substrate for Alouatta trichromacy Contrast sensitivity of Alouatta retinal ganglion cells Contrast sensitivity of Alouatta retinal ganglion cells Temporal MTFs (contrast gain as a function of temporal frequency) obtained from Alouatta MC and PC cells followed the same general pattern of temporal MTFs obtained from macaques [34] and Cebus [9]. The low luminance contrast gain of PC cells observed in trichromatic primates have been considered to be largely due to mutual cancellation of the opponent cone signals [33], [34], [37]. PC cells of dichromatic and trichromatic Cebus monkeys showed similar low gain [9]. PC cells of dichromatic platyrrhines might be described as color blind versions of cone- opponent PC cells of trichromatic primates. The functional significance played by such a numerous, luminance contrast insensitive cell class remains uncertain. In any event, PC cells in male Alouatta appear to have properties as in the macaque. November 2014 \| Volume 9 \| Issue 11 \| e113321 Physiological properties of Alouatta retinal ganglion cells Physiological properties of Alouatta retinal ganglion cells Alouatta MC and PC ganglion cells exhibit similarities with MC and PC ganglion cells of catarrhine primates. To list these similarities: MC cells were sensitive to luminance contrast, exhibited phasic responses to luminance steps and responded to the phase protocol and HFP protocols as in the macaque. PC cells were less sensitive to luminance contrast, exhibited tonic responses to chromatic pulses, responded to the phase protocol with a minimum response when the green and red lights were in phase November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org 8 Alouatta Color Vision Figure 5. Ganglion cell response to stimulus contrast. (A–B) Response amplitude (left panels) and phase (right panels) as a function of stimulus luminance contrast for Alouatta MC cell (A) and PC cell (B). The results obtained with three temporal frequencies are given: 1.2 Hz (filled squares), 9.8 Hz (filled triangles), and 39 Hz (filled diamonds) for the MC cell; 1.2 Hz (filled squares), 9.8 Hz (filled triangles), and 19.4 Hz (filled diamonds) for the PC cell. Data representing response amplitude as a function of contrast have been fitted with Naka-Rushton functions. MC cell vigorously responded to low levels of luminance contrast, but responses rapidly saturated accompanied by advancement in response phase, especially at intermediate and high temporal frequencies. PC cells were quite insensitive to low levels of luminance contrast, but the responses did not exhibit saturation or phase advancement. (C) Temporal modulation transfer functions (temporal MTFs) for Alouatta MC cells (average of 7 cells) stimulated with luminance sinusoids (filled squares) and a PC cell stimulated with luminance (empty diamonds) and averaged responses of 4 cells to red-green (filled diamonds) sinusoids. Contrast gain was defined as the initial slope of the Naka-Rushton functions fitted to the amplitude versus contrast data such as those illustrated in the left panels (A–B). Michelson contrast and cone contrast were used for the luminance and red-green chromatic temporal MTFs, respectively. MC cells were much more sensitive than PC cells to temporal luminance modulation at all the temporal frequencies range and vigorously responded to very high temporal frequencies. On the other hand, PC cells were very sensitive to red-green contrast, especially at low and intermediate temporal frequencies. doi:10.1371/journal.pone.0113321.g005 Figure 5. Ganglion cell response to stimulus contrast. Physiological properties of Alouatta retinal ganglion cells (A–B) Response amplitude (left panels) and phase (right panels) as a function of stimulus luminance contrast for Alouatta MC cell (A) and PC cell (B). The results obtained with three temporal frequencies are given: 1.2 Hz (filled squares), 9.8 Hz (filled triangles), and 39 Hz (filled diamonds) for the MC cell; 1.2 Hz (filled squares), 9.8 Hz (filled triangles), and 19.4 Hz (filled diamonds) for the PC cell. Data representing response amplitude as a function of contrast have been fitted with Naka-Rushton functions. MC cell vigorously responded to low levels of luminance contrast, but responses rapidly saturated accompanied by advancement in response phase, especially at intermediate and high temporal frequencies. PC cells were quite insensitive to low levels of luminance contrast, but the responses did not exhibit saturation or phase advancement. (C) Temporal modulation transfer functions (temporal MTFs) for Alouatta MC cells (average of 7 cells) stimulated with luminance sinusoids (filled squares) and a PC cell stimulated with luminance (empty diamonds) and averaged responses of 4 cells to red-green (filled diamonds) sinusoids. Contrast gain was defined as the initial slope of the Naka-Rushton functions fitted to the amplitude versus contrast data such as those illustrated in the left panels (A–B). Michelson contrast and cone contrast were used for the luminance and red-green chromatic temporal MTFs, respectively. MC cells were much more sensitive than PC cells to temporal luminance modulation at all the temporal frequencies range and vigorously responded to very high temporal frequencies. On the other hand, PC cells were very sensitive to red-green contrast, especially at low and intermediate temporal frequencies. doi:10.1371/journal.pone.0113321.g005 References Yeh T, Lee BB, Kremers J, Cowing JA, Hunt DM, et al. (1995a) Visual responses in the lateral geniculate of dichromatic and trichromatic marmosets (Callithrix jacchus). J Neurosci 15: 7892–7904. 30. 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Stockholm, Sweden. 1–60. 14. Matsushita Y, Oota H, Welker BJ, Pavelka MS, Kawamura S (2014) Color vision variation as evidenced by hybrid L/M opsin genes in wild populations of trichromatic Alouatta New World monkeys. Int J Primatol 35: 71–87. 36. Lee BB, Martin PR, Valberg A (1988) The physiological basis of heterochro- matic flicker photometry demonstrated in the ganglion cells of the macaque retina. References 1. Nathans J, Thomas D, Hogness DS (1986) Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232: 193– 202. 1. Nathans J, Thomas D, Hogness DS (1986) Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232: 193– 202. 25. Solomon SG, White AJ, Martin PR (1999) Temporal contrast sensitivity in the lateral geniculate nucleus of a New World monkey, the marmoset Callithrix jacchus. J Physiol (Lond) 517: 907–917. 2. 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References J Physiol (Lond) 404: 323–347. 15. Dulai KS, von Dornum M, Mollon JD, Hunt DM (1999). The evolution of trichromatic color vision by opsin gene duplication in New World and Old World primates. Genome Res 9: 629–638. y 37. Lee BB, Martin PR, Valberg A (1989b) Amplitude and phase of responses of macaque retinal ganglion cells to flickering stimuli. J Physiol (Lond) 414: 245– 263. 16. Silveira LCL, Picanc¸o-Diniz CW, Sampaio LFS, Oswaldo-Cruz E (1989) Retinal ganglion cell distribution in the Cebus monkey: a comparison with the cortical magnification factors. Vision Res 29: 1471–1483. 38. Lindsey DT, Pokorny J, Smith VC (1986) Phase dependent sensitivity to heterochromatic flicker. J Opt Soc Am A 3: 921–927. 17. Franco ECS, Finlay BL, Silveira LCL, Yamada ES, Crowley JC (2000) Conservation of absolute foveal area in New World primates: a constraint on eye size and conformation. Brain Behav Evol 56: 276–286. 39. Smith VC, Lee BB, Pokorny J, Martin PR, Valberg A (1992) Responses of macaque ganglion cells to the relative phase of heterochromatically modulated lights. J Physiol (Lond) 458: 191–221. 18. Silveira LCL (2004) Comparative study of the primate retina. In: Kaas JH, Collins CE, editors. The Primate Visual System. Boca Raton, Florida, USA: CRC Press. 29–51. 40. Silveira LCL, Lee BB, Yamada ES, Kremers J, Hunt DM, et al. (1999) Ganglion cells of a short wavelength sensitive cone pathway in New World monkeys: morphology and physiology. Vis Neurosci 16: 333–343. 19. Silveira LCL, Gru¨nert U, Kremers J, Lee BB, Martin PR (2005) Comparative anatomy and physiology of the primate retina. In: Kremers J, editor. The primate visual system: A comparative approach. Chichester, England, UK: John Wiley & Sons. 127–160. 41. Brainard DH, Roorda A, Yamauchi Y, Calderone JB, Metha AB, et al. (2000) Functional consequences of the relative numbers of L and M cones. J Opt Soc Am A 17: 607–614. 42. Kremers J, Scholl HPN, Knau H, Berendschot TTJM, Usui T, et al. (2000) L/ M-cone ratios in human trichromats assessed by psychophysics, electroretinog- raphy and retinal densitometry. J Opt Soc Am A 17: 517–526. y 20. Jacobs GH, De Valois RL (1965) Chromatic opponent cells in squirrel monkey lateral geniculate nucleus. Nature 206: 487–489. raphy and retinal densitometry. J Opt Soc Am A 17: 517–526. g 21. Kremers J, Weiss S (1997). Receptive field dimensions of lateral geniculate cells in the common marmoset (Callithrix jacchus). Vision Res 37: 2171–2181. Evolution of primate trichromacy generally related to foraging behavior. Stephen Polyak was probably the first to explicitly point out that detecting orange and red fruits against the green foliage would benefit from trichromacy [58]. There have been several studies measuring fruits and leaves surface reflectance and quantifying primate foraging behavior in natural conditions which were then used to provide quantitative data for theories of trichromacy evolution [59–64]. These ideas focused in the evolutionary advantage of acquiring a better distinction of reddish fruits against green foliage [65], [66], yellowish dappled with red young nutritive leaves against mature green leaves [62], and reddish falling leaves against healthy green leaves [67]. These possibilities are not mutually exclusive. It should be observed that both catarrhines and Alouatta distinguish themselves from the majority of platyrrhines by having a diet mainly based on fruits and on leaves [67], [68]. For the Alouatta, there have been reports from field studies of their foraging behavior associated with quantitative analysis in the color domain Among the possible scenarios for the evolution of trichromatic vision in primates [9], [55], a common view is that polymorphic color vision arose in prosimians and passed to anthropoids before the split between catarrhines and platyrrhines. Full trichromacy then arose independently at least twice, in catarrhines and in the Alouatta [4], [56], [57]. The absorption peaks of the two LWS cone opsins found in the two groups are usually similar (Jacobs et al. [12] and the present study), possibly indicating an optimization for red-green color opponency in regular trichro- mats. Another possibility, originally proposed by Mollon [55], and supported by others based on molecular data [4], [14], [15], is that trichromatic vision in platyrrhines and catarrhines evolved separately. However, the close similarities in retinal anatomy and physiology between the two groups, further underlined here, make, in our view, parallel evolution unlikely. There are several hypotheses about the evolutionary pressures that might have driven primates to trichromacy and they are November 2014 \| Volume 9 \| Issue 11 \| e113321 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 Alouatta Color Vision of surface reflectance underlying the importance of selecting both the appropriate fruits [60], [61] and leaves [64] for their diet. Checklist S1 The ARRIVE Guidelines Checklist. (DOC) Checklist S1 The ARRIVE Guidelines Checklist. (DOC) Checklist S1 The ARRIVE Guidelines Checklist. (DOC) Appendix S1 Alouatta housing conditions including feeding regimens and environmental enrichment. (DOC) Conceived and designed the experiments: LCLS JK JKB BBL. Performed the experiments: LCLS CAS MSF JK JKB BBL. Analyzed the data: LCLS CAS JK JKB BBL. Contributed reagents/materials/analysis tools: LCLS CAS MSF JK JKB BBL. Contributed to the writing of the manuscript: LCLS CAS MSF JK JKB BBL. Author Contributions Appendix S1 Alouatta housing conditions including feeding regimens and environmental enrichment. (DOC) References Silveira LCL, Lee BB, Yamada ES, Kremers J, Hunt DM (1998) Post-receptoral mechanisms of colour vision in New World primates. Vision Res 38: 3329–3337. 62. Lucas PW, Darvell BW, Lee PKD, Yuen TDB, Choong MF (1998) Colour cues for leaf food selection by long-tailed macaques (Macaca fascicularis) with a new suggestion for the evolution of trichromatic colour vision. Folia Primatologica 69: 139–154. 51. Chan TL, Martin PR, Clunas N, Gru¨nert U (2001) Bipolar cell diversity in the primate retina: morphologic and immunocytochemical analysis of a New World monkey, the marmoset Callithrix jacchus. J Comp Neurol 437: 219–239. 52. Martin PR, Lee BB, White AJR, Solomon SG, Ru¨ttiger L (2001) Chromatic sensitivity of ganglion cells in the peripheral primate retina. Nature 410: 933– 936. 63. Lucas PW, Dominy NJ, Riba-Hernandez P, Stoner KE, Yamashita N, et al. (2003) Evolution and function of routine trichromatic vision in primates. Evolution 57: 2636–2643. 53. Solomon SG, Lee BB, White AJ, Ru¨ttiger L, Martin PR (2005) Chromatic organization of ganglion cell receptive fields in the peripheral retina. J Neurosci 25: 4527–4539. 64. Stoner KE, Riba-Hernandez P, Lucas PW (2005) Comparative use of color vision for frugivory by sympatric species of platyrrhines. Am J Primatology 67: 399–409. 54. Szmajda BA, Gru¨nert U, Martin PR (2005) Mosaic properties of midget and parasol ganglion cells in the marmoset retina. Vis Neurosci 22: 395–404. 54. Szmajda BA, Gru¨nert U, Martin PR (2005) Mosaic properties 65. Snodderly DM (1979) Visual discriminations encountered in food foraging by a neotropical primate: implications for the evolution of color vision. In: Burtt EHJ, editor. The behavioral significance of color. New York, New York, USA: Garland STMP Press. 238–279. 55. Mollon JD (1991) Uses and evolutionary origins of primate color vision. In: Cronly-Dillon JR, Gregory RL, editors. Evolution of the eye and visual system. Houndmills, Basingstoke, Hampshire, England, UK: MacMillan Press. 306–319. 66. Mollon JD, Jordan G (1988/1989) Eine evolutiona¨re Interpretation des menschlichen Farbensehens. Die Farbe 35/36: 139–170. g p g 56. Kainz PM, Neitz J, Neitz M (1998) Recent evolution of uniform trichromacy in a New World monkey. Vision Res 38: 3315–3320. 67. Kremers J, Silveira LCL, Yamada ES, Lee BB (1999) The ecology and evolution of primate colour vision. In: Gegenfurtner KR, Sharpe LT, editors. Color vision: From genes to perception. Cambridge, England, UK: Cambridge University Press. 123–142. 57. Bowmaker JK (2008) Evolution of vertebrates visual pigments. Vision Res 48: 2022–2041. 58. References 43. Smith VC, Pokorny J (1975). Spectral sensitivity of the foveal cone photopigments between 400 and 500 nm. Vision Res 15: 161–171. 43. Smith VC, Pokorny J (1975). Spectral sensitivity of the in the common marmoset (Callithrix jacchus). Vision Res 37: 217 photopigments between 400 and 500 nm. Vision Res 15: 161–17 22. Kremers J, Weiss S, Zrenner E (1997) Temporal properties of marmoset lateral geniculate cells. Vision Res 37: 2649–2660. 44. Naka KI, Rushton WAH (1966) S-potentials from colour units in the retina of fish (Cyprinidae). J Physiol (Lond) 185: 536–555. 23. White AJR, Wilder HD, Goodchild AK, Sefton AJ, Martin PR (1998) Segregation of receptive field properties in the lateral geniculate nucleus of a New-World Monkey, the marmoset Callithrix jacchus. J Neurophysiol 80: 2063– 2076. 45. 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J Opt Soc Am A 12, 456–464. 60. Regan BC, Julliot C, Simmen B, Vie´not F, Charles-Dominique P, et al. (1998) Frugivory and colour vision in Alouatta seniculus, a trichromatic platyrrhine monkey. Vision Res 38: 3321–3327. 49. Boycott BB, Wa¨ssle H (1991) Morphological classification of bipolar cells of the primate retina. Eur J Neurosci 3: 1069–1088. y 61. Regan BC, Julliot C, Simmen B, Vie´not F, Charles-Dominique P, et al. (2001) Fruits, foliage and the evolution of primate colour vision. Phil Trans R Soc Lond B 356: 229–283. p J 50. 68. Fleagle JG (1988) Primate adaptation and evolution. San Diego, California, U.S.A.: Academic Press. 486 p. References Polyak S (1957) The vertebrate visual system. Chicago, Illinois, USA: Chicago University Press. 1390 p. 68. Fleagle JG (1988) Primate adaptation and evolution. San Diego, California, U.S.A.: Academic Press. 486 p. 59. Osorio D, Vorobyev M (1996) Colour vision as an adaptation to frugivory in primates. Proc R Soc Lond B 263: 593–539. 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https://openalex.org/W2946940667	https://bmcmedinformdecismak.biomedcentral.com/track/pdf/10.1186/s12911-019-0815-y	English	null	Correction to: Automatically identifying social isolation from clinical narratives for patients with prostate Cancer	BMC medical informatics and decision making	2,019	cc-by	283	Correction to: BMC Med Inform Decis Mak https://doi.org/10.1186/s12911-019-0795-y Following publication of the original article [1], the au- thors reported an error in one of the authors’ names. In this Correction the incorrect and correct author name are shown. The original publication of this article has been corrected. Originally the author name was published as: – Chanita A Hughes-Halbert The correct author name is: Correction to: Automatically identifying social isolation from clinical narratives for patients with prostate Cancer Vivienne J. Zhu1* , Leslie A. Lenert1, Brian E. Bunnell1, Jihad S. Obeid1, Melanie Jefferson2 and Chanita Hughes Halbert2 Author details 1 Author details 1Biomedical Informatics Center, Medical University of South Carolina, Chalrleston, SC, USA. 2Holling Cancer Center and Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC, USA. Received: 16 April 2019 Accepted: 16 April 2019 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Reference 1. Zhu, et al. Automatically identifying social isolation from clinical narratives for patients with prostate Cancer. BMC Med Inform Decis Mak. 2019;19:43 https://doi.org/10.1186/s12911-019-0795-y. * Correspondence: zhuv@musc.edu 1Biomedical Informatics Center, Medical University of South Carolina, Chalrleston, SC, USA Full list of author information is available at the end of the article (2019) 19:89 (2019) 19:89 Zhu et al. BMC Medical Informatics and Decision Making https://doi.org/10.1186/s12911-019-0815-y Open Access
https://openalex.org/W3117684457	https://zenodo.org/records/6406642/files/Discovery%20of%20Monoacylglycerol%20Lipase%20(MAGL)%20Inhibitors%20Based%20on%20a%20Pharmacophore-Guided%20Virtual%20Screening%20Study.pdf	English	null	Discovery of Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Pharmacophore-Guided Virtual Screening Study	Molecules/Molecules online/Molecules annual	2,020	cc-by	8,952	Keywords: virtual screening; MAGL; pharmacophore model; hit identiﬁcation Citation: Jha, V.; Biagi, M.; Spinelli, V.; Di Stefano, M.; Macchia, M.; Minutolo, F.; Granchi, C.; Poli, G.; Tuccinardi, T. Discovery of Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Pharmacophore-Guided Virtual Screening Study. Molecules 2021, 26, 78. https://dx.doi.org/10.3390/ molecules26010078 Article Discovery of Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Pharmacophore-Guided Virtual Screening Study a, Marzia Biagi, Valeria Spinelli, Miriana Di Stefano , Marco Macchia, Filippo Minutolo , Granchi , Giulio Poli * and Tiziano Tuccinardi * Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy; vibhu.jha@farm.unipi.it (V.J.); marziabiagi@outlook.com (M.B.); madavale83@libero.it (V.S.); miriana.distefano@phd.unipi.it (M.D.S.); marco.macchia@unipi.it (M.M.); ﬁlippo.minutolo@unipi.it (F.M.); carlotta.granchi@unipi.it (C.G.) * Correspondence: giulio.poli@unipi.it (G.P.); tiziano.tuccinardi@unipi.it (T.T.); Tel.: +39-050-2219603 (G.P.); +39-050-2219595 (T.T.) Abstract: Monoacylglycerol lipase (MAGL) is an important enzyme of the endocannabinoid system that catalyzes the degradation of the major endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL is associated with pathological conditions such as pain, inﬂammation and neurodegenerative diseases like Parkinson’s and Alzheimer’s disease. Furthermore, elevated levels of MAGL have been found in aggressive breast, ovarian and melanoma cancer cells. Due to its different potential therapeutic implications, MAGL is considered as a promising target for drug design and the discovery of novel small-molecule MAGL inhibitors is of great interest in the medicinal chemistry ﬁeld. In this context, we developed a pharmacophore-based virtual screening protocol combined with molecular docking and molecular dynamics simulations, which showed a ﬁnal hit rate of 50% validating the reliability of the in silico workﬂow and led to the identiﬁcation of two promising and structurally different reversible MAGL inhibitors, VS1 and VS2. These ligands represent a valuable starting point for structure-based hit-optimization studies aimed at identifying new potent MAGL inhibitors. molecules molecules 1. Introduction The endocannabinoid system (ECS) is composed of the two cannabinoid recep- tors CB1 and CB2, the endogenous cannabinoid ligands (endocannabinoids, eCBs) N- arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), and the enzymes involved in endocannabinoid synthesis and degradation [1]. 2-AG is broadly considered as a major eCB, as it is about 100–1000 fold more abundant than AEA in the brain and peripheral organs [2]. 2-AG fully activates CB1 and CB2 receptors as an agonist, while AEA acts as partial agonist for both receptors [3]. The cannabinoid ligands AEA and 2-AG are synthesized on-demand from phospholipid precursors of the plasma membrane and released into the extracellular environment via facilitated diffusion. Fatty acid amide hydrolase (FAAH) is the enzyme that principally accounts for intracellular degradation of AEA, whereas monoacylglycerol lipase (MAGL) and α/β hydrolase-6 and -12 (ABHD6 and ABHD12) are involved in the intracellular degradation of 2-AG [4]. MAGL is respon- sible for the hydrolysis of about 85% of 2-AG in the brain and it is thus considered as the main hydrolytic enzyme of 2-AG [5,6]. MAGL-catalyzed hydrolysis of 2-AG leads to the formation of arachidonic acid (AA), which constitutes the precursor of eicosanoids that promote neuroinﬂammation and are involved in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease [7]. The modulation of the 2-AG levels obtained via MAGL inhibition is considered as a promising pharmacological strategy to activate the ECS without the typical side effects associated with direct CB1 receptor agonists. Furthermore, MAGL plays a key role in the development and spread of cancer, as it was found to be upregulated in aggressive cancer cells and primary tumors. MAGL-mediated hydrolysis of Received: 7 December 2020 Accepted: 25 December 2020 Published: 26 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional afﬁliations. Copyright: © 2020 by the authors. Li- censee 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/molecules Molecules 2021, 26, 78. https://dx.doi.org/10.3390/molecules26010078 Molecules 2021, 26, 78 2 of 13 monoacylglycerols in adipose tissue and liver leads to the production of free fatty acids that are used by rapidly growing tumor cells as building blocks for the formation of new cellular membranes and for the synthesis of pro-tumorigenic signaling factors [8]. 1. Introduction Due to the broad-spectrum of its therapeutic implications, MAGL has drawn increased attention as a promising drug target [9] and the therapeutic potential of selective MAGL inhibitors has been highlighted in animal models of inﬂammation, pain, anxiety and other neuroinﬂam- matory diseases [10,11]. A signiﬁcant number of MAGL inhibitors have been reported till date; however, most of them are characterized as irreversible inhibitors that form a covalent bond with MAGL. Several studies suggested that the genetic deletion or chronic inhibition of MAGL by irreversible MAGL inhibitors results in desensitization of CB1 receptors and ultimately impairs the beneﬁts associated to the indirect CB1 stimulation [12,13]. Therefore, the identiﬁcation of reversible MAGL inhibitors represents the basis for the development of an alternative promising therapeutic approach able to circumvent the drawbacks associated with irreversible MAGL inhibitors. In the last decades, many academic research groups and pharmaceutical industries have focused their research on the discovery of new MAGL inhibitors [14–18]. In this context, we developed a pharmacophore-based virtual screening (VS) protocol aimed at the identiﬁcation of new reversible MAGL inhibitors, which was based on the latest X-ray structure of MAGL in complex with a reversible non-covalent inhibitor. Our vs. study, which combined pharmacophore screening, molecular docking with multiple approaches and MD simulations led to the discovery of two novel compounds endowed with micromolar inhibitory activity against MAGL, thus conﬁrming the reliability of the vs. workﬂow and providing a starting point for future structure-based hit optimization studies aimed at designing new and more potent MAGL inhibitors. 2. Results The latest X-ray structure of human MAGL in complex with the piperazinyl-pyrrolidine 3l reversible inhibitor (Figure 1, PDB code: 5ZUN) [16] was used as a reference in the present study. The X-ray complex shows that the co-crystallized inhibitor forms key H- bond and hydrophobic interactions within MAGL binding site (Figure 1). The piperazine- carbonyl-thiazole portion of the inhibitor nicely occupies the bottom of the receptor pocket, while the biphenyl-pyrrolidin-2-one moiety resides at the entrance of the hydrophobic tunnel. The pyrrolidine carbonyl of the co-crystallized inhibitor establishes H-bond inter- actions with the backbone nitrogen of A51 and M123, within the oxyanion hole of MAGL catalytic site, that are considered as fundamental for MAGL inhibitory activity. Further- more, the carbonyl group linked to the thiazole ring of the ligand forms an H-bond with R57; notably, the same carbonyl group also shows an interaction with a structural water molecule that forms an H-bond network with E53 and H272. These water-bridged H-bonds between the co-crystallized inhibitor and the two residues are supposed to contribute to the strong binding afﬁnity for the target. A signiﬁcant number of hydrophobic interactions were also noted between the co-crystallized ligand and the enzyme binding site. The phenyl ring of the inhibitor is encircled by the sidechains of A51, I179, L213 and L241, establishing strong van der Waals contacts with these residues. The terminal o-chlorophenyl ring of the inhibitor forms hydrophobic contacts with I179 and L205, while the electron-rich chlorine was found to be in proximity to I179, L205 and L213, thus forming additional van der Waals interactions. Finally, the thiazole ring of the inhibitor forms a face-to-face π-π stacking with Y194 that contributes to the ligand inhibitory activity. g y y After analyzing the main protein-ligand interactions in the enzyme binding site, as a ﬁrst step of our study, the X-ray structure of MAGL in complex with its piperazinyl- pyrrolidine inhibitor was subjected to a preliminary MD simulation of 20 ns in order to evaluate the stability of the above-mentioned H-bond interactions, with particular attention to the H-bond network mediated by the structural water molecule. In fact, the H-bond interaction between the thiazole carbonyl and the structural water was believed to be of remarkable importance, due to the presence of this water molecule in various MAGL X-ray Molecules 2021, 26, 78 3 of 13 3 of 13 structures [19]. 2. Results Therefore, we envisioned that targeting the structural water mediating the H-bond network with E53 and H272 could provide a novel strategy for the development of potent and reversible MAGL inhibitors. The MD analysis highlighted a remarkable stability of the ligand disposition throughout the simulation, possessing an average RMSD of 0.4 Å with respect to the starting coordinates and thus well maintaining the hydrophobic and van der Waals interactions identiﬁed in the X-ray complex. Similarly, the structural water was found to maintain its disposition, with an average RMSD of 0.5 Å during the MD (Figure S1). The H-bond interactions of the ligand with A51 and M123 were maintained for more than 85% of the MD, whereas the interactions with R57 and the structural water molecule were observed for about 70% of the MD. EVIEW 3 of 14 Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand-binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines. Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand- binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines. Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand-binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines. Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand- binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines. Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). 2. Results The three optional features of the pharmacophore included three additional hydrophobic features representing: (a) the chlorine of the co-crystallized inhibitor forming van der Waals contacts with I179, L205 and L213, (b) the terminal phenyl ring showing hydrophobic contacts with I179 and L205, (c) the ligand thiazole ring exhibiting face-to-face π-π stacking with Y194 (Figure 2). feature representing the phenyl ring of the ligand involved in van der Waals contacts with A51, I179, L213 and L241 residues, was considered as the fifth mandatory feature. The three optional features of the pharmacophore included three additional hydrophobic features representing: (a) the chlorine of the co-crystallized inhibitor forming van der Waals contacts with I179, L205 and L213, (b) the terminal phenyl ring showing hydrophobic contacts with I179 and L205, (c) the ligand thiazole ring exhibiting face-to- face π-π stacking with Y194 (Figure 2). I179, L213 and L241 residues, was considered as the ﬁfth mandatory feature. The three optional features of the pharmacophore included three additional hydrophobic features representing: (a) the chlorine of the co-crystallized inhibitor forming van der Waals contacts with I179, L205 and L213, (b) the terminal phenyl ring showing hydrophobic contacts with I179 and L205, (c) the ligand thiazole ring exhibiting face-to-face π-π stacking with Y194 (Figure 2). feature representing the phenyl ring of the ligand involved in van der Waals contacts with A51, I179, L213 and L241 residues, was considered as the fifth mandatory feature. The three optional features of the pharmacophore included three additional hydrophobic features representing: (a) the chlorine of the co-crystallized inhibitor forming van der Waals contacts with I179, L205 and L213, (b) the terminal phenyl ring showing hydrophobic contacts with I179 and L205, (c) the ligand thiazole ring exhibiting face-to- face π-π stacking with Y194 (Figure 2). Figure 2. Receptor-based pharmacophore model with mandatory features 1–5 and optional features 6–8 superimposed with MAGL inhibitor 3l in the X-ray complex. Figure 2. Receptor-based pharmacophore model with mandatory features 1–5 and optional features 6–8 superimposed with MAGL inhibitor 3l in the X-ray complex. Figure 2. Receptor-based pharmacophore model with mandatory features 1–5 and optional features 6–8 superimposed with MAGL inhibitor 3l in the X-ray complex. Figure 2. Receptor-based pharmacophore model with mandatory features 1–5 and optional features 6–8 superimposed with MAGL inhibitor 3l in the X-ray complex. 2. Results The pharmacophore model was screened against a combination of four commercial databases (i.e., Enamine, ChemBridge, Pharmeks and Vitas-M) that comprise around 4 million compounds. The pharmacophore screening step allowed to select a large set of diverse compounds showing the desirable protein-ligand interactions in the MAGL binding site, which were ranked according to the number of satisfied features. In total, 276,150 compounds matched at least the 5 mandatory features of the pharmacophore, while 5707 molecules showed to satisfy all 8 features of the model (Table 1). Given the large number of pharmacophore hits identified, only those matching at least seven features were selected for the subsequent stage of the vs. workflow. The pharmacophore model was screened against a combination of four commercial databases (i.e., Enamine, ChemBridge, Pharmeks and Vitas-M) that comprise around 4 million compounds. The pharmacophore screening step allowed to select a large set of diverse compounds showing the desirable protein-ligand interactions in the MAGL binding site, which were ranked according to the number of satisﬁed features. In total, 276,150 compounds matched at least the 5 mandatory features of the pharmacophore, while 5707 molecules showed to satisfy all 8 features of the model (Table 1). Given the large number of pharmacophore hits identiﬁed, only those matching at least seven features were selected for the subsequent stage of the vs. workﬂow. Table 1. Results of the receptor-based pharmacophore screening. Number of Matched Features Number of Compounds 8 5707 7 19,314 6 182,236 5 276,150 Table 1. Results of the receptor-based pharmacophore screening. Number of Matched Features Number of Compounds 8 5707 7 19,314 6 182,236 5 276,150 Table 1. Results of the receptor-based pharmacophore screening. Table 1. Results of the receptor-based pharmacophore screening. The next step was the validation of the docking procedures, which involved the assessment of various programs to identify the methods able to reproduce the binding orientation of the X-ray ligand. The co-crystallized inhibitor 3l was thus subjected to self- docking studies using 13 docking methods. The root mean-square deviation (RMSD) between the position of the co-crystallized ligand predicted by docking and its The next step was the validation of the docking procedures, which involved the assessment of various programs to identify the methods able to reproduce the binding orientation of the X-ray ligand. The co-crystallized inhibitor 3l was thus subjected to self-docking studies using 13 docking methods. 2. Results (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand-binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines Figure 1. Piperazinyl-pyrrolidine inhibitor 3l in the MAGL binding site (PDB code: 5ZUN). (A) The inner surface of the protein surrounding the ligand (green sticks), displaying the shape of the ligand- binding cavity, is shown in gray. (B) The protein residues surrounding the ligand, constituting the binding site, are show in blue sticks, while hydrogen bonds are shown as black dashed lines. After analyzing the main protein-ligand interactions in the enzyme binding site, as a first step of our study, the X-ray structure of MAGL in complex with its piperazinyl- pyrrolidine inhibitor was subjected to a preliminary MD simulation of 20 ns in order to evaluate the stability of the above-mentioned H-bond interactions, with particular attention to the H-bond network mediated by the structural water molecule. In fact, the H-bond interaction between the thiazole carbonyl and the structural water was believed to be of remarkable importance, due to the presence of this water molecule in various MAGL X-ray structures [19]. Therefore, we envisioned that targeting the structural water di ti th H b d t k ith E53 d H272 ld id l t t f th The MD simulation thus conﬁrmed the stability of all the interactions observed in the reference ligand-protein X-ray structure, which were subsequently exploited to develop a pharmacophore model. A receptor-based pharmacophore model was constructed using Ligandscout program [20], considering both ligand-protein interactions and the excluded volume of the binding pocket. A total of eight pharmacophore features were considered, including ﬁve mandatory and three optional features (Figure 2). The four H-bond acceptor features corresponding to the interactions of both ligand’s carbonyl groups with A51, M123, R57 and the structural water (Figure 1) were set as mandatory. A hydrophobic feature representing the phenyl ring of the ligand involved in van der Waals contacts with A51, Molecules 2021, 26, 78 4 of 13 cceptor h A51 I179, L213 and L241 residues, was considered as the ﬁfth mandatory feature. 2. Results The root mean-square deviation (RMSD) between the position of the co-crystallized ligand predicted by docking and its experimental disposition was calculated. The comprehensive results of preliminary self-docking studies on 5ZUN X-ray structure are shown in Table 2: 9 out of 13 docking methods showed good results, with RMSD values below 2.0 Å. The docking solutions were also visually checked to conﬁrm that the fundamental ligand-protein interactions with A51, R57, M123 and the structural water molecule within MAGL binding pocket were maintained. After this analysis, only 6 docking methods Molecules 2021, 26, 78 5 of 13 (i.e., Dock6, Glamdock, Glide SP and Gold with ASP, PLP and Goldscore ﬁtness func- tions) were selected for the next steps, since only the binding poses produced with these procedures showed all fundamental ligand-protein interactions. The 19,314 compounds retrieved from the pharmacophore screening were eventually docked in the X-ray structure of MAGL using the six selected docking methods. The top ranked docking solutions pro- duced for each ligand by each docking method were subjected to a pose ﬁltering analysis in search for compounds maintaining the interactions represented by the pharmacophore model. In particular, only compounds forming the H-bonds with A51, R57, M123 and the structural water molecule and respecting at least two out of the four hydrophobic features, where retained. By intersecting the six different subsets of compounds obtained by ﬁltering the results of the six different docking methods, we identiﬁed 113 compounds showing the desired interactions in the binding poses predicted by all methods. Finally, these ligands were subjected to a pose consensus analysis in which the six docking results obtained for each compound were clustered together in search for common binding modes [21,22]. Only the 40 compounds for which a full pose consensus among all six docking procedures was observed (i.e., compounds for which all predicted binding modes had reciprocal RMSDs below 2.0 Å) were selected and considered as potential MAGL inhibitors. The selected compounds were then subjected to 20 ns of MD simulations with the same parameters and conditions as used in the preliminary MD study of the reference X-ray complex. The Gold Goldscore pose of each ligand was used as the starting ligand disposition within MAGL binding site, since Goldscore procedure showed the best RMSD value in the preliminary self-docking analysis. 2. Results The trajectories of the 40 protein-ligand complexes were analyzed in terms of H-bond stability and ligand RMSD with respect to the starting coordinates. The compounds that showed an average RMSD value lower than 2.0 Å and the maintenance of the H-bonds with R57, the structural water molecule and at least A51 or M123 for more than 70% of the simulation were retained. Out of the 40 analyzed compounds, seven ligands were ultimately selected by the MD ﬁlter and thus subjected to enzyme inhibition assays in order to test their inhibitory activity against MAGL (see Materials and methods for details). As shown in Table 3, four compounds out of the seven tested showed a MAGL inhibition activity; considering the high similarity between VS2 and VS4, the vs. study results in a hit rate of 50% with the two best MAGL inhibitors (i.e., VS1 and VS2) with an IC50 of 34.7 µM and 48.9 µM. A similarity search for compounds VS1 and VS2 against the already published hydrolase inhibitors revealed that there are no hydrolase inhibitors with a similarity score greater than 80 (score = 100 means that the two compounds are identical). Table 2. Preliminary self-docking analyses on 5ZUN X-ray structure. Docking Method RMSD (Å) Autodock 4.2 1.5 Dock6 0.5 Fred 3.8 Glamdock 1.3 Glide SP 0.6 Glide XP 1.5 Gold ASP 1.4 Gold Chemscore 3.1 Gold Goldscore 0.6 Gold PLP 0.7 Plants 3.2 rDock 1.6 Vina 9.4 Table 2. Preliminary self-docking analyses on 5ZUN X-ray structure. Table 2. Preliminary self-docking analyses on 5ZUN X-ray structure. Molecules 2021, 26, 78 6 of 13 Table 3. Structure and MAGL inhibition activity of the tested compounds. Compound 17b [19] and compound 3l [16] were used as reference compounds. 2. Results # Structure IC50 (µM) # Structure IC50 (µM) VS1 34.7 ± 2.4 7 of 21 9 of 21 IC50 (µM) # Structure IC50 (µM) VS1 34.7 ± 2.4 7 of 21 9 of 21 # Structure IC50 (µM) VS1 VS1 34.7 ± 2.4 34.7 ± 2.4 VS2 W 7 of 21 VS2 48.9 ± 4.2 48.9 ± 4.2 VS3 EW 8 of 21 VS3 79.4 ± 6.3 79.4 ± 6.3 VS4 W 9 of 21 VS4 138 ± 11 138 ± 11 VS5 VS5 >200 >200 VS6 W 11 of 21 >200 VS7 W 12 of 21 VS7 >200 >200 VS1 VS2 48.9 ± 4.2 W 8 of 21 12 of 21 34.7 ± 2.4 79.4 ± 6.3 3 VS5 VS6 7 of 13 Molecules 2021, 26, 78 Table 3. Cont. Table 3. Cont. # Structure IC50 (µM) 17b 17b 0.90 ± 0.08 0.90 ± 0.08 3l 3l 0.042 ± 0.001 0.042 ± 0.001 REVIEW 7 of 14 17b 0.90 ± 0.08 3l 0.042 ± 0.001 3l 0.042 ± 0.001 3l 0.042 ± Figure 3 shows the minimized average structure of compound VS1 in complex with GL obtained by the last 10 ns of MD simulation. The sulphonamide moiety of VS1 plays multiple H-bond interactions in the oxyanion hole of MAGL binding site. The onamide oxygen of the ligand forms an H-bond with the backbone nitrogen of A51 erved for 82% of the simulation. Although the interaction with the backbone nitrogen M123 was lost during the MD, the sulphonamide nitrogen of the ligand establishes an itional stable H-bond with the backbone oxygen of A51 maintained for 97% of the MD, ch effectively anchors the ligand core to the protein binding site. The carbonyl oxygen he ligand pyrimidinone moiety forms an H-bond interaction with R57 maintained for % of the simulation and an H-bond network with the structural water molecule, E53 H272 observed for 94% of the MD. Moreover, the pyrimidinone nitrogen forms an itional direct H-bond interaction with E53 maintained throughout the whole ulation. Finally, the o-chloro-p-fluorophenyl ring of VS1 shows van der Waals contacts h L205, L213, L241 and A51, while a face-to-face π-π stacking is observed between the imidinone ring of VS1 and Y194. 2. Results These hydrophobic interactions, which well mimic se identified in the reference X ray structure most likely contribute to the binding Figure 3 shows the minimized average structure of compound VS1 in complex with MAGL obtained by the last 10 ns of MD simulation. The sulphonamide moiety of VS1 displays multiple H-bond interactions in the oxyanion hole of MAGL binding site. The sulfonamide oxygen of the ligand forms an H-bond with the backbone nitrogen of A51 observed for 82% of the simulation. Although the interaction with the backbone nitrogen of M123 was lost during the MD, the sulphonamide nitrogen of the ligand establishes an additional stable H-bond with the backbone oxygen of A51 maintained for 97% of the MD, which effectively anchors the ligand core to the protein binding site. The carbonyl oxygen of the ligand pyrimidinone moiety forms an H-bond interaction with R57 maintained for 98% of the simulation and an H-bond network with the structural water molecule, E53 and H272 observed for 94% of the MD. Moreover, the pyrimidinone nitrogen forms an additional direct H-bond interaction with E53 maintained throughout the whole simulation. Finally, the o-chloro-p-ﬂuorophenyl ring of VS1 shows van der Waals contacts with L205, L213, L241 and A51, while a face-to-face π-π stacking is observed between the pyrimidinone ring of VS1 and Y194. These hydrophobic interactions, which well mimic those identiﬁed in the reference X-ray structure, most likely contribute to the binding afﬁnity of the ligand for MAGL. Figure 3 shows the minimized average structure of compound VS1 in complex with MAGL obtained by the last 10 ns of MD simulation. The sulphonamide moiety of VS1 displays multiple H-bond interactions in the oxyanion hole of MAGL binding site. The sulfonamide oxygen of the ligand forms an H-bond with the backbone nitrogen of A51 observed for 82% of the simulation. Although the interaction with the backbone nitrogen of M123 was lost during the MD, the sulphonamide nitrogen of the ligand establishes an additional stable H-bond with the backbone oxygen of A51 maintained for 97% of the MD, which effectively anchors the ligand core to the protein binding site. The carbonyl oxygen of the ligand pyrimidinone moiety forms an H-bond interaction with R57 maintained for 98% of the simulation and an H-bond network with the structural water molecule, E53 and H272 observed for 94% of the MD. 2. Results The m-chlorophenyl ring of the inhibitor forms van der Waals contacts with L213, L241 residues while the furan ring shows a π-π stacking with Y194 (Figure 4). The possible explanation behind the lower IC50 value of VS1 as compared to VS2, could be ascribed to the presence of a direct H- bond between VS1 and the carboxyl group of E53, which is instead absent in the binding mode predicted for VS2. Compounds VS3 and VS4 show a predicted pattern of ligand- protein interactions like that described for VS2. However, both VS3 and VS4 show only a single H-bond with the residues of the oxyanion hole (Figure S2), which can probably explain their lower inhibitory activity with respect to VS2. Figure 4. Minimized average structure of VS2 within MAGL binding site. Figure 4. Minimized average structure of VS2 within MAGL binding site. Figure 4. Minimized average structure of VS2 within MAGL binding site. Figure 4. Minimized average structure of VS2 within MAGL binding site. In order to verify whether the most promising compounds could interact with cysteine residues of MAGL enzyme, the activity of the two most potent inhibitors VS1 and VS2 was also tested in presence of the thiol-containing agent 1,4-dithio-D,L-threitol (DTT). As shown in Figure 5, the IC50 value of the two compounds was only very slightly, but not significantly, influenced by the presence of DTT, shifting for VS1 from 35.4 µM when assayed in the absence of DTT to 34.1 µM when assayed in the presence of 10 µM DTT and for VS2 from 47.5 µM to 49.5 µM, thus excluding any significant interaction of the two ligands with MAGL cysteine residues. Furthermore, with the aim of establishing whether the mechanism of inhibition was reversible or irreversible, the effects of preincubation on the inhibitory activity of compounds VS1 and VS2 were evaluated. In this assay, the compounds were preincubated with the enzyme for 0, 30 and 60 min before adding the substrate to start the enzymatic reaction. An irreversible inhibitor should show a higher potency after longer incubation times, whereas a reversible inhibitor should display a constant inhibition potency that is independent from the incubation time. As shown in Figure 5, this assay confirmed the reversible character of both compounds, as they did not show any significant increase in inhibitory potency at longer incubation times. 2. Results Moreover, the pyrimidinone nitrogen forms an additional direct H-bond interaction with E53 maintained throughout the whole simulation. Finally, the o-chloro-p-fluorophenyl ring of VS1 shows van der Waals contacts with L205, L213, L241 and A51, while a face-to-face π-π stacking is observed between the pyrimidinone ring of VS1 and Y194. These hydrophobic interactions, which well mimic those identified in the reference X-ray structure, most likely contribute to the binding affinity of the ligand for MAGL. y y inity of the ligand for MAGL. Figure 3. Minimized average structure of VS1 within MAGL binding site. Figure 3. Minimized average structure of VS1 within MAGL binding site. Figure 3. Minimized average structure of VS1 within MAGL binding site. Figure 3. Minimized average structure of VS1 within MAGL binding site. Molecules 2021, 26, 78 8 of 13 Besides VS1, the hit compound VS2 showed a promising MAGL inhibitory activity with an IC50 below 50 µM and also VS3 and VS4 demonstrated MAGL inhibition with IC50 values of 79.4 µM and 138 µM, respectively (Table 3). As shown in Figure 4, the carbonyl group of VS2 core ring establishes both H-bonds with A51 and M123, maintained for 95% and 74%, respectively, during the simulation. The furan carbonyl of VS2 is involved in H-bond interactions with R57 and the structural water molecule observed for more than 80% of the simulation, whereas the water-bridged interactions with E53 and H272 were maintained for about 70% of the MD. The m-chlorophenyl ring of the inhibitor forms van der Waals contacts with L213, L241 residues while the furan ring shows a π-π stacking with Y194 (Figure 4). The possible explanation behind the lower IC50 value of VS1 as compared to VS2, could be ascribed to the presence of a direct H-bond between VS1 and the carboxyl group of E53, which is instead absent in the binding mode predicted for VS2. Compounds VS3 and VS4 show a predicted pattern of ligand-protein interactions like that described for VS2. However, both VS3 and VS4 show only a single H-bond with the residues of the oxyanion hole (Figure S2), which can probably explain their lower inhibitory activity with respect to VS2. VIEW 8 of 14 VS2 is involved in H-bond interactions with R57 and the structural water molecule observed for more than 80% of the simulation, whereas the water-bridged interactions with E53 and H272 were maintained for about 70% of the MD. ions 3. Conclusions armacophore-based vs. protocol was developed and performed to identify new hibitors. The key protein-ligand interactions detected from the latest X-ray of MAGL in complex with a non-covalent piperazinyl-pyrrolidine inhibitor oited to build a receptor-based pharmacophore model. In particular, an H-bond n with a structural water molecule was taken into consideration, as it was o contribute to the binding affinity of the reference MAGL inhibitor. In fact, GL X-ray structures present a water molecule located in the polar portion of nding site that forms a network of interactions with E53 and H272. These two emonstrated to represent effective anchoring points for MAGL ligands, since ds predicted to displace the structural water molecule and form direct ns with E53 and H272, such as our reference compound 17b, showed nanomolar hibitory activity. However, compound 3l, the co-crystallized ligand in the hibitor X-ray complex used as a reference for our vs. protocol, displayed ns with E53 and H272 through an H-bond network mediated by the structural ecule and showed a very high MAGL inhibitory potency. For this reason, we d that such water molecule could be considered in our vs. in order to search for ential ligands that could exploit the structural water as an indirect anchoring MAGL binding site. The whole vs. workflow, comprising pharmacophore multiple parallel molecular docking studies and a final MD simulation analysis selection of seven final compounds that were tested for MAGL inhibition Out of the seven selected molecules, two compounds showed a promising potency (with IC50 below 50 µM) and two additional compounds demonstrated hibition activity in the micromolar range. The positive outcomes from this in y provided validation of the reliability of the vs. workflow, which may be plied to screen additional commercial databases for identifying new MAGL particular, the obtained vs. result confirmed that targeting the structural water within MAGL binding site, accounting for the H-bond network with E53 and resents a profitable strategy for identifying novel hit compounds. The water- H-bonds with E53 and H272 were confirmed to represent key MAGL-anchoring ns that should be considered in structure-based drug design campaigns aimed ping potent MAGL inhibitors. In this context, despite VS1 and VS2 are zed by a micromolar MAGL inhibition, the discovery of these two structurally A pharmacophore-based vs. protocol was developed and performed to identify new MAGL inhibitors. 2. Results Taken together, these experimental studies, aimed at better characterizing the mode of action of compounds VS1 and VS2 further supported the reliability of the binding In order to verify whether the most promising compounds could interact with cysteine residues of MAGL enzyme, the activity of the two most potent inhibitors VS1 and VS2 was also tested in presence of the thiol-containing agent 1,4-dithio-D,L-threitol (DTT). As shown in Figure 5, the IC50 value of the two compounds was only very slightly, but not signiﬁcantly, inﬂuenced by the presence of DTT, shifting for VS1 from 35.4 µM when assayed in the absence of DTT to 34.1 µM when assayed in the presence of 10 µM DTT and for VS2 from 47.5 µM to 49.5 µM, thus excluding any signiﬁcant interaction of the two ligands with MAGL cysteine residues. Furthermore, with the aim of establishing whether the mechanism of inhibition was reversible or irreversible, the effects of preincubation on the inhibitory activity of compounds VS1 and VS2 were evaluated. In this assay, the compounds were preincubated with the enzyme for 0, 30 and 60 min before adding the substrate to start the enzymatic reaction. An irreversible inhibitor should show a higher potency after longer incubation times, whereas a reversible inhibitor should display a constant inhibition potency that is independent from the incubation time. As shown in Figure 5, this assay conﬁrmed the reversible character of both compounds, as they did not show any signiﬁcant increase in inhibitory potency at longer incubation times. Taken together, these experimental studies, aimed at better characterizing the mode of action of Molecules 2021, 26, 78 9 of 13 compounds VS1 and VS2, further supported the reliability of the binding mode predicted for the two ligands. 9 of 14 compounds VS1 and VS2, further supported the reliability of the binding mode predict for the two ligands. Figure 5. Compound VS1- and VS2-MAGL inhibition analysis. (A) Effect of DTT on MAGL inhibition activity. (B) IC50 (µM) values of VS1 and VS2 at different preincubation times with MAGL (0 min, 30 min and 60 min). Figure 5. Compound VS1- and VS2-MAGL inhibition analysis. (A) Effect of DTT on MAGL inhibition activity. (B) IC50 (µM) values of VS1 and VS2 at different preincubation times with MAGL (0 min, 30 min and 60 min). Figure 5. Compound VS1- and VS2-MAGL inhibition analysis. (A) Effect of DTT on MAGL inhibition activity. 2. Results (B) IC50 (µM) values of VS1 and VS2 at different preincubation times with MAGL (0 min, 30 min and 60 min). Figure 5. Compound VS1- and VS2-MAGL inhibition analysis. (A) Effect of DTT on MAGL inhibition activity. (B) IC50 (µM) values of VS1 and VS2 at different preincubation times with MAGL (0 min, 30 min and 60 min). 4.2. Database Generation and Pharmacophore Screening 4.2. Database Generation and Pharmacophore Screening Approximately 4 million compounds from the commercial Enamine, Vitas-M, Chem- Bridge and Pharmeks databases were used as the screening database. The software iCon [23] implemented in LigandScout was used to carry out ligand conformational sam- pling and to set up the screening database. The generated pharmacophore model including ﬁve mandatory features, three optional features and the excluded volume spheres was used to screen the generated screening database and to search for desirable compounds. Only compounds matching at least ﬁve mandatory features of the model and respecting the excluded volume constraints were retrieved in this search. ions 3. Conclusions The key protein-ligand interactions detected from the latest X- ray structure of MAGL in complex with a non-covalent piperazinyl-pyrrolidine inhibitor were exploited to build a receptor-based pharmacophore model. In particular, an H- bond interaction with a structural water molecule was taken into consideration, as it was believed to contribute to the binding afﬁnity of the reference MAGL inhibitor. In fact, many MAGL X-ray structures present a water molecule located in the polar portion of MAGL binding site that forms a network of interactions with E53 and H272. These two residues demonstrated to represent effective anchoring points for MAGL ligands, since compounds predicted to displace the structural water molecule and form direct interactions with E53 and H272, such as our reference compound 17b, showed nanomolar MAGL inhibitory activity. However, compound 3l, the co-crystallized ligand in the MAGL-inhibitor X-ray complex used as a reference for our vs. protocol, displayed interactions with E53 and H272 through an H-bond network mediated by the structural water molecule and showed a very high MAGL inhibitory potency. For this reason, we envisioned that such water molecule could be considered in our vs. in order to search for novel potential ligands that could exploit the structural water as an indirect anchoring point to MAGL binding site. The whole vs. workﬂow, comprising pharmacophore screening, multiple parallel molecular docking studies and a ﬁnal MD simulation analysis led to the selection of seven ﬁnal compounds that were tested for MAGL inhibition activity. Out of the seven selected molecules, two compounds showed a promising inhibitory potency (with IC50 below 50 µM) and two additional compounds demonstrated MAGL inhibition activity in the micromolar range. The positive outcomes from this in silico study provided validation of the reliability of the vs. workﬂow, which may be further applied to screen additional commercial databases for identifying new MAGL ligands. In particular, the obtained vs. result conﬁrmed that targeting the structural water molecule within MAGL binding site, accounting for the H-bond network with E53 and H272, represents a proﬁtable strategy for identifying novel hit compounds. The water-mediated H-bonds with E53 and H272 were conﬁrmed to represent key MAGL-anchoring interactions that should be considered in structure-based drug design campaigns aimed at developing potent MAGL inhibitors. 4.1. Pharmacophore Model Generation The pharmacophore model was constructed using LigandScout 4.2 [20]. The pharma- cophore hypothesis was generated from the X-ray structure of human MAGL in complex with piperazinyl-pyrrolidine inhibitor (PDB code: 5ZUN) [16]. A complete model that represents all the possible pharmacophore features of the ligand, including the interaction with the structural water molecule, recognized by the program was generated; all the features were taken into consideration in the ﬁnal pharmacophore model, for a total of four H-bond acceptor features and four hydrophobic features. Of these, ﬁve pharmacophore features (four H-bond and a hydrophobic feature) were set as mandatory and the other three (hydrophobic) features were set as optional. The excluded volume spheres that resemble regions of space in the proximity of the pharmacophore model occupied by the enzyme, were also taken into consideration for the pharmacophore model development. 4.3. Molecular Docking and Pose Filtering All docking calculations were carried out using the X-ray structure of human MAGL in complex with piperazinyl-pyrrolidine inhibitor (PDB code: 5ZUN) [16] already employed for pharmacophore modeling. In total, 13 different docking procedures were used in this study: Autodock 4.2.3, Autodock Vina 1.1, Dock 6.7, Fred 3.0, GlamDock, Gold 5.1 with its four ﬁtness functions (i.e., ChemScore, GoldScore, ChemPLP and Astex Statistical Potential), Glide 5.0 with standard precision (SP) and extra precision (XP) methods, Plants and rDock, following the procedures described in our previous studies [21,24]. A self-docking analysis for each docking method was performed by calculating the root-mean-square deviation (RMSD) between the position of the crystallographic ligand predicted by docking and its known experimental disposition, using the rms_analysis software of Gold suite. The ﬁltering of the docking results was performed by superimposing the docked compounds to the pharmacophore model directly from the supplied poses, without changing their coordinates. The pose consensus analysis was performed using our in-house complete- linkage clustering procedure, as described in our precious studies [25]. ions 3. Conclusions In this context, despite VS1 and VS2 are characterized by a micromolar MAGL inhibition, the discovery of these two structurally unrelated molecules could serve as a starting point for 10 of 13 Molecules 2021, 26, 78 10 of 13 structure-based hit optimization studies aimed at designing new and more potent MAGL ligands. structure-based hit optimization studies aimed at designing new and more potent MAGL ligands. 4.4. Molecular Dynamics Simulations All simulations were carried out using Amber 16 [26]. General amber force ﬁeld (GAFF) parameters were assigned to the ligands, while partial charges were determined using the AM1-BCC method as implemented in the Antechamber suite of Amber 16. All ligand-protein complexes were placed in a rectangular parallelepiped water-box, by using TIP3P explicit solvent model and solvated with a 20.0 Å water cap. Either Na+ or Cl− ions were added as counterions in order to neutralize the systems. Before MD simulations, two stages of energy minimization were carried out; in the ﬁrst step, a position restraint of 100 kcal/(mol·Å2) was applied to the complex, thus minimizing only the position of the water molecules through 5000 steps of steepest descent followed by conjugate gradient, until a convergence of 0.05 kcal/(mol·Å2). Successively, the whole system was energy Molecules 2021, 26, 78 11 of 13 11 of 13 minimized imposing a harmonic force constant of 10 kcal/(mol·Å2) only on the protein α-carbons. The minimized complexes were used as starting conformations for the MD simulations. Periodic boundary conditions and particle mesh Ewald (PME) electrostatics were employed in the simulations. The time step of the simulations was 2 fs, a cut-off of 10.0 Å was set for the non-bonded interactions and SHAKE algorithm was used to keep all bonds involving hydrogen atoms rigid. Constant-volume MD simulation was performed for the ﬁrst 0.5 ns, during which the temperature of the system was raised from 0 to 300 K. The system was then equilibrated through 3 ns of constant pressure periodic boundary MD, employing the Langevin thermostat in order to keep the temperature of the system constant. At last, additional 16.5 ns of constant pressure MD production were performed. Thus, a total of 20 ns MD simulation was carried out for each protein-ligand complex analyzed in this study. In all MD steps the α-carbons of the protein were restrained with a harmonic force constant of 10 kcal/mol·Å2. All the obtained MD trajectories were analyzed using the cpptraj program implemented in Amber 16 [27]. 4.5. Similarity Search The test compounds were compared with published hydrolase inhibitors. This com- parison was made by using the Similarity Search task of SciFinder. Similarity search locates structures that are similar to the query, based on a two-dimensional small-molecule com- parison using a Tanimoto similarity metric. A score of 100 means that the two structures are identical. 4.6. Enzymatic Assays Compounds VS1–VS7 were bought from their source databases (ChemBridge, Enam- ine, Pharmeks and Vitas-M corporation). Human recombinant MAGL and 4-nitrophenyl acetate substrate (4-NPA) were bought from Cayman Chemical (Ann Arbor, MI, USA). The reversible compound 17b reported by Granchi et al. in 2016 was synthesized in our laboratory and used as reference compound [19]. Compound 3l was synthesized in our laboratory according to the procedure reported by Aida et al. [16] and used as reference compound. The IC50 values were produced in 96-well microtiter plates. The MAGL reac- tion was carried out at rt at a ﬁnal volume of 200 µL in 10 mM Tris buffer, pH 7.2, containing 1 mM EDTA and 0.1 mg/mL BSA. A total of 150 µL of 4-NPA 133.3 µM was added to 10 µL of DMSO containing the appropriate amount of compound. The reaction was started by adding 40 µL of MAGL (11 ng/well) in such a way that the assay was linear over 30 min. After the reaction had progressed for 30 min, absorbance values were measured by using a VictorX3 instrument (PerkinElmer, Waltham, MA, USA) at 405 nm. Two more reactions were carried out: one reaction containing no compounds and the second one containing neither enzyme nor inhibitor. To remove possible false positive results, a blank analysis was performed for each compound concentration and the ﬁnal absorbance results were obtained detracting the absorbance produced by the presence of all the components except MAGL in the same conditions [28]. Supplementary Materials: The following are available online, Figure S1: MD simulation analysis of the reference X-ray complex, Figure S2: Predicted binding mode of VS3 and VS4 within MAGL. Author Contributions: Conceptualization, T.T. and G.P.; methodology, T.T.; validation, M.D.S. and C.G.; investigation, V.J., M.B. and V.S.; resources, C.G.; writing—original draft preparation, V.J. and G.P.; writing—review and editing, F.M., M.M. and C.G.; supervision, T.T.; funding acquisition, C.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MIUR (PRIN 2017, project 2017SA5837) and the Italian Ministry of Health—Ricerca Finalizzata 2016—NET-2016-02363765. Funding: This research was funded by MIUR (PRIN 2017, project 2017SA5837) and the Italian Ministry of Health—Ricerca Finalizzata 2016—NET-2016-02363765. Funding: This research was funded by MIUR (PRIN 2017, project 2017SA5837) and the Italian Ministry of Health—Ricerca Finalizzata 2016—NET-2016-02363765. Data Availability Statement: All data comes from the author. Data Availability Statement: All data comes from the author. 4.6. Enzymatic Assays Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Sample Availability: Samples of the compounds are available from the authors. Sample Availability: Samples of the compounds are available from the authors. 12 of 13 Molecules 2021, 26, 78 References The Endocannabinoid System as an Emerging Target of Pharmacotherapy. Pharmacol. Rev. 2006 ef] 11. Deng, H.; Li, W. Monoacylglycerol lipase inhibitors: Modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders. Acta Pharm. Sin. B 2020, 10, 582–602. [CrossRef] [PubMed] 12. 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https://openalex.org/W3177326348	https://europepmc.org/articles/pmc8494431?pdf=render	English	null	Genome-wide homozygosity and risk of four non-Hodgkin lymphoma subtypes	null	2,021	cc-by	12,381	Abstract Author Manuscript This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, 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. Correspondence to: Sonja Berndt, Division of Cancer Epidemiology & Genetics, National Cancer Institute, NIH, 9609 Medical Center Drive, Room 6E610, MSC 9771, Rockville, MD 20850, USA. berndts@mail.nih.gov. HHS Public Access A h i Author Manuscript Published in final edited form as: J Transl Genet Genom. 2021 ; 5: 200–217. doi:10.20517/jtgg.2021.08. A full list of authors and affiliations appears at the end of the article. A full list of authors and affiliations appears at the end of the article. Abstract Authors’ contributions O ib d h d d i d l i M A M hi l MJ M h d M W SS K E Sl SL Oversaw or contributed to the study design and analysis: Moore A, Machiela MJ, Machado M, Wang SS, Kane E, Slager SL, Carrington MN, Lan Q, Rothman N, Cerhan JR, Dean M, Camp NJ, Yeager M, Berndt SI Performed or assisted in analyses or writing the initial draft of the manuscript: Moore A, Machiela MJ, Machado M, Wang SS, Kane E, Slager SL, Zhou W, Birmann BM, Milne RL, Cerhan JR, Dean M, Camp NJ, Yeager M, Berndt SI RL, Cerhan JR, Dean M, Camp NJ, Yeager M, Berndt SI Collected/contributed study data or samples: Wang SS, Kane E, Slager SL, Lan Q, Adami HO, Albanes D, Arslan AA, Becker N, Benavente Y, Birmann BM, Bisanzi S, Boffetta P, Bracci PM, Brennan P, Brooks-Wilson AR, Canzian F, Caporaso NE, Clavel J, Cocco P, Conde L, Cox DG, Cozen W, Curtin K, De Vivo I, de Sanjose S, Foretova L, Gapstur SM, Ghesquières H, Giles GG, Glenn M, Glimelius B, Gao C, Habermann TM, Hjalgrim H, Jackson RD, Liebow M, Link BK, Maynadie M, McKay J, Melbye M, Miligi L, Milne RL, Molina TJ, Monnereau A, Nieters A, North KE, Offit K, Patel AV, Piro S, Ravichandran V, Riboli E, Salles G, Severson RK, Skibola CF, Smedby KE, Southey MC, Spinelli JJ, Staines A, Stewart C, Teras LR, Tinker LF, Travis RC, Vajdic CM, Vermeulen RCH, Joseph V, Weiderpass E, Weinstein S, Wong Doo N, Zhang Y, Zheng T, Chanock SJ, Rothman N, Cerhan JR, Camp NJ, Berndt SI Author Manuscript Read, revised and/or provided feedback on the manuscript: Moore A, Machiela MJ, Machado M, Wang SS, Kane E, Slager SL, Zhou W, Carrington M, Lan Q, Milne RL, Birmann BM, Adami HO, Albanes D, Arslan AA, Becker N, Benavente Y, Bisanzi S, Boffetta P, Bracci PM, Brennan P, Brooks-Wilson AR, Canzian F, Caporaso N, Clavel J, Cocco P, Conde L, Cox DG, Cozen W, Curtin K, De Vivo I, de Sanjose S, Foretova L, Gapstur SM, Ghesquières H, Giles GG, Glenn M, Glimelius B, Gao C, Habermann TM, Hjalgrim H, Jackson RD, Liebow M, Link BK, Maynadie M, McKay J, Melbye M, Miligi L, Molina TJ, Monnereau A, Nieters A, North KE, Offit K, Patel AV, Piro S, Ravichandran V, Riboli E, Salles G, Severson RK, Skibola CF, Smedby KE, Southey MC, Spinelli JJ, Staines A, Stewart C, Teras LR, Tinker LF, Travis RC, Vajdic CM, Vermeulen RCH, Joseph V, Weiderpass E, Weinstein S, Wong Doo N, Zhang Y, Zheng T, Chanock SJ, Rothman N, Cerhan JR, Dean M, Camp NJ, Yeager M, Berndt SI Availability of data and materials Availability of data and materials NCI NHL GWAS data used in this manuscript has been deposited in the dbGaP repository with accession code phs000801.v2.p1. Abstract Other relevant data used in this study are available upon request. HL GWAS data used in this manuscript has been deposited in the dbGaP repository with accession code phs000801.v2.p1. elevant data used in this study are available upon request. All authors declare that there are no conflicts of interest. Ethical approval and consent to participate Author Manuscript All studies obtained informed consent from participants and were approved by their respective Institutional Review Boards as listed. These are ATBC: (NCI Special Studies Institutional Review Board); BCCA: UBC BC Cancer Agency Research Ethics Board; CPS-II: American Cancer Society; ELCCS: Northern and Yorkshire Research Ethics Committee; ENGELA: IRB00003888 - Comite d’ Evaluation Ethique de l’Inserm IRB # 1; EPIC: Imperial College London; EpiLymph: International Agency for Research on Cancer, HPFS: Harvard School of Public Health (HSPH) Institutional Review Board; Iowa-Mayo SPORE: University of Iowa Institutional Review Board; Italian GxE: Comitato Etico Azienda Ospedaliero Universitaria di Cagliari; Mayo Clinic Case-Control: Mayo Clinic Institutional Review Board; Mayo GEC: Mayo Clinic Institutional Review Board, #07-005788-03; MCCS: Cancer Council Victoria’s Human Research Ethics Committee; MD Anderson: University of Texas MD Anderson Cancer Center Institutional Review Board; MSKCC: Memorial Sloan-Kettering Cancer Center Institutional Review Board; NCI-SEER (NCI Special Studies Institutional Review Board); NHS: Partners Human Research Committee, Brigham and Women’s Hospital, NSW: NSW Cancer Council Ethics Committee; NYU-WHS: New York University School of Medicine Institutional Review Board; PLCO: (NCI Special Studies Institutional Review Board); SCALE: Scientific Ethics Committee for the Capital Region of Denmark; SCALE: Regional Ethical Review Board in Stockholm (Section 4) IRB#5; UCSF1/UCSF2: University of California San Francisco Committee on Human Research; UTAH: University of Utah; WHI: Fred Hutchinson Cancer Research Center; Yale: Human Investigation Committee, Yale University School of Medicine. Page 2 Page 2 Moore et al. Aim: Recessive genetic variation is thought to play a role in non-Hodgkin lymphoma (NHL) etiology. Runs of homozygosity (ROH), defined based on long, continuous segments of homozygous SNPs, can be used to estimate both measured and unmeasured recessive genetic variation. We sought to examine genome-wide homozygosity and NHL risk. Aim: Recessive genetic variation is thought to play a role in non-Hodgkin lymphoma (NHL) etiology. Runs of homozygosity (ROH), defined based on long, continuous segments of homozygous SNPs, can be used to estimate both measured and unmeasured recessive genetic variation. We sought to examine genome-wide homozygosity and NHL risk. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Abstract Author Manuscript Methods: We used data from eight genome-wide association studies of four common NHL subtypes: 3061 chronic lymphocytic leukemia (CLL), 3814 diffuse large B-cell lymphoma (DLBCL), 2784 follicular lymphoma (FL), and 808 marginal zone lymphoma (MZL) cases, as well as 9374 controls. We examined the effect of homozygous variation on risk by: (1) estimating the fraction of the autosome containing runs of homozygosity (FROH); (2) calculating an inbreeding coefficient derived from the correlation among uniting gametes (F3); and (3) examining specific autosomal regions containing ROH. For each, we calculated beta coefficients and standard errors using logistic regression and combined estimates across studies using random- effects meta-analysis. Author Manuscript Results: We discovered positive associations between FROH and CLL (β = 21.1, SE = 4.41, P = 1.6 × 10−6) and FL (β = 11.4, SE = 5.82, P = 0.02) but not DLBCL (P = 1.0) or MZL (P = 0.91). For F3, we observed an association with CLL (β = 27.5, SE = 6.51, P = 2.4 × 10−5). We did not find evidence of associations with specific ROH, suggesting that the associations observed with FROH and F3 for CLL and FL risk were not driven by a single region of homozygosity. Conclusion: Our findings support the role of recessive genetic variation in the etiology of CLL and FL; additional research is needed to identify the specific loci associated with NHL risk. Keywords Non-Hodgkin lymphoma; homozygosity; chronic lymphocytic leukemia; follicular lymphoma; diffuse large B-cell lymphoma; marginal zone lymphoma Non-Hodgkin lymphoma; homozygosity; chronic lymphocytic leukemia; follicular lymphoma; diffuse large B-cell lymphoma; marginal zone lymphoma Author Manuscript INTRODUCTION Disentangling the heritable component of non-Hodgkin lymphoma (NHL) and its subtypes is an active area of research. An early study of familial aggregation in NHL reported an increased risk of NHL among siblings, but not parents or offspring, of an index NHL case[1]. Several subsequent studies found an elevated risk of NHL associated with a first-degree family history of NHL with the highest risks for siblings[2,3]. Studies have also reported higher NHL subtype-specific risks for first-degree relatives of cases affected with a given NHL subtype[4,5], suggesting a degree of subtype specificity. In general, these findings suggest genetic factors are important in NHL etiology and, in particular, the potential role of recessively acting genetic risk alleles, but they also underscore the potential for genetic heterogeneity in susceptibility to different NHL subtypes. Part of the difficulty in characterizing risk and inheritance patterns of NHL subtypes is the limited study sample size, especially when examining specific subtypes. Genome-wide association studies (GWAS) have identified multiple susceptibility loci associated with four major subtypes of NHL[6-11], but a substantial fraction of the disease heritability remains unexplained. Most GWAS performed assume an additive model of genetic risk, which has statistical power to detect allelic associations acting through a variety of mechanisms but may not efficiently detect recessive effects, particularly as minor allele frequency and imputation Author Manuscript Page 3 Moore et al. quality decrease[12]. Therefore, recessively acting loci, particularly those with low minor allele frequency, could be missed by current genome-wide scans and represent potential novel disease-associated loci. Author Manuscript Aut Author Manuscript The widespread use of dense genotyping arrays has led to the identification of sizeable genomic regions consisting of consecutive homozygous SNPs in non-consanguineous populations[13]. These runs of homozygosity (ROH) vary in length, with short ROH persisting from ancient relatedness and long ROH of several megabases arising from recent parental relatedness[14]. The use of ROH as a measure of the burden of homozygosity has been demonstrated to perform better at identifying rare, recessive mutations than a conventional SNP-by-SNP analysis[15]. Furthermore, studies incorporating whole-exome sequencing have uncovered an enrichment of deleterious variants in ROH[16]. In recent years, studies have examined the association between ROH and various cancers[17-21], among other complex common diseases and traits[22]. Although no association was observed with the cumulative distribution of ROH, individual ROH were associated with the risk of childhood acute lymphoblastic leukemia[19]. INTRODUCTION Hodgkin lymphoma has been inconsistently associated with specific ROH and overall homozygosity[20,21]. To our knowledge, no studies have examined ROH in association with adult NHL. Author Manuscript The goal of the present study was to investigate the association of homozygosity with the risk of four major NHL subtypes: chronic lymphocytic leukemia/small chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Several measures of homozygosity were tested against NHL risk using data from eight GWAS. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. METHODS Author Manuscript Author Manuscript We used data from eight previous GWAS of NHL[6,8,10,11] composed of cases and controls of European ancestry [Supplementary Table 1 and 2]. The National Cancer Institute (NCI) NHL GWAS included cases with one of four common NHL subtypes and controls from 22 studies of NHL: 9 prospective cohort studies, 8 population-based case-control studies, and 5 hospital- or clinic-based case-control studies or case series. These 22 studies comprising the NCI NHL GWAS were genotyped using the Illumina OmniExpress or Omni2.5 arrays and analyzed as a single study. The other seven GWAS were the University of California at San Francisco Molecular Epidemiology of Non-Hodgkin Lymphoma study (UCSF2)[23], the University of California at San Francisco Molecular Epidemiology of Non-Hodgkin Lymphoma study (UCSF1) combined with controls from the Nurses’ Health Study (NHS)[24], the Scandinavian Lymphoma Etiology Study (SCALE)[25], the Groupe d’Etude des Lymphomes de l’Adulte (GELA) study combined with controls from the European Prospective Investigation into Cancer, Chronic Diseases, Nutrition, and Lifestyles (EPIC), the Mayo Clinic Case-Control Study of Diffuse Large B-cell Lymphoma (Mayo), the Genetic Epidemiology of CLL Consortium (GEC), and the Utah Chronic Lymphocytic Leukemia Study (Utah). Genotyping was performed on commercially available Illumina and Affymetrix platforms [Supplementary Table 1]. Details, including information on quality control and data cleaning, have been previously reported[6,8,10,11]. All studies obtained Author Manuscript Page 4 Moore et al. Page 4 informed consent from participants and were approved by their appropriate Institutional Review Boards. Author Manuscript A Author Manuscript Prior to analysis, additional quality control and filtering were applied to each GWAS separately, including removal of SNPs with a minor allele frequency < 0.05, > 3% missing, or Hardy-Weinberg P-value < 1 × 10−6 among controls, and removal of subjects with call rates < 97%. After quality control metrics, genotype data were available for 10,467 NHL cases, including 3061 CLL, 3814 DLBCL, 2784 FL, and 808 MZL cases, as well as 9374 controls [Supplementary Table 2]. We used PLINK1.9[26,27] to identify ROH; specifically, we used the two-step command --homozyg. In the first step, PLINK1.9 identifies directly genotyped SNPs that are possibly within an ROH by looking at 50-SNP sliding windows across the genome and flagging all SNPs that are encompassed by at least 5% of fully homozygous windows. METHODS For this step, we allowed one heterozygous SNP and up to five SNPs with no calls within each window to account for a small amount of possible genotyping error and loss. In the second step, ROH are identified from these sliding windows by requiring a minimum number of consecutive homozygous SNPs. We required at least 100 consecutive homozygous SNPs for each ROH and that these SNPs span at least 1500 kilobases (kb), with at least one SNP every 50 kb and the maximum gap between SNPs of 5000 kb. These parameters were selected with reference to the “ROH_1.5Mb” ROH calling parameters used by Gazal et al.[28] We restricted analyses to the autosomal chromosomes. Author Manuscript Author Manuscript Au To estimate the extent of homozygosity across the genome, we calculated the fraction of the autosome covered by ROH (FROH) by summing the lengths of ROH and dividing by 3 × 109 base pairs as the approximate size of the autosome for all GWAS. As another measure to assess homozygosity, we also quantified and tested differences in relatedness across the genome in our study using a variant of the inbreeding coefficient, F3[29]. F3, which estimates the correlation between uniting gametes, is an alternative to ROH-based estimates with potentially reduced bias and standard errors[30]. We estimated F3 using the -ibc command in PLINK1.9. To estimate the association of FROH and F3 with NHL, we then estimated beta coefficients and standard errors for each GWAS using logistic regression, adjusting for age, sex (except in the UCSF1/NHS study, where all controls were female), fraction of missing SNPs, and the ten principal components of ancestry to account for population stratification. The fraction of missing SNPs was calculated for each participant as the number of SNPs without calls divided by the total number of SNPs genotyped on the array that passed quality control metrics. Associations were combined across GWAS for each subtype of NHL using random-effects meta-analysis implemented with the command “metan” in STATA v15. Author Manuscript Author Manuscript After determining ROH as described above, we also tested whether specific genomic regions encompassed by ROH were associated with risk of each of the four NHL subtypes. We divided each autosomal chromosome into “bins” of 500 kb in length. We then calculated the midpoint of each identified ROH and assigned it to the corresponding bin. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. RESULTS Table 1 presents summary statistics for the ROH, FROH, and F3 by GWAS. Among participants, the median total length of ROH ranged from 11,535 to 23,014 kb depending on the GWAS. The median number of ROH per individual ranged from 4 in the UCSF2 GWAS, which used an older and less dense GWAS chip, to 8 in the GEC GWAS, which included familial CLL cases. Median FROH ranged from 0.38% to 0.77% of the autosome. Median F3 ranged from −6.99 × 10−4 to 3.02 × 10−3 [Table 1]. Table 1 presents summary statistics for the ROH, FROH, and F3 by GWAS. Among participants, the median total length of ROH ranged from 11,535 to 23,014 kb depending on the GWAS. The median number of ROH per individual ranged from 4 in the UCSF2 GWAS, which used an older and less dense GWAS chip, to 8 in the GEC GWAS, which included familial CLL cases. Median FROH ranged from 0.38% to 0.77% of the autosome. Median F3 ranged from −6.99 × 10−4 to 3.02 × 10−3 [Table 1]. Author Manuscript We discovered a positive association between the risk of CLL and increased homozygosity as measured by FROH (β = 21.1, 95% SE = 4.41, P = 1.6 × 10−6) and F3 (β = 27.5, SE = 6.51, P = 2.4 × 10−5) [Table 2] with limited evidence of between-study heterogeneity (Phet = 0.42 and 0.11, respectively) [Supplementary Table 3]. As CLL is an indolent lymphoma and there is a potential for tumor DNA contamination in the blood drawn for genotyping, we performed a sensitivity analysis using only CLL cases and controls from prospective nested case-control studies (ATBC, CPS-II, EPIC, HPFS, MCCS, NHS, NYU-WHS, PLCO, and WHI) from the NCI NHL GWAS, where the DNA was often collected many years prior to diagnosis. Despite the reduction in the number of CLL cases (n = 2140 to 889), the estimated association parameters for FROH and F3 were similar (FROH: β = 21.3, SE = 7.76, P = 6.04 × 10−3; F3: β = 22.5, SE = 5.45, P = 3.57 × 10−5). Figure 1A shows the P-values (−log10) from the meta-analysis assessing the associations of CLL with ROH centered in each 500 kb bin in the autosome. METHODS Each study participant in the analysis was therefore categorized as either homozygous (exposed) or heterozygous (unexposed) at each bin across the autosome. We calculated beta coefficients Moore et al. Page 5 and standard errors for the association between presence of an ROH in each bin and risk of NHL subtype within each GWAS using logistic regression, adjusting for age, sex (except in the UCSF1/NHS study), fraction of missing SNPs, and ten principal components of ancestry. Results were combined across GWAS using METAL[31], and multiple-testing adjustment was performed using a Bonferroni correction. Author Manuscript J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. RESULTS No bins reached statistical significance after correction for multiple testing (CLL Bonferroni alpha level = 0.05/45,590 bins = 1.1 × 10−5); the most significantly associated bin was located at chromosome 22q12.2 (P = 5.92 × 10−4). However, one of the top ten associated bins overlapped with the chromosome 13q14 region, a region where a somatic deletion is often seen in CLL[32]. To test whether the association between FROH and CLL was due to mosaicism at 13q14, we excluded the 45 CLL cases identified to have this deletion from the NCI NHL GWAS. After removal of these 45 cases, the association between FROH and CLL was slightly attenuated but remained statistically significant (β = 16.34, SE = 4.74, P = 5.71 × 10−4), similar to the results from the entire NCI NHL GWAS for CLL (β = 18.9, SE = 4.76, P = 6.73 × 10−5). Author Manuscript As structural alterations in chromosomes, such as trisomy 12, are a hallmark of CLL and could potentially be mistakenly called as ROH using this method, we conducted a sensitivity analysis excluding chromosomes 12 and 13, which are frequently altered in CLL, from the meta-analysis[33]. After excluding these two chromosomes, the results for the association between FROH and CLL were found to be similar (β = 19.3, SE = 4.4, P = 9.0 × 10−6) to previous results for all 22 autosomal chromosomes. Similarly, for F3, exclusion of chromosomes 12 and 13 led to results that are similar in magnitude and remain statistically J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 6 Page 6 significant (β = 17.6, SE = 7.4, P = 0.02). Between-study heterogeneity (I2 = 62.2%, Phet = 0.05) was detected in this sensitivity analysis, largely attributable to the GEC GWAS. significant (β = 17.6, SE = 7.4, P = 0.02). Between-study heterogeneity (I2 = 62.2%, Phet = 0.05) was detected in this sensitivity analysis, largely attributable to the GEC GWAS. Author Manuscript A Author Manuscript We also identified positive associations between FL risk and increased FROH (β = 11.4, SE = 5.82, P = 0.02) but not F3 (β = 13.2, SE = 8.01, P = 0.10) [Table 2]. RESULTS There was evidence of between-study heterogeneity in the meta-analysis of F3 and FL (I2 = 64.2%, P = 0.04) due to the USCF1/NHS GWAS, but not in the meta-analysis of FROH and FL (I2 = 5.3%, P = 0.37) [Supplementary Table 3]. We performed a sensitivity analysis using only cases and controls collected from prospective nested case-control studies (ATBC, CPS-II, EPIC, HPFS, MCCS, NHS, NYU-WHS, PLCO, and WHI) as part of the NCI NHL GWAS. This resulted in a reduction of the number of cases (n = 2085 to 521) and statistical power, but the association between FROH and risk of FL was qualitatively similar (β = 14.1, SE = 11.4, P = 0.21). Given the importance of the HLA region in FL risk, we conducted a sensitivity analysis removing chromosome 6 from estimations of FROH and F3 and repeating the meta-analysis of the four GWAS. The results for both FROH (β = 12.5, SE = 6.29, P = 0.047) and F3 (β = 11.1, SE = 9.26, P = 0.23) were found to be similar to the between-study heterogeneity observed for F3 (I2 = 73.1%, P = 0.01) due to the UCSF1/NHS GWAS. Author Manuscript Figure 1B shows the P-values (−log10) from the meta-analysis of logistic regression models estimating the associations of FL with ROH centered in each 500-kb bin in the autosome. No bins reached statistical significance assuming a Bonferroni-corrected alpha level. The most significant association was for a bin overlapping the HLA region 6p21.33 (P = 2.78 × 10−3). No associations were observed with either FROH or F3 and the risk of DLBCL or MZL [Table 2]; however, between-study heterogeneity in DLBCL risk was present for both FROH (I2 = 72.5%, Phet = 0.01) and F3 (I2 = 82.4%, Phet = 0.001) [Supplementary Table 3]. In a leave-one-out sensitivity analysis [Supplementary Table 4], removal of either the NCI NHL GWAS or the GELA/EPIC GWAS, which had association results in opposite directions, reduced the I2 among the remaining three GWAS to 0%. Without the GELA/EPIC GWAS, there were significant associations between FROH and DLBCL (β = 13.1, SE = 4.62, P = 0.004) and between F3 and DLBCL (β = 13.4, SE = 3.18, P = 2.4 × 10−5). Author Manuscript J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. DISCUSSION To our knowledge, the present study represents the first analysis of the association between homozygosity and risk of four major NHL subtypes. We assessed genome-wide homozygosity using F3 and FROH, where the beta coefficient represents the log odds for a one-unit change in the fraction of ROH. We discovered that increased genome-wide homozygosity was associated with an increased risk of CLL and FL. We estimated that a 0.1% increase in the fraction of ROH was associated with a 2% increase in CLL risk and 1% increase in FL risk compared to those without any ROH. While we did not identify specific regions containing novel associations with these four subtypes of NHL, our analyses suggest that recessive genetic variation is likely to play a role in the risk of these two NHL subtypes and may account for a fraction of the unexplained heritability of these diseases. Our study did not find convincing evidence that recessive variation is a significant contributor to the Author Manuscript Moore et al. Page 7 risk of DLBCL and MZL, further supporting the hypothesis that each NHL subtype is likely to have its own genetic architecture of disease susceptibility. Author Manuscript A Author Manuscript Evidence suggests that CLL has high heritability with first-degree relatives having a 6-8.5- fold increase in risk of CLL[5,34]. Approximately 17%-25% of the familial relative risk of CLL is explained by currently known risk loci[6,9], leaving a sizable fraction that remains undiscovered. We discovered strong associations between CLL and genome-wide homozygosity as measured by FROH and F3. These findings suggest that recessive genetic variants are likely to contribute to risk. CLL is a hematologic malignancy characterized by large-scale chromosomal alterations[35] and is often preceded by a long-lasting pre-malignant stage of monoclonal B-cell lymphocytosis (MBL)[36]. It is possible that cases with diagnosed CLL at the time of enrollment or those with unidentified MBL had tumor DNA in their blood samples, and that some mosaic events may have been erroneously picked up as ROH or homozygous genotypes due to the calling method utilized. This could have inflated our association results, but our sensitivity analyses suggested that any influence of mosaicism on our findings was likely to be small. We excluded CLL cases with DNA collected after diagnosis in the NCI NHL GWAS and saw similar results to our primary analysis, suggesting that tumor contamination is unlikely to be responsible for the observed association. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. DISCUSSION Further sensitivity analysis excluding individuals with known 13q14 deletion in NCI NHL GWAS showed that the associations between FROH and F3 and CLL were only slightly attenuated. We also removed chromosomes 12 and 13 from the calculation of FROH and F3, as these two chromosomes are frequently altered in newly diagnosed CLL[33], and again saw results similar to the primary analysis. While we cannot exclude the possibility that some of the association could be due to erroneous homozygosity calling of structural variation, the robustness of the associations indicates that potential bias from these sources probably does not explain the observed association. Author Manuscript We did not identify any specific homozygous regions that were associated with CLL risk after adjustment for multiple comparisons, but we observed nominal associations with ROH at chromosomes 22q12.2 and 13q14.2. The chromosome 22q12.2 region includes POZ/BTB and AT hook containing zinc finger 1 (PATZ1), which is a transcription factor that interacts with p53 and may modulate p53-mediated apoptosis[37]. The region also contains phosphoinositide-3-kinase interacting protein 1 (PIK3IP1), which inhibits antitumor immunity[38]. The chromosome 13q14.2 region contains several genes of potential interest, including RB transcriptional corepressor 1 (RB1) and cysteinyl-leukotriene receptor 2 (CYSLTR2). RB1 is a known tumor suppressor, which is frequently somatically deleted in CLL[32]. CYSLTR2 is part of the leukotriene pathway and thought to play a role in lymphocyte proliferation and maturation[39]. Author Manuscript For FL, we discovered a positive association between FROH and risk of FL, suggesting recessive genetic variation is a contributor to risk. Previous epidemiologic work has shown that family history of NHL is a risk factor for FL[40], and first-degree relatives have a 4-6- fold increase risk of FL[4,5]. The largest GWAS of FL to date detected multiple genetic loci associated with risk, with the strongest associations seen in the HLA region[10]. Consistent J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 8 Page 8 with our findings, a previous study of first-degree relatives suggested that FL may follow an autosomal recessive mode of inheritance[5]. In sensitivity analyses, we showed that results for FROH are similar after excluding the entirety of chromosome 6, suggesting that our findings were not due to the HLA region. DISCUSSION This finding supports the role of non-HLA variation in the etiology of FL and provides evidence that there may be additional rare, recessive loci that are associated with the risk of FL. We observed similar results after limiting to cases with prospectively collected DNA, suggesting that tumor contamination is unlikely. Although we did not find any individual regions (based on 500-kb bins of ROH) that were significantly associated with the risk of FL after adjustment for multiple testing, we observed nominal evidence for homozygosity overlapping the HLA region at chromosome 6p21.33. We previously reported that homozygosity for classical HLA class II alleles, such as HLA-DRB1, was associated with an increased risk of FL compared to individuals with heterozygosity[41]. These findings are consistent with the hypothesis that HLA homozygosity may increase risk by reducing an individual’s ability to recognize a diverse set of foreign antigens. Author Manuscript Author Manuscript Author Manuscript DLBCL shows a strong association with family history of NHL in epidemiologic studies[42], and it has been previously estimated that 16% of the variation in DLBCL is due to common genetic variants[8]. Overall, we did not observe an association between DLBCL and homozygosity, suggesting the recessive variation may not have a strong role in DLBCL susceptibility, a finding consistent with a previous study of DLBCL among first-degree relatives[5]. The meta-analysis for DLBCL was affected by substantial between-study heterogeneity (Phet = 0.01 for FROH and Phet = 0.001 for F3). In the leave-one-out meta- analysis, removing either the NCI NHL GWAS or the GELA/EPIC GWAS reduced the I2 among the remaining three GWAS to 0%, and significant associations were observed with FROH and F3 after removing GELA/EPIC. The GELA/EPIC GWAS was different from the other studies in that it included patients from clinical trials, who were slightly younger and less likely to be female than the other studies [Supplementary Table 2]. In addition, this GWAS combined cases from French clinical trials with controls from various European countries in the EPIC cohort. It is possible that the degree of population matching, while adequate for a GWAS, is not sufficient for analyses of F3 and FROH, which are known to be especially sensitive to population stratification[43]. Even after adjustment using principal components, some population substructure may have affected the GELA/EPIC study, leading to results that differ from the other DLBCL studies. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. DISCUSSION Our analysis of FL similarly included one GWAS that combined the cases and controls from 2 different United States studies. Although we did observe some heterogeneity among the FL studies for F3, we did not observe such heterogeneity for FROH and FL, suggesting the F3 may be more sensitive to population substructure than FROH. The patients in the UCSF1/NHS study were slightly younger and less likely to be female than the cases in the NCI GWAS, which could have also contributed to the heterogeneity in results. Author Manuscript DLBCL is known to have substantial disease heterogeneity[44,45]. It is possible that a greater understanding of the heterogeneity of DLBCL and a further molecular subtype-specific analysis, which was not possible for the present study, may allow a better elucidation of any association between ROH and disease risk. While the analysis of F3 and DLBCL was similarly affected by between-study heterogeneity [Supplementary Table 3], a potential Moore et al. Page 9 advantage of F3 over FROH as a measure of homozygosity is its expected smaller variance and bias[30]. In addition, the method of calculating FROH used in this study does not count homozygosity below a length threshold of 1500 kb[28]. It is possible that a portion of the recessive genetic variation in DLBCL resides in short, common ROH of ancient ancestry[14] that was unmeasured by our approach. Author Manuscript Autho Author Manuscript Although family history of NHL or other hematologic cancers has been associated with increased risk of MZL[46], we did not find evidence linking FROH or F3 to MZL. Our published GWAS of MZL found two independent loci conferring risk, both in the HLA region of the genome[11], and a follow-up study reported that homozygosity at class I HLA-B and -C and class II HLA-DRB1 loci was associated with increased risk of MZL[41]. Although our study did not detect an association with homozygosity more broadly across the genome, local homozygosity, at least in select regions, is still likely to play a role in risk. Of the four subtypes of NHL examined in the present study, MZL is the least common. Our power to detect an association with MZL was limited due to the small sample size, which was possibly compounded by the known heterogeneity within MZL, with multiple recognized subtypes[47]. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. DISCUSSION Given that we expect ROH to capture the effects of small, scattered, recessive genotypes, a larger sample size is likely needed to elucidate the genetic etiology of MZL. As other highly heritable traits, such as certain autoimmune diseases and atopy, are associated with the risk of MZL[46], identifying shared genetic architecture with these more common phenotypes may be a complementary strategy to further elucidate the underlying genetic architecture. Author Manuscript Author Manuscript A This study had both limitations and strengths. The use of FROH as a measure of homozygosity is known to require large sample sizes[30]. We were able to combine eight GWAS across four subtypes of NHL to increase our sample size and provide a fairly comprehensive analysis of recessive inheritance. However, as discussed, we may have had insufficient sample size to detect associations for MZL. Further, we could not separately examine clinically relevant molecular subtypes of DLBCL. ROH patterns are exquisitely sensitive to population history[48]. Thus, we restricted our study population to individuals of European ancestry and adjusted for principal components of ancestry. This limited the potential for problematic population stratification in our study sample, but it also reduced the generalizability of the findings to individuals of other ancestries. To date, the optimal approach to analyze specific ROH to identify genomic regions with novel, recessively acting risk alleles has not been established, but both the ROH calling procedure and the use of F3 are accepted as means of identifying recent relatedness in outbred European populations[28,30] and can provide evidence for the presence of recessive loci. Although it is possible that the associations between FROH and F3 and CLL are due in part to DNA contamination by tumor cells, our sensitivity analyses suggest that germline homozygosity is likely an independent risk factor for CLL. Finally, although our study provides clues as to the genetic etiology of NHL, the clinical value of these findings is uncertain. Additional studies are needed to further elucidate the role of recessive variation in NHL risk. Author Manuscript Author Manuscript In conclusion, we provide new evidence for the role of recessive genetic variation in the risk of CLL and FL in outbred European-ancestry populations. The knowledge that recessive variation in disease susceptibility to NHL is likely to be present suggests that further studies Moore et al. Page 10 should be undertaken to identify the specific loci responsible for the associations reported here. DISCUSSION As GWAS increase in sample size, they will have greater statistical power to identify recessive genetic variants-associated risk and further characterize the underlying genetic architecture of specific NHL subtypes. Author Manuscript Authors Amy Moore1, Mitchell J. Machiela1, Moara Machado1,2, Sophia S. Wang3, Eleanor Kane4, Susan L. Slager5, Weiyin Zhou1,6, Mary Carrington7,8, Qing Lan1, Roger L. Milne9,10,11, Brenda M. Birmann12, Hans-Olov Adami13,14,15, Demetrius Albanes1, Alan A. Arslan16,17,18, Nikolaus Becker19, Yolanda Benavente20,21, Simonetta Bisanzi22, Paolo Boffetta23,24, Paige M. Bracci25, Paul Brennan26, Angela R. Brooks-Wilson27,28, Federico Canzian29, Neil Caporaso1, Jacqueline Clavel30, Pierluigi Cocco31, Lucia Conde32, David G. Cox33, Wendy Cozen34,35, Karen Curtin36, Immaculata De Vivo12,14, Silvia de Sanjose1,21, Lenka Foretova37, Susan M. Gapstur38, Hervè Ghesquières39,40, Graham G. Giles9,10,11, Martha Glenn36, Bengt Glimelius41, Chi Gao14, Thomas M. Habermann42, Henrik Hjalgrim43, Rebecca D. Jackson44, Mark Liebow42, Brian K. Link45, Marc Maynadie46, James McKay26, Mads Melbye43,47, Lucia Miligi48, Thierry J. Molina49, Alain Monnereau30,50, Alexandra Nieters51, Kari E. North52,53, Kenneth Offit54, Alpa V. Patel38, Sara Piro48, Vignesh Ravichandran54, Elio Riboli55, Gilles Salles40,56,57, Richard K. Severson58, Christine F. Skibola59, Karin E. Smedby60,61, Melissa C. Southey62, John J. Spinelli63,64, Anthony Staines65, Carolyn Stewart54, Lauren R. Teras38, Lesley F. Tinker66, Ruth C. Travis67, Claire M. Vajdic68, Roel C. H. Vermeulen69,70, Joseph Vijai54, Elisabete Weiderpass26, Stephanie Weinstein1, Nicole Wong Doo71, Yawei Zhang72, Tongzhang Zheng73, Stephen J. Chanock1, Nathaniel Rothman1, James R. Cerhan5, Michael Dean1, Nicola J. Camp36, Meredith Yeager1,6, Sonja I. Berndt1 Author Manuscript Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. Refer to Web version on PubMed Central for supplementary material. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Affiliations 1Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA. 2Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil. Author Manuscript 3Division of Health Analytics, City of Hope Beckman Research Institute, Duarte, CA 91010, USA. 4Department of Health Sciences, University of York, York YO10 5DD, UK. 5Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 11 Page 11 6Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Gaithersburg, MD 20877, USA. Author Manuscript 7Basic Science Program, Frederick National Laboratory for Cancer Research in the Laboratory of Integrative Cancer Immunology, National Cancer Institute, Bethesda, MD 20892, USA. 8Ragon Institute of MGH, Cambridge, MA 02139, USA. 9Cancer Epidemiology Division, Cancer Council Victoria, Melbourne, Victoria 3004, Australia. 10Centre for Epidemiology and Biostatistics, School of Population and Global Health, The University of Melbourne, Melbourne, Victoria 3010, Australia. 11Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, Victoria 3800, Australia. Author Manuscript 12Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. 13Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm 17176, Sweden. 14Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA. 15Institute of Health and Society, Clinical Effectiveness Research Group, University of Oslo, Oslo 0315, Norway. 16Department of Obstetrics and Gynecology, New York University School of Medicine, New York, NY 10016, USA. 17Department of Population Health, New York University School of Medicine, New York, NY 10016, USA. 18Perlmutter Comprehensive Cancer Center, NYU Langone Health, New York, NY 10016, USA. 19Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Baden-Württemberg 69120, Germany. 20Cancer Epidemiology Research Programme, Catalan Institute of Oncology- IDIBELL, L'Hospitalet de Llobregat, Barcelona 08908, Spain. 21Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona 08036, Spain. Author Manuscript 22Regional Cancer Prevention Laboratory, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence 50139, Italy. 23Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY 11794, USA. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 12 Page 12 24Department of Medical and Surgical Sciences, University of Bologna, Bologna 41026, Italy. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Affiliations Author Manuscript 25Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA 94118, USA. 26International Agency for Research on Cancer (IARC), Lyon 69372, France. 27Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z1L3, Canada. 28Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A1S6, Canada. 29Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany. 30Center of Research in Epidemiology and Statistics Sorbonne Paris Cité (CRESS), UMR1153, INSERM, Villejuif 75004, France. 31Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Monserrato, Cagliari 09042, Italy. 32Bill Lyons Informatics Centre, UCL Cancer Institute, University College London, London WC1E 6DD, UK. 33INSERM U1052, Cancer Research Center of Lyon, Centre Léon Bérard, Lyon 69008, France. 34Department of Preventive Medicine, USC Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. 35Norris Comprehensive Cancer Center, USC Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. 36Department of Internal Medicine and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. 37Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno 656 53, Czech Republic. 38Department of Population Science, American Cancer Society, Atlanta, GA 30303, USA. 39Department of Hematology, Centre Léon Bérard, Lyon 69008, France. Author Manuscript 40INSERM U1052, Cancer Research Center of Lyon, Lyon-1 University, Pierre- Bénite Cedex 69008, France. 41Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala 75105, Sweden. 42Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA. 43Department of Epidemiology Research, Division of Health Surveillance and Research, Statens Serum Institut, Copenhagen 2300, Denmark. Moore et al. Page 13 44Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, Columbus, OH 43210, USA. 44Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, Columbus, OH 43210, USA. Author Manuscript 45Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA. 46U1231, Registre des Hémopathies Malignes de Côte d’Or, University of Burgundy and Dijon University Hospital, Dijon 21070, France. 47Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. 48Environmental and Occupational Epidemiology Branch-Cancer Risk Factors and Lifestyle Epidemiology Unit, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence 50139, Italy. Author Manuscript 49Department of Pathology, AP-HP, Necker Enfants Malades, Université Paris Descartes, EA 7324, Sorbonne Paris Cité 75015, France. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Affiliations 50Registre des Hémopathies Malignes de la Gironde, Institut Bergonié, Bordeaux Cedex 33076, France. 51Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Baden-Württemberg 79108, Germany. 52Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 53Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Author Manuscript 54Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 55School of Public Health, Imperial College London, London W2 1PG, UK. 56Department of Hematology, Hospices Civils de Lyon, Pierre Benite Cedex 69495, France. 57Department of Hematology, Université Lyon-1, Pierre Benite Cedex 69495, France. 58Department of Family Medicine and Public Health Sciences, Wayne State University, Detroit, MI 48201, USA. 59Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA. Author Manuscript 60Department of Medicine, Solna, Karolinska Institutet, Stockholm 17176, Sweden. 61Hematology Center, Karolinska University Hospital, Stockholm 17176, Sweden. 62Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne Melbourne Victoria 3010 Australia 60Department of Medicine, Solna, Karolinska Institutet, Stockholm 17176, Sweden. 61Hematology Center, Karolinska University Hospital, Stockholm 17176, Sweden. 60Department of Medicine, Solna, Karolinska Institutet, Stockholm 17176, Sweden. 61Hematology Center, Karolinska University Hospital, Stockholm 17176, Sweden. 61Hematology Center, Karolinska University Hospital, Stockholm 17176, Sweden. 62Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia. 62Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 14 63Cancer Control Research, BC Cancer Agency, Vancouver, British Columbia V5Z1L3, Canada. Author Manuscript 64School of Population and Public Health, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada. 65School of Nursing, Psychotherapy and Community Health, Dublin City University, Dublin 9, Ireland. 66Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98117, USA. 67Cancer Epidemiology Unit, University of Oxford, Oxford OX3 7LF, UK. 68Centre for Big Data Research in Health, University of New South Wales, Sydney, New South Wales 2052, Australia. Author Manuscript 69Institute for Risk Assessment Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands. 70Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands. 71Concord Clinical School, University of Sydney, Concord, New South Wales 2139, Australia. Affiliations 72Department of Environmental Health Sciences, Yale School of Public Health, New Haven, CT 06520, USA. 73Department of Epidemiology, Brown University, Providence, RI 02903, USA. Author Manuscript Acknowledgements ATBC - The ATBC Study is supported by the Intramural Research Program of the United States National Cancer Institute, National Institutes of Health, Department of Health and Human Services. BCCA - Canadian Institutes for Health Research (CIHR); Canadian Cancer Society; Michael Smith Foundation for Health Research. CPS-II - The Cancer Prevention Study-II (CPS-II) Nutrition Cohort is supported by the American Cancer Society. Genotyping for all CPS-II samples were supported by the Intramural Research Program of the National Institutes of Health, NCI, Division of Cancer Epidemiology and Genetics. The authors would also like to acknowledge the contribution to this study from central cancer registries supported through the Centers for Disease Control and Prevention National Program of Cancer Registries, and cancer registries supported by the National Cancer Institute Surveillance Epidemiology and End Results program. CPS-II - The Cancer Prevention Study-II (CPS-II) Nutrition Cohort is supported by the American Cancer Society. Genotyping for all CPS-II samples were supported by the Intramural Research Program of the National Institutes of Health, NCI, Division of Cancer Epidemiology and Genetics. The authors would also like to acknowledge the contribution to this study from central cancer registries supported through the Centers for Disease Control and Prevention National Program of Cancer Registries, and cancer registries supported by the National Cancer Institute Surveillance Epidemiology and End Results program. ELCCS - Blood Cancer UK, United Kingdom. Author Manuscript ENGELA - Association pour la Recherche contre le Cancer (ARC), Institut National du Cancer (INCa), Fondation de France, Fondation contre la Leucémie, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES). EPIC - Coordinated Action (Contract #006438, SP23-CT-2005-006438); HuGeF (Human Genetics Foundation), Torino, Italy; Cancer Research UK. The coordination of EPIC is financially supported by the European Commission (DG-SANCO) and the International Angency for Research on Cancer. The national cohorts are supported by the Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l'Education National, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Centre (DKFZ), Federal Ministry of Education and Research (BMBF), Deutsche Krebshilfe, Deutsches Krebsforschungszentrum and Federal Ministry of Education and J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Acknowledgements Page 15 Page 15 Research (Germany); the Hellenic Health Foundation (Greece); Associazione Italiana per la Ricerca sul Cancro- AIRC-Italy and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); ERC-2009-AdG 232997 and Nordforsk, Nordic Centre of Excellence programme on Food, Nutrition and Health (Norway); Health Research Fund (FIS), PI131/00061 to Granada, PI13/01162 to EPIC-Murcia, Regional Governments of Andalucía, Asturias, Basque Country, Murcia (no. 6236) and Navarra, ISCIII RETIC ((RD06/0020) (Spain); Swedish Cancer Society, Swedish Research Council and Country Councils of Skåne and Västerbotten (Sweden); Cancer Research UK [14136 to K.T. Khaw, N.J. Wareham; C570/A16491 to RCT and C8221/A19170 to T. Key (EPIC-Oxford)], Medical Research Council [10001433 to K.T. Khaw, N.J. Wareham, MR/M012190/1 to T. Key (EPIC-Oxford)] (United Kingdom). Author Manuscript Author Manuscript EpiLymph - European Commission (grant references QLK4-CT-2000-00422 and FOOD-CT-2006-023103); We thank CERCA programme/Generalitat de Catalunya for institutional support. This work was supported by Spanish Ministry of Economy and Competitiveness - Carlos III Institute of Health cofunded by FEDER funds/ European Regional Develpment Fund (ERDF) - a way to build Europe (grant references CIBERESP, PI17/01280, PI20/00288) with the support of the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia (2017SGR1085) who had no role in the data collection, analysis or interpretation of the results; the NIH (contract NO1-CO-12400); the Compagnia di San Paolo-Programma Oncologia; the Federal Office for Radiation Protection grants StSch4261 and StSch4420, the José Carreras Leukemia Foundation grant DJCLS-R12/23, the German Federal Ministry for Education and Research (BMBF-01- EO-1303); the Health Research Board, Ireland and Cancer Research Ireland; Czech Republic supported by MH CZ - DRO (MMCI, 00209805) and MEYS - NPSI - LO1413; Fondation de France and Association de Recherche Contre le Cancer. Author Manuscript Auth Author Manuscript GEC/Mayo GWAS - National Institutes of Health (CA118444, CA148690, CA92153). Intramural Research Program of the NIH, National Cancer Institute. Veterans Affairs Research Service. Data collection for Duke University was supported by a Leukemia & Lymphoma Society Career Development Award, the Bernstein Family Fund for Leukemia and Lymphoma Research, and the National Institutes of Health (K08CA134919), National Center for Advancing Translational Science (UL1 TR000135). HPFS (Walter C. Acknowledgements Willet) - The HPFS was supported in part by National Institutes of Health grants U01 CA167552, R01 CA149445, and R01 CA098122. We would like to thank the participants and staff of the Health Professionals Follow-up Study for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and interpretation of these data. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health, and those of participating registries as required. Author Manuscript Iowa-Mayo SPORE - NCI Specialized Programs of Research Excellence (SPORE) in Human Cancer (P50 CA97274); National Cancer Institute (P30 CA086862, P30 CA15083); Henry J. Predolin Foundation. Italian GxE - Italian Association for Cancer Research (AIRC, Investigator Grant 11855) (PC); Fondazione Banco di Sardegna 2010-2012, and Regione Autonoma della Sardegna (LR7 CRP-59812/2012) (MGE). Mayo Clinic Case-Control - National Institutes of Health (R01 CA92153 and CA200703); National Cancer Institute (P30 CA015083). MCCS - The Melbourne Collaborative Cohort Study recruitment was funded by VicHealth and Cancer Council Victoria. The MCCS was further supported by Australian NHMRC grants 209057, 396414 and 1074383 and by infrastructure provided by Cancer Council Victoria. Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index and the Australian Cancer Database. Author Manuscript MSKCC - Geoffrey Beene Cancer Research Grant, Lymphoma Foundation (LF5541); Barbara K. Lipman Lymphoma Research Fund (74419); Robert and Kate Niehaus Clinical Cancer Genetics Research Initiative (57470); U01 HG007033; ENCODE; U01 HG007033. NCI-SEER - Intramural Research Program of the National Cancer Institute, National Institutes of Health, and Public Health Service (N01-PC-65064, N01-PC-67008, N01-PC-67009, N01-PC-67010, N02-PC-71105). NHS (Meir J. Stampfer) - The NHS was supported in part by National Institutes of Health grants CA186107, CA87969, CA49449, CA149445, CA098122 and CA134958. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Acknowledgements The collection of cancer incidence data used in this study was supported by the California Department of Health Services as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885; the National Cancer Institute’s Surveillance, Epidemiology, and End Results Program under contract HHSN261201000140C awarded to the Cancer Prevention Institute of California, contract HHSN261201000035C awarded to the University of Southern California, and contract HHSN261201000034C awarded to the Public Health Institute; and the Centers for Disease Control and Prevention’s National Program of Cancer Registries, under agreement #1U58 DP000807-01 awarded to the Public Health Institute. The ideas and opinions expressed herein are those of the authors, and endorsement by the State of California, the California Department of Health Services, the National Cancer Institute, or the Centers for Disease Control and Prevention or their contractors and subcontractors is not intended nor should be inferred. Author Manuscript UTAH/Sheffield - National Institutes of Health CA134674. Partial support for data collection at the Utah site was made possible by the Utah Population Database (UPDB) and the Utah Cancer Registry (UCR). Partial support for all datasets within the UPDB is provided by the Huntsman Cancer Institute (HCI) and the HCI Cancer Center Support grant, P30 CA42014. The UCR is supported in part by NIH contract HHSN261201000026C from the National Cancer Institute SEER Program with additional support from the Utah State Department of Health and the University of Utah. Partial support for data collection in Sheffield, UK was made possible by funds from Yorkshire Cancer Research and the Sheffield Experimental Cancer Medicine Centre. We thank the NCRI Haemato- oncology Clinical Studies Group, colleagues in the North Trent Cancer Network the North Trent Haemato-oncology Database. Author Manuscript UTAH-MM - National Institutes of Health CA134674, the Leukemia Lymphoma Society 6067-09, and pilot funds from the Huntsman Cancer Institute (HCI). Partial support for data collection at the Utah site was made possible by the Utah Population Database (UPDB) and the Utah Cancer Registry (UCR). Partial support for all datasets within the UPDB is provided by the HCI Comprehensive Cancer Center Support grant, P30 CA42014 (M Beckerle). The UCR is supported in part by NIH contract HHSN261201000026C from the National Cancer Institute SEER Program with additional support from the Utah State Department of Health and the University of Utah. Acknowledgements We would like to thank the participants and staff of the Nurses' Health Study for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, NHS (Meir J. Stampfer) - The NHS was supported in part by National Institutes of Health grants CA186107, CA87969, CA49449, CA149445, CA098122 and CA134958. We would like to thank the participants and staff of the Nurses' Health Study for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 16 Page 16 ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and nterpretation of these data. ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and interpretation of these data. Author Manuscript NSW - NSW was supported by grants from the Australian National Health and Medical Research Council (ID990920), the Cancer Council NSW, and the University of Sydney Faculty of Medicine. NYU-WHS - National Cancer Institute (R01 CA098661, P30 CA016087); National Institute of Environmental Health Sciences (ES000260). PLCO - This research was supported by the Intramural Research Program of the National Cancer Institute and by contracts from the Division of Cancer Prevention, National Cancer Institute, NIH, DHHS. SCALE - Swedish Cancer Society (2009/659). Stockholm County Council (20110209) and the Strategic Research Program in Epidemiology at Karolinska Institutet. National Institutes of Health (5R01 CA6966902); Plan Denmark. UCSF2 - The UCSF studies were supported by the NCI, National Institutes of Health, CA1046282 and CA154643. Acknowledgements WHI - WHI investigators are: Program Office - (National Heart, Lung, and Blood Institute, Bethesda, Maryland) Jacques Rossouw, Shari Ludlam, Dale Burwen, Joan McGowan, Leslie Ford, and Nancy Geller; Clinical Coordinating Center - (Fred Hutchinson Cancer Research Center, Seattle, WA) Garnet Anderson, Ross Prentice, Andrea LaCroix, and Charles Kooperberg; Investigators and Academic Centers - (Brigham and Women's Hospital, Harvard Medical School, Boston, MA) JoAnn E. Manson; (MedStar Health Research Institute/Howard University, Washington, DC) Barbara V. Howard; (Stanford Prevention Research Center, Stanford, CA) Marcia L. Stefanick; (The Ohio State University, Columbus, OH) Rebecca Jackson; (University of Arizona, Tucson/ Phoenix, AZ) Cynthia A. Thomson; (University at Buffalo, Buffalo, NY) Jean Wactawski-Wende; (University of Florida, Gainesville/Jacksonville, FL) Marian Limacher; (University of Iowa, Iowa City/Davenport, IA) Robert Wallace; (University of Pittsburgh, Pittsburgh, PA) Lewis Kuller; (Wake Forest University School of Medicine, Winston-Salem, NC) Sally Shumaker; Women’s Health Initiative Memory Study - (Wake Forest University School of Medicine, Winston-Salem, NC) Sally Shumaker. The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, United States Department of Health and Human Services through contracts HHSN268201100046C, HHSN268201100001C, HHSN268201100002C, HHSN268201100003C, HHSN268201100004C, and HHSN271201100004C. Author Manuscript YALE - National Cancer Institute (CA62006); National Cancer Institute (CA165923). REFERENCES 1. Chatterjee N, Hartge P, Cerhan JR, et al. 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Disclaimer: This project was funded by the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH and in part with federal funds from the Frederick National Laboratory for Cancer J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. Page 17 Page 17 Research, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. This Research was supported in part by the Intramural Research Program of the NIH, Frederick National Lab, Center for Cancer Research. Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. Research, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. This Research was supported in part by the Intramural Research Program of the NIH, Frederick National Lab, Center for Cancer Research. Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. Author Manuscript REFERENCES Quantification of inbreeding due to distant ancestors and its detection using dense single nucleotide polymorphism data. Genetics 2011;189:237–49. [PubMed: 21705750] 16. Szpiech ZA, Xu J, Pemberton TJ, et al. Long runs of homozygosity are enriched for deleterious variation. Am J Hum Genet 2013;93:90–102. [PubMed: 23746547] 17. Enciso-Mora V, Hosking FJ, Houlston RS. Risk of breast and prostate cancer is not associated with increased homozygosity in outbred populations. Eur J Hum Genet 2010;18:909–14. [PubMed: 20407466] J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Moore et al. 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Monti S, Savage KJ, Kutok JL, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood 2005;105:1851–61. [PubMed: 15550490] Author Manuscript 46. Bracci PM, Benavente Y, Turner JJ, et al. Medical history, lifestyle, family history, and occupational risk factors for marginal zone lymphoma: the InterLymph Non-Hodgkin Lymphoma Subtypes Project. J Natl Cancer Inst Monogr 2014;2014:52–65. [PubMed: 25174026] 47. Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Geneva: IARC Press; 2008. 48. McQuillan R, Leutenegger AL, Abdel-Rahman R, et al. Runs of homozygosity in European populations. Am J Hum Genet 2008;83:359–72. [PubMed: 18760389] Author Manuscript Author Manuscript Moore et al. Page 20 Page 20 Figure 1. Manhattan plots of log10(P) values for the association between runs of homozygosity (ROH) and the risk of chronic lymphocytic leukemia (CLL) (A) and follicular lymphoma (FL) (B). Here, ROH were divided into 500-kb bins across each chromosome, and each bin was tested for its association with CLL or FL. No bins reached statistical significance after correction for multiple testing. Author Manuscript Author Manuscript Figure 1. Figure 1. Manhattan plots of log10(P) values for the association between runs of homozygosity (ROH) and the risk of chronic lymphocytic leukemia (CLL) (A) and follicular lymphoma (FL) (B). Here, ROH were divided into 500-kb bins across each chromosome, and each bin was tested for its association with CLL or FL. No bins reached statistical significance after correction for multiple testing. Figure 1. Manhattan plots of log10(P) values for the association between runs of homozygosity (ROH) and the risk of chronic lymphocytic leukemia (CLL) (A) and follicular lymphoma (FL) (B). Here, ROH were divided into 500-kb bins across each chromosome, and each bin was tested for its association with CLL or FL. No bins reached statistical significance after correction for multiple testing. Author Manuscript Page 21 Moore et al. g Subtype Study No. cases No. Figure 1. controls Median ROH total length (kb) IQR ROH total length (kb) Median number of ROH IQR number of ROH Median FROH IQR FROH Median F3 IQR F3 CLL NCI Cases 2140 18593 13057-25486 7 5-9 0.62% 0.44%-0.79% 2.57 × 10−3 −1.34 × 10−3−7.49 × 10−3 Controls 6105 18045 12674-24396 7 5-9 0.60% 0.42%-0.81% 1.48 × 10−3 −2.07 × 10−3−5.36 × 10−3 GEC Cases 387 23014 16496-29054 8 6-10 0.77% 0.55%-0.97% 6.41 × 10−4 −3.66 × 10−3−4.85 × 10−3 Controls 294 21920 14852-28391 8 6-10 0.73% 0.50%-0.95% −6.99 × 10−4 −4.90 × 10−3−3.06 × 10−3 UCSF2 Cases 213 12993 7893-19530 4 3-6 0.43% 0.26%-0.65% 2.73 × 10−3 −0.63 × 10−3−8.83 × 10−3 Controls 746 12173 7523-17111 4 3-6 0.41% 0.25%-0.57% 1.51 × 10−3 −2.25 × 10−3−5.72 × 10−3 Utah Cases 321 18806 13535-24431 7 5-9 0.63% 0.45%-0.81% 3.69 × 10−4 −2.79 × 10−3−4.71 × 10−3 Controls 403 17452 12288-22866 6 5-8 0.58% 0.41%-0.76% 9.47 × 10−4 −2.09 × 10−3−3.89 × 10−3 DLBCL NCI Cases 2621 18046 12671-24990 7 5-9 0.60% 0.42%-0.83% 2.11 × 10−3 −1.48 × 10−3−6.54 × 10−3 Controls 6105 18199 12753-24519 7 5-9 0.61% 0.43%-0.82% 1.46 × 10−3 −2.05 × 10−3−5.39 × 10−3 Mayo Cases 393 16586 12167-21774 6 5-8 0.55% 0.41%-0.73% 1.67 × 10−4 −3.03 × 10−3−3.07 × 10−3 Controls 172 16451 11368-23319 6 4-8 0.55% 0.38%-0.78% 2.19 × 10−4 −2.55 × 10−3−2.94 × 10−3 UCSF2 Cases 253 11535 5957-19153 4 3-6 0.38% 0.20%-0.64% 1.69 × 10−3 −1.62 × 10−3−6.60 × 10−3 Controls 745 12182 7554-17158 4 3-6 0.41% 0.25%-0.57% 1.48 × 10−3 −2.20 × 10−3−5.82 × 10−3 GELA/ EPIC Cases 547 15526 10391-21466 6 4-7 0.52% 0.35%-0.72% 1.27 × 10−3 −2.27 × 10−3−5.00 × 10−3 Controls 525 17327 11782-23437 6 4-8 0.58% 0.39%-0.78% 2.56 × 10−3 −0.87 × 10−3−6.14 × 10−3 FL NCI Cases 2085 17838 12387-24319 7 5-9 0.59% 0.41%-0.81% 1.41 × 10−3 −2.14 × 10−3−6.15 × 10−3 Controls 6105 18110 12753-24512 7 5-9 0.60% 0.43%-0.82% 1.49 × 10−3 −2.06 × 10−3−5.41 × 10−3 UCSF1/NH S Cases 119 17058 12645-22150 7 5-8 0.57% 0.42%-0.74% 8.51 × 10−5 −3.57 × 10−3−3.05 × 10−3 Controls 340 17144 12100-24513 6.5 5-9 0.57% 0.40%-0.82% 6.35 × 10−4 −3.05 × 10−3−4.29 × 10−3 UCSF2 Cases 209 13621 8911-19655 5 3-7 0.45% 0.30%-0.66% 3.02 × 10−3 −1.34 × 10−3−7.58 × 10−3 Controls 745 12071 7517-17115 4 3-6 0.40% 0.25%-0.57% 1.50 × 10−3 −2.16 × 10−3−5.76 × 10−3 SCALE Cases 371 12582 7643-19776 5 3-7 0.42% 0.25%-0.66% 3.02 × 10−3 −0.50 × 10−3−7.52 × 10−3 Controls 790 12132 7506-17654 5 3-6 0.40% 0.25%-0.59% 1.22 × 10−3 −1.60 × 10−3−5.00 × 10−3 MZL NCI Cases 808 18097 12831-24555 7 5-9 0.60% 0.43%-0.82% 1.83 × 10−3 −1.54 × 10−3−7.30 × 10−3 Author Manuscript Author Manuscript J Transl Genet Genom. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Figure 1. Author manuscript; available in PMC 2021 October 06. Author Manuscript Author Manuscript Author Manuscript Author Manuscr Moore et al. Subtype Study No. cases No. controls Median ROH total length (kb) IQR ROH total length (kb) Median number of ROH IQR number of ROH Median FROH IQR FROH Median F3 IQR F3 Controls 6102 18268 12811-24623 7 5-9 0.61% 0.43%-0.82% 1.46 × 10−3 −2.01 × 10−3−5.38 × 10−3 Median and interquartile range are provided for runs of homozygosity, fraction of runs of homozygosity, and correlation between uniting gametes (F3). GWAS: Genome-wide association studies; IQR: interquartile range; ROH: runs of homozygosity; FROH: fraction of runs of homozygosity; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MZL: marginal zone lymphoma. Page 22 Page 22 Author Manuscript Author Manuscript J Transl Genet Genom. Author manuscript; available in PMC 2021 October 06. Page 23 Page 23 Moore et al. Risk of NHL subtypes associated with measures of genome-wide homozygosity, FROH and F3 Risk of NHL subtypes associated with measures of genome-wide homozygosity, FROH and F3* Author Manuscript Risk of NHL subtypes associated with measures of genome wide homozygosity, FROH and F3 FROH F3 Subtype β SE P-value I 2 P het β SE P-value I 2 P het CLL 21.14 4.41 1.59 × 10−6 0.0% 0.42 27.46 6.51 2.44 × 10−5 49.7% 0.11 DLBCL 0.04 10.89 1.0 72.5% 0.01 1.96 9.57 0.84 82.4% 0.001 FL 11.39 5.82 0.02 5.3% 0.37 13.19 8.01 0.10 64.2% 0.04 MZL −0.87 7.88 0.91 6.40 5.20 0.22 Estimates of the log odds (β), standard error (SE), and P-value are provided for the association between FROH and F3 and each subtype, adjusted for age, sex (except UCSF1/NHS), percentage of missing SNPs, and principal components and combined using random effects meta-analysis. The I2 statistic provides an estimate of heterogeneity in association estimates across GWAS, and Phet is the P-value for heterogeneity among studies. FROH: Fraction of runs of homozygosity; NHL: non-Hodgkin lymphoma; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MZL: marginal zone lymphoma. FROH F3 Subtype β SE P-value I 2 P het β SE P-value I 2 P het CLL 21.14 4.41 1.59 × 10−6 0.0% 0.42 27.46 6.51 2.44 × 10−5 49.7% 0.11 DLBCL 0.04 10.89 1.0 72.5% 0.01 1.96 9.57 0.84 82.4% 0.001 FL 11.39 5.82 0.02 5.3% 0.37 13.19 8.01 0.10 64.2% 0.04 MZL −0.87 7.88 0.91 6.40 5.20 0.22 Estimates of the log odds (β), standard error (SE), and P-value are provided for the association between FROH and F3 and each subtype, adjusted for age, sex (except UCSF1/NHS), percentage of missing SNPs, and principal components and combined using random effects meta-analysis. The I2 statistic provides an estimate of heterogeneity in association estimates across GWAS, and Phet is the P-value for heterogeneity among studies. FROH: Fraction of runs of homozygosity; NHL: non-Hodgkin lymphoma; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MZL: marginal zone lymphoma. Estimates of the log odds (β), standard error (SE), and P-value are provided for the association between FROH and F3 and each subtype, adjusted for age, sex (except UCSF1/NHS), percentage of missing SNPs, and principal components and combined using random effects meta-analysis. The I2 statistic provides an estimate of heterogeneity in association estimates across GWAS, and Phet is the P-value for heterogeneity among studies. Estimates of the log odds (β), standard error (SE), and P-value are provided for the association between FROH and F3 and each subtype, adjusted for age, sex (except UCSF1/NHS), percentage of missing SNPs, and principal components and combined using random effects meta-analysis. The I2 statistic provides an estimate of heterogeneity in association estimates across GWAS, and Phet is the P-value for heterogeneity among studies. FROH: Fraction of runs of homozygosity; NHL: non-Hodgkin lymphoma; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MZL: marginal zone lymphoma. Risk of NHL subtypes associated with measures of genome-wide homozygosity, FROH and F3* FROH: Fraction of runs of homozygosity; NHL: non-Hodgkin lymphoma; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MZL: marginal zone lymphoma. Author Manuscript Author Manuscript Author Manuscript
https://openalex.org/W2154266265	https://inria.hal.science/hal-01286399/document	English	null	Efficient Cardinality/Mean-Variance Portfolios	IFIP advances in information and communication technology	2,014	cc-by	9,713	To cite this version: R. Pedro Brito, Luís N. Vicente. Eﬀicient Cardinality/Mean-Variance Portfolios. 26th Conference on System Modeling and Optimization (CSMO), Sep 2013, Klagenfurt, Austria. pp.52-73, 10.1007/978- 3-662-45504-3_6. hal-01286399 Eﬀicient Cardinality/Mean-Variance Portfolios R. Pedro Brito, Luís N. Vicente To cite this version: R. Pedro Brito, Luís N. Vicente. Eﬀicient Cardinality/Mean-Variance Portfolios. 26th Conference on System Modeling and Optimization (CSMO), Sep 2013, Klagenfurt, Austria. pp.52-73, 10.1007/978- 3-662-45504-3_6. hal-01286399 To cite this version: R. Pedro Brito, Luís N. Vicente. Eﬀicient Cardinality/Mean-Variance Portfolios. 26th Conference on System Modeling and Optimization (CSMO), Sep 2013, Klagenfurt, Austria. pp.52-73, 10.1007/978- 3-662-45504-3_6. hal-01286399 Distributed under a Creative Commons Attribution 4.0 International License ⋆Support for this research was provided by FCT under the grant PTDC/MAT/098214/2008. Eﬃcient cardinality/mean-variance portfolios R. Pedro Brito1 and Lu´ıs N. Vicente2⋆ 1 Department of Mathematics, University of Coimbra 3001-454 Coimbra, Portugal rpedro.brito@gmail.com. 2 CMUC, Department of Mathematics, University of Coimbra 3001-454 Coimbra, Portugal lnv@mat.uc.pt Abstract. We propose a novel approach to handle cardinality in portfo- lio selection, by means of a biobjective cardinality/mean-variance prob- lem, allowing the investor to analyze the eﬃcient tradeoﬀbetween return- risk and number of active positions. Recent progress in multiobjective op- timization without derivatives allow us to robustly compute (in-sample) the whole cardinality/mean-variance eﬃcient frontier, for a variety of data sets and mean-variance models. Our results show that a signiﬁcant number of eﬃcient cardinality/mean-variance portfolios can overcome (out-of-sample) the naive strategy, while keeping transaction costs rela- tively low. Keywords: portfolio selection, cardinality, sparse portfolios, multiob- jective optimization, eﬃcient frontier, derivative-free optimization HAL Id: hal-01286399 https://inria.hal.science/hal-01286399v1 Submitted on 10 Mar 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction One knows since the pioneer work of Markowitz [19] that a rational investor has typically two goals in mind: to maximize the portfolio return (given, e.g., by the portfolio expected return) and to minimize the portfolio risk (described, e.g., by the portfolio variance). Traditionally, the Markowitz mean-variance optimization model is taken as a quadratic program (QP), intended to minimize the portfolio risk (variance) for a given level of expected return, over a set of feasible portfolios. By varying the level of expected return, the Markowitz model determines the so-called eﬃcient frontier, as the set of nondominated portfolios regarding the two goals (variance and mean of the return). The rational investor can thus make choices, by analyzing the tradeoﬀbetween expected return and variability of the investment, over a set of appropriate portfolios. Several modiﬁcations to the classical Markowitz model or alternative method- ologies have since then been proposed. One resulting from a simple observation was suggested in an article by DeMiguel, Garlappi, and Uppal [14]. These authors analyzed a number of methodologies inspired on the classic model of Markowitz 54 R.P. Brito, L.N. Vicente and showed that none were able to signiﬁcantly and consistently overcome the naive strategy, that is to say, the one in which the available investor’s wealth is divided equally among the available securities. One possible explanation is related to the ill conditioning of the objective function of the Markowitz model (given by the variance of the return). One of the important issues to consider in portfolio selection is how to handle transaction costs. There are well known modiﬁcations that can be made in the Markowitz model to incorporate transaction costs, such as to bound the turnover, which basically amount to further linear constraints in the QP. A recent tech- nique to keep transaction costs low consists of selecting sparse portfolios, i.e., portfolios with few active positions, by imposing a cardinality constraint. Such a constraint, however, changes the classical QP into a MIQP (mixed-integer quadratic programming), which can no longer be solved in polynomial time. In this paper, we suggest an alternative approach to the cardinality con- strained Markowitz mean-variance optimization model, reformulating it directly as a biobjective problem, allowing the investor to analyze the tradeoﬀbetween cardinality and mean-variance, in a general scenario where short-selling is per- mitted. 1 Introduction Such an approach allows us to ﬁnd the set of nondominated points of biobjective problems in which an objective is smooth and combines mean and variance and the other is nonsmooth (the cardinality or ℓ0 norm of the vector of portfolio positions). The mean-variance objective function can take a num- ber of forms. A parameter free possibility is given by proﬁt per unity of risk (a nonlinear function obtained by dividing the expected return by its variance). Given the lack of derivatives of the cardinality function, we decided then to apply a directional derivative-free algorithm for the solution of the biobjective optimization problem. Such methods do not require derivatives, although their convergence results typically assume some weak form of smoothness such as Lip- schitz continuity. Direct multisearch is a derivative-free multiobjective method- ology for which one can show some type of convergence in the discontinuous case. More importantly, it exhibited excellent numerical performance on a com- parison to a number of other multiobjective optimization solvers. We applied direct multisearch to determine (in-sample) the set of eﬃcient or nondominated cardinality/mean-variance portfolios. To illustrate our approach, we gathered several data sets from the FTSE 100 index (for returns of single securities) and from the Fama/French benchmark collection (for returns of portfolios), computed the eﬃcient cardinality/mean- variance portfolios using (in-sample) optimization, and measured their out-of- sample performance using a rolling-sample approach. We found that a large number of sparse portfolios for the FTSE 100 data sets, among the eﬃcient cardinality/mean-variance ones, consistently overcome the naive strategy in terms of out-of-sample performance measured by the Sharpe ratio. This eﬀect is also clearly visible for the FF data sets, where the performance of a large portion of the cardinality/mean-variance eﬃcient frontier outperforms, in most of the in- stances, the naive strategy. The transactions costs are shown to be relatively low Eﬃcient cardinality/mean-variance portfolios 55 for all eﬃcient cardinality/mean-variance portfolios, with a moderate increase with cardinality. The organization of our paper is as follows. In the next section, we formulate the classical Markowitz model for portfolio selection, describe the naive strat- egy, and formulate the problem with cardinality constraint. In Section 3, we reformulate the cardinality constrained Markowitz mean-variance optimization model as a biobjective problem for application of multiobjective optimization. In Section 4, we present the empirical results. Finally, in Section 5 we summarize our ﬁndings and discuss future research. 2.1 The classical Markowitz mean-variance model Portfolios consist of securities (shares or bonds, for example, or classes or indices of the same). Suppose the investor has a certain wealth to invest in a set of N securities. The return of each security i is described by a random variable Ri, whose average can be computed (from estimation based on historical data). Let µi = E(Ri), i = 1, . . . , N, denote the expected returns of the securities. Let also wi, i = 1, . . . , N, represent the proportions of the total investment to allocate in the individual securities. The portfolio return is assumed linear in w1, . . . , wN, and thus the portfolio expected return can be written as E(R) = E(wiR1 + · · · + wNRN) = w1µ1 + · · · + wNµN = µ⊤w with µ = (µ1, . . . , µN)⊤ and w = (w1, . . . , wN)⊤. The portfolio variance, in turn, is calculated by V (R) = E N X i=1 wiRi −E N X i=1 wiRi 2 . So, So, V (R) = N X i=1 N X j=1 E[(Ri −µi)(Rj −µj)]wiwj. Representing each entry i, j of the covariance matrix Q by Representing each entry i, j of the covariance matrix Q by σij = E[(Ri −µi)(Rj −µj)], one has one has V (R) = w⊤Qw, V (R) = w⊤Qw, where Q is symmetric and positive semi-deﬁnite (and typically assumed positive deﬁnite). As said before, a portfolio is deﬁned by an N × 1 vector w of weights R.P. Brito, L.N. Vicente 56 representing the proportion of the total funds invested in the N securities. This vector of weights is thus required to satisfy the constraint N X i=1 wi = e⊤w = 1, where e is the N × 1 vector of entries equal to 1. Lower bounds on the variables, of the form wi ≥0, i = 1, . . . , n, can be also considered if short selling is undesirable. In general, we will say that Li ≤wi ≤Ui, i = 1, . . . , N, for given lower Li and upper Ui bounds on the variables. Markowitz’s model [19,20] is based on the formulation of a mean-variance optimization problem. 2.1 The classical Markowitz mean-variance model By solving this problem, we identify a portfolio of mini- mum variance among all which provide an expected return not below a certain target value r. The aim is thus to minimize the risk from a given level of return. The formulation of this problem can be described as: min w∈RN w⊤Qw subject to µ⊤w ≥r, e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. (1) (1) e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. Problem (1) is a convex quadratic programming problem (QP), for which the ﬁrst order necessary conditions are also suﬃcient for (global) optimality. See [12,21] for a survey of portfolio optimization. The classical Markowitz mean-variance model can be seen as way of solving the biobjective problem which consists of simultaneously minimizing the portfolio risk (variance) and maximizing the portfolio proﬁt (expected return) min w∈RN w⊤Qw max w∈RN µ⊤w subject to e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. (2) min w∈RN w⊤Qw max w∈RN µ⊤w subject to e⊤w = 1, L ≤w ≤U i = 1 N (2) (2) subject to e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. Li ≤wi ≤Ui, i = 1, . . . , N. In fact, it is easy to prove that a solution of (1) is nondominated, eﬃcient or Pareto optimal for (2). Eﬃcient portfolios are thus the ones which have the minimum variance among all that provide at least a certain expected return, or, alternatively, those that have the maximal expected return among all up to a certain variance. The eﬃcient frontier (or Pareto front) is typically represented as a 2-dimensional curve, where the axes correspond to the expected return and the standard deviation of the return of an eﬃcient portfolio. 2.2 The naive strategy 1/N The naive strategy is the one in which the available investor’s wealth is divided equally among the securities available wi = 1 N , i = 1, . . . , N. Eﬃcient cardinality/mean-variance portfolios 57 This strategy has diversiﬁcation as its main goal, it does not involve optimization, and it completely ignores the data. Although a number of theoretical models have been developed in the last years, many investors pursuing diversiﬁcation revert to the use of the naive strategy to allocate their wealth (see [4]). DeMiguel, Garlappi, and Uppal [14] evaluated fourteen models across seven empirical data sets and showed that none is consistently better than the naive strategy. A possible explanation for this phe- nomenon lies on the fact that the naive strategy does not involve estimation and promotes ‘optimal’ diversiﬁcation. The naive strategy is therefore an excellent benchmarking strategy. 2.3 The cardinality constrained Markowitz mean-variance model Some of these heuristics vary among evolutionary algorithms, tabu search, and simulated an- nealing (see [15,26]). nonzeros entries) is N K = N!/[(N −K)!K!]. Although there are exact algorithms for the solution of MIQPs (see [5,6,7,25]), many researchers and portfolio man- agers prefer to use heuristics approaches (see [3,9,11,15,17,26]). Some of these heuristics vary among evolutionary algorithms, tabu search, and simulated an- nealing (see [15,26]). Promotion of sparsity is also used in the ﬁeld of signal and imaging pro- cessing, where a new technique called compressed sensing has been intensively studied in the recent years. Essentially one aims at recovering a desired signal or image with the least possible amount of basis components. The major de- velopments in compressed sensing have been achieved by replacing the ℓ0 norm by the ℓ1 one, the latter being a convex relation of the former and known to also promote sparsity. The use of the ℓ1 norm leads to recovering optimization problems solvable in polynomial time (in most of the cases equivalent to linear programs), and a number of sparse optimization techniques have been developed for the numerical solution of such problems. These ideas have already been used in portfolio selection primarily to promote regularization of ill conditioning (of the estimation of data or of the variance of the return itself). DeMiguel et al. [13] constrained the Markowitz classical model by imposing a bound on the ℓ1 norm of the vector of portfolio positions, among other possibilities. Brodie et al. [8] focus on a modiﬁcation to the Markowitz mean-variance classical model by the incorporation of a term involving a multiple of the ℓ1 norm of the vector of port- folio positions. Inspired by sparse reconstruction (see, for instance, [7]), they also proposed an heuristic for the solution of the problem. 2.3 The cardinality constrained Markowitz mean-variance model Since the appearance of the classical Markowitz mean-variance model, a number of methodologies have been proposed to render it more realistic. The classical Markowitz model assumes a perfect market without transaction costs or taxes, but such costs are an important issue to consider as far as the portfolio selec- tion is concerned, especially for small investors. Recently, it has been studied the addition of a constraint that sets an upper bound on the number of ac- tive positions taken in the portfolio, in an attempt to improve performance and reduce transactions costs. Such a cardinality constraint is deﬁned by limiting card(x) = \|{i ∈{1, ..., N} : xi ̸= 0}\| and leads to cardinality constrained port- folio selection problems. In particular, the cardinality constrained Markowitz mean-variance optimization problem has the form: min w∈RN w⊤Qw subject to µ⊤w ≥r, card(w) ≤K, e⊤w = 1, Li ≤wi ≤Ui i = 1 N (3) min w∈RN w⊤Qw subject to µ⊤w ≥r, (3) e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N, where K ∈{1, . . . , N}. Although card(x) is not a norm, it is frequently called the ℓ0 norm in the literature, ∥x∥0 = card(x). By introducing binary variables, one can rewrite the problem as a mixed-integer quadratic programming (MIQP) problem: where K ∈{1, . . . , N}. Although card(x) is not a norm, it is frequently called the ℓ0 norm in the literature, ∥x∥0 = card(x). By introducing binary variables, one can rewrite the problem as a mixed-integer quadratic programming (MIQP) problem: min w,y∈RN w⊤Qw subject to µ⊤w ≥r, e⊤y ≤K, e⊤w = 1, Liyi ≤wi ≤Uiyi, i = 1, . . . , N, yi ∈{0, 1}, i = 1, . . . , N. (4) e⊤y ≤K, (4) Liyi ≤wi ≤Uiyi, i = 1, . . . , N, yi ∈{0, 1}, i = 1, . . . , N. yi ∈{0, 1}, i = 1, . . . , N. However such MIQPs are known to be hard combinatorial problems. The num- ber of sparsity patterns in w (i.e., number of diﬀerent possibilities of having K 58 R.P. Brito, L.N. Vicente nonzeros entries) is N K = N!/[(N −K)!K!]. Although there are exact algorithms for the solution of MIQPs (see [5,6,7,25]), many researchers and portfolio man- agers prefer to use heuristics approaches (see [3,9,11,15,17,26]). 3 The cardinality/mean-variance biobjective model Although the cardinality constrained Markowitz mean-variance model described in (3) provides an alternative to the classical Markowitz model in the sense of realistically limiting the number of active positions in a portfolio, it is dependent on the parameter K, the maximum number of such positions. Thus, one has to vary K to obtain various levels of cardinality or sparsity, and for each value of K solve an MIQP of the form (4). The alternative suggested in this paper is to consider the cardinality func- tion as an objective function itself. At a ﬁrst glance, one could see the prob- lem as a triobjective optimization problem by minimizing the variance of the return, maximizing the expected return, and minimizing the cardinality over the set of feasible portfolios. Such a framework was taken into account in the studies [1,2,10,18]. However, these authors did not investigate the eﬀects of car- dinality constraints on portfolio models in terms of out-of-sample performance, a subject still poorly analyzed in the literature. On the other hand, investors may ﬁnd it useful to directly analyze the tradeoﬀbetween cardinality and mean- variance. A parameter-free possibility is to consider a Sharpe ratio type objec- tive function, by maximizing expected return per variance and minimizing the cardinality, over the set of feasible portfolios. In this case, the cardinality/mean- Eﬃcient cardinality/mean-variance portfolios 59 variance biobjective optimization problem is posed as variance biobjective optimization problem is posed as min w∈RN −µ⊤w w⊤Qw min w∈RN card(w) (5) min w∈RN card(w) ⊤ subject to e⊤w = 1, subject to e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. By solving (5), we identify a cardinality/mean-variance eﬃcient frontier. A port- folio in this frontier is such that there exists no other feasible one which simulta- neously presents a lower cardinality and a lower mean-variance measure. Given such an eﬃcient frontier and a mean-variance target, an investor may directly ﬁnd the answers to the questions of what is the optimal (lowest) cardinality level that can be chosen and what are the portfolios leading to such a cardinal- ity level. Problem (5) has two objective functions and linear constraints. The ﬁrst objective f1(w) = −µ⊤w/w⊤Qw is nonlinear but smooth. However, the second objective function f2(w) = card(w) = \|{i ∈{1, ..., N} : wi ̸= 0}\| is piece- wise linear discontinuous, consequently nonlinear and nonsmooth. 3 The cardinality/mean-variance biobjective model We have thus decided to solve the biobjective optimization problem (5) using a derivative-free solver, based on direct multisearch. 4 Empirical performance of eﬃcient cardinality/mean- variance portfolios Now we report a number of experiments made to numerically determine and assess the eﬃcient cardinality/mean-variance frontier. We applied direct mul- tisearch to determine the Pareto front or eﬃcient frontier of the biobjective optimization problem (5) (according to Appendix A). We tested three data sets collected from the FTSE 100 index and three others from the Fama/French benchmark collection (see Subsection 4.1). The eﬃcient frontiers obtained by the initial in-sample optimization are given in Subsection 4.2. The out-of-sample performance of the cardinality/mean-variance eﬃcient portfolios, measured by a rolling-sample approach, is described in Subsection 4.3. In Subsection 4.4 we measure the out-of-sample performance by the Sharpe ra- tio, in Subsubsection 4.5 we report the proportional transaction costs, and in Subsection 4.6 we measure the out-of-sample performance by the Sharpe ratio of returns net of transaction costs, all of this for each cardinality/mean-variance eﬃcient portfolio. To better assess the robustness of our results, we also con- sidered, using the FTSE 100 data, a sample including the ﬁnancial crisis years 2008–2010, and the corresponding results are reported in Subsection 4.7. The section is ended with a discussion of the overall obtained results. 4.1 Data sets For the ﬁrst three data sets we collected daily data for securities from the FTSE 100 index, from 01/2003 to 12/2007 (ﬁve years). Such data is public and avail- able from the site http://www.bolsapt.com. The three data sets are referred R.P. Brito, L.N. Vicente 60 to as DTS1, DTS2, and DTS3, and are formed by 12, 24, and 48 securities, respectively. The composition of these data sets is given in Table 1. We used the daily continuous returns for the in-sample optimization (estimation of Q and µ) and the daily discrete returns for the out-of-sample analysis. We also included in our experiments three data sets from the Fama/French benchmark collection (FF10, FF17, and FF48, with cardinalities 10, 17, and 48), using the monthly returns from 07/1971 to 06/2011 (forty years) given there for a number of indus- try security sectors. More information on these security sectors (or portfolios of securities) can be found in http://mba.tuck.dartmouth.edu/pages/faculty/ ken.french/data_library.html. SECURITIES 3 I GROUP (1,2,3) JOHNSON MATTHEY P (3) AMEC (1,2,3) LEGAL & GENERAL (3) ANGLO AMERICAN (1,2,3) LLOIDS BANKING GR (3) ANTOFAGASTA (1,2,3) LONMIN (3) ASSOCIAT BRIT FOO (1,2,3) MARKS & SPENCER (3) ASTRAZENECA (1,2,3) MORRINSON SUPERMKT (3) AVIVA (1,2,3) NEXT (3) B SKY B GROUP (1,2,3) OLD MUTUAL (3) BAE SYSTEMS (1,2,3) PEARSON (3) BARCLAYS (1,2,3) PRUDENTIAL (3) BG GROUP (1,2,3) REED ELSEVIER PLC (3) BHP BILLITON (1,2,3) RENTOKIL INITIAL (3) BP (2,3) REXAM (3) BRIT AMER TOBACCO (2,3) RIO TINTO (3) BRIT LAND CO REIT (2,3) ROYAL BK SCOTL GR (3) BRITISH AIRWAYS (2,3) RSA INSUR GRP (3) CAB & WIRE WRLD (2,3) SABMILLER (3) CAPITA GRP (2,3) SAGE GRP (3) COBHAM (2,3) SAINSBURY (3) DIAGEO (2,3) SCHRODERS (3) HAMMERSON REIT (2,3) SEVERN TRENT (3) IMPERIAL TOBACCO (2,3) SHIRE (3) INTERNATIONAL POW (2,3) UNITED UTILITIES (3) INVENSYS (2,3) VODAFONE GRP (3) Table 1. Composition of the three data sets from the FTSE 100 index. In brackets we indicate the data set to which each security belongs to. ( , ) ( ) Table 1. Composition of the three data sets from the FTSE 100 index. In brackets we indicate the data set to which each security belongs to. 4.2 In-sample optimization We then applied the solver dms (version 0.2) to compute the eﬃcient frontier (or Pareto front) of the cardinality/mean-variance biobjective optimization prob- lem (5). A few modiﬁcations to (5) were made before applying the solver as well as a few changes to the solver default parameters (the details are described in Appendix A). We present results for the initial in-sample optimization. For the FTSE 100 data sets this sample is from 01/2003 to 12/2006 and for the FF data sets is from 07/1971 to 06/1996. Figures 1–3 and Figures 4–6 contain the plots of the eﬃcient frontiers calculated for, respectively, the FTSE 100 and FF data sets. In all these plots we also marked three other portfolios. The ﬁrst one is the 1/N portfolio corresponding to the naive strategy. A second one is obtained maximizing expected return per variance min w∈RN −µ⊤w w⊤Qw subject to e⊤w = 1. (6) (6) 61 Eﬃcient cardinality/mean-variance portfolios This portfolio corresponds to the extreme point (of maximum cardinality) of the eﬃcient frontier (or Pareto front) of the cardinality/mean-variance biobjective optimization problem (5). The third one is a classical Markowitz related portfolio and is obtained by minimizing variance under no short-selling min w∈RN w⊤Qw subject to e⊤w = 1, w ≥0. (7) (7) w ≥0. This instance was solved using the quadprog function from the MATLAB [24] Optimization Toolbox. Regarding problem (7), it is known that not allowing short-sale has a regularizing eﬀect on minimum-variance Markowitz portfolio selection (see [16]) and leads to portfolios of low cardinality. Since we know that minimum variance portfolios outperform mean-variance portfolios (the estimate error of the expected returns is eliminated, see [16]), we considered the following cardinality constrained minimum variance model (instead of the one introduced in Section 2.3) min w∈RN w⊤Qw subject to card(w) ≤K, e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. min w∈RN w⊤Qw subject to card(w) ≤K, e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. Li ≤wi ≤Ui, i = 1, . . . , N. By introducing binary variables, one can rewrite this problem as a mixed-integer quadratic programming (MIQP) problem: min w,y∈RN w⊤Qw subject to e⊤y ≤K, e⊤w = 1, Liyi ≤wi ≤Uiyi, i = 1, . . . , N, yi ∈{0, 1}, i = 1, . . . , N. 4.2 In-sample optimization (8) (8) e⊤w = 1, We also mark in the plots the portfolios that result from solving problem (8) for each value of K ∈[1, N]. For this purpose we used the solver cplexmiqp from ILOG IBM CPLEX for MATLAB [22]. 4.5 Transaction costs Since one is rebalancing portfolios for each out-of-sample period, one can com- pute the transaction costs of such a trade. We set the proportional transaction cost equal to 50 basis points per transaction (as usually assumed in the litera- ture). Thus the cost of a trade over all assets is given by TC = T −1 X t=1 0.5% N X i=1 \| wi,t+1 −wi,t \|, (9) (9) with T = 12 for the FTSE 100 data sets and T = 15 for the FF data sets. The results are given in Figures 13–15 for the FTSE 100 portfolios and in Figures 16– 18 for the FF ones. 4.4 Out-of-sample performance measured by the Sharpe ratio In each period of evaluation, the out-of-sample performance was then measured by the Sharpe ratio S = m −rf σ , where m is the mean return, rf is the return of the risk-free asset3, and σ is the standard deviation. The results (over all the periods of evaluation) are given in Figures 7–9 for the FTSE 100 portfolios and in Figures 10–12 for the FF ones. Using IBM SPSS Statistics [23] we calculated the p-values for the statistical signiﬁcance of the diﬀerence between Sharpe ratios of the benchmark naive portfolio and all the others computed portfolios. We did not report them here because they are not statistically signiﬁcant. where m is the mean return, rf is the return of the risk-free asset3, and σ is the standard deviation. The results (over all the periods of evaluation) are given in Figures 7–9 for the FTSE 100 portfolios and in Figures 10–12 for the FF ones. Using IBM SPSS Statistics [23] we calculated the p-values for the statistical signiﬁcance of the diﬀerence between Sharpe ratios of the benchmark naive portfolio and all the others computed portfolios. We did not report them here because they are not statistically signiﬁcant. 4.3 Out-of-sample performance The analysis of out-of-sample performance relies on a rolling-sample approach. For the FTSE 100 data sets we considered 12 periods (months) of evalua- tion. We begin by computing the eﬃcient frontier (or Pareto front) of the cardinality/mean-variance biobjective optimization problem (5) for the in-sample time window from 01/2003 to 12/2006 (see Subsection 4.2). We then held ﬁxed R.P. Brito, L.N. Vicente 62 each portfolio and observed its returns over the next period (January 2007). Then we discarded January 2003 and brought January 2007 into the sample. We repeated this process until exhausting the 12 months of 2007. We applied the same rolling-sample approach to the FF data sets, considering an initial in-sample time window from 07/1971 to 06/1996 (see Subsection 4.2) and 15 periods of evaluation (the 15 next years). 3 For the FTSE 100 data sets we used the 3 month Treasury-Bills UK. Such data is public and made available by the Bank of England, at the site http://www. bankofengland.co.uk. For the FF data sets we used the 90-day Treasury-Bills US. Such data is public and made available by the Federal Reserve, at the site http://www.federalreserve.gov. 4 We used as a risk-free asset the daily startling certiﬁcate of deposit interest rate. Such data is public and made available by the Bank of England, at the site http: //www.bankofengland.co.uk. 4.7 Results including the ﬁnancial crisis years 2008–2010 The FTSE 100 data set used covered the period 2003–2007. With the aim of testing the robustness of the results, we also tried a FTSE 100 data set that covers the time window 2003–2010 (including thus the ﬁnancial crisis years 2008– 2010). The data sets were formed as described in Section 4.1, but excluding British Airways (see Table 1) due to missing data during the period considered, and including Wolseley (following an arbitrary alphabetic order). We performed an out-of-sample analysis as described in Section 4.3. We used daily periods of evaluation. We began by computing the eﬃcient frontier (or Pareto front) of the cardinality/mean-variance biobjective optimization problem (5) for the in- sample time window from 01/2003 to 12/2010 (using daily data). We then held ﬁxed each portfolio and observed its returns over the next period (ﬁrst trading day of January 2011). Then we discarded this ﬁrst trading day of January 2011 and brought this into the sample. We repeated this process until exhausting the ﬁrsts 15 trading days of 2011. The results of the out-of-sample performance measured by the Sharpe ratio4 are given in Figures 25–27. The results of the proportional transaction costs, are given in Figures 28–30. The results of the out-of-sample performance measured by the Sharpe ratio of returns net of transaction costs, are given in Figures 31–33. 4.6 Out-of-sample performance measured by the Sharpe ratio of returns net of transaction costs In the presence of transaction costs we calculated the Sharpe ratio of returns net of transaction costs C SR = m −TC −rf σ , SR = m −TC −rf σ , 3 For the FTSE 100 data sets we used the 3 month Treasury-Bills UK. Such data is public and made available by the Bank of England, at the site http://www. bankofengland.co.uk. For the FF data sets we used the 90-day Treasury-Bills US. Such data is public and made available by the Federal Reserve, at the site http://www.federalreserve.gov. 3 For the FTSE 100 data sets we used the 3 month Treasury-Bills UK. Such data is public and made available by the Bank of England, at the site http://www. bankofengland.co.uk. For the FF data sets we used the 90-day Treasury-Bills US. Such data is public and made available by the Federal Reserve, at the site http://www.federalreserve.gov. Eﬃcient cardinality/mean-variance portfolios 63 where m is the mean return, TC is the proportional transaction cost in (9), rf is the return of the risk-free asset, and σ is the standard deviation. The out- of-sample performance was then measured by the Sharpe ratio of returns net of transaction costs. The results are given in Figures 19–21 for the FTSE 100 portfolios and in Figures 22–24 for the FF ones. 4.8 Discussion of the results Contrary to one could think, given the intractability of f2(w) = card(w) and the fact that no derivatives are being used for f1(w) = −µ⊤w/w⊤Qw, direct multisearch (the solver dms) was capable of quickly determining (in-sample) the eﬃcient frontier for the biobjective optimization problem (5). For instance, for the data sets of roughly 50 assets, a regular laptop takes a few dozens of seconds to produce the eﬃcient frontiers. We have a direct way of dealing with sparsity, which oﬀers a complete determination of an eﬃcient frontier for all cardinalities. According to a priori preferences, one could choose (in-sample) the desired cardinality. For the portfolios constructed using the FTSE 100 index data (portfolios of individual securities), a large number of our sparse portfolios, among the eﬃcient cardinality/mean-variance ones, consistently overcame the naive strategy and at least one of the two related classical Markowitz models, in terms of out-of-sample performance measured by the Sharpe ratio. This eﬀect R.P. Brito, L.N. Vicente 64 has even happened for the largest data set (DTS3 with 48 securities), where the demand for sparsity is more relevant. For the portfolios constructed using the Fama/French benchmark collection (where securities are portfolios rather than individual securities), the scenario is diﬀerent since the behavior of the naive strategy is even more diﬃcult to outperform. Still, a large number of sparse eﬃcient cardinality/mean-variance portfolios consistently overcame the naive strategy. In both cases, FTSE 100 and FF data, the transaction costs of the eﬃ- cient cardinality/mean-variance portfolios are lower than the mean per variance portfolio (solution of problem (6)) and higher than the minimum-variance port- folio (solution of problem (7)). Note that the minimum-variance portfolio does not allow short-selling, and so the weights at the outset are much more lim- ited, thus leading to better results. Evaluating the performance out-of-sample by the Sharpe ratio of returns net of transaction costs (take into account the transaction costs), the eﬃcient cardinality/mean-variance portfolios do not over- came the naive strategy for FTSE 100 data, but for FF data a large number of sparse eﬃcient cardinality/mean-variance portfolios still consistently overcame the naive strategy. When we compare the performance results between the eﬃ- cient cardinality/mean-variance portfolios and the cardinality constrained min- imum variance portfolios (solution of (8)), without considering the transaction costs, we observed better results for the FTSE 100 and worse for the FF. 5 Conclusions and perspectives for future work In this paper we have developed a new methodology to deal with the compu- tation of mean-variance Markowitz portfolios with pre-speciﬁed cardinalities. Instead of imposing a bound on the maximum cardinality or including a penal- ization or regularization term into the objective function (in classical Markowitz mean-variance models), we took the more direct approach of explicitly consider- ing the cardinality as a separate goal. This led us to a cardinality/mean-variance biobjective optimization problem (5) whose solution is given in the form of an ef- ﬁcient frontier or Pareto front, thus allowing the investor to tradeoﬀamong these two goals when having transaction costs and portfolio management in mind. In addition, and surprisingly, a signiﬁcant portion of the eﬃcient cardinality/mean- variance portfolios (with cardinality values considerably lower than the num- ber N of securities) have exhibited superior out-of-sample performance (under reasonably low transaction costs that only increase moderately with cardinality). We solved the biobjective optimization problem (5) using a derivative-free solver running direct multisearch. Direct-search methods based on polling are known in general to be slow but extremely robust due their directional properties. Such a feature is crucial given the diﬃculty of the problem (one discontinuous objective function, the cardinality, and discontinuous Pareto fronts). We have observed the robustness of direct multisearch, in other words, its capability of successfully solving a vast majority of the instances (all in our case) even if at the expense of a large budget of function evaluations. Direct multisearch was applied oﬀ-the-shelf to determine the cardinality/mean-variance eﬃcient frontier. The structure of problem (5), or of its practical counterpart (10), was essentially ignored. One can use the fact that the ﬁrst objective function is smooth and of known derivatives to speed up the optimization and reduce even further the budget of function evaluations. Moreover, we also point out that it is trivial to run the poll step of direct multisearch in a parallel mode. The use of derivative-free single or multiobjective optimization opens the research range of future work in sparse or dense portfolio selection. In fact, since derivative-free algorithms only rely on zero order information, they are applicable to any objective function of black-box type. One can thus use any measure to quantify the proﬁt and risk of a portfolio. The classical Markowitz model assumes that the return of a portfolio is a linear combination of the returns of the individual securities. 4.8 Discussion of the results The MIQP performed better in terms of Sharpe ratio of returns net of transaction costs since the cost of transaction costs are lower, one possible explanation for this is the fact of not taking into account the estimation of the expected returns. Moreover, our cardinality/mean-variance portfolios are truly eﬃcient whereas the cardinality constrained minimum variance do not necessarily exhibit Pareto eﬃciency. For the FTSE 100 data set, the analysis including the ﬁnancial crisis years 2008–2010 shows that the results are robust. Finally, we also computed the cardinality/mean-variance eﬃcient frontier for the data set FF100, where portfolios are formed on size and book-to-market (see Figure 34). (This time we needed a budget of the order of 107 function evaluations, see Appendix A.) We remark that FF48 and FF100 are the data sets also used in [8]. In this paper, as we said before, the authors focus on a modiﬁcation to the Markowitz classical model by the incorporation of a term involving a multiple of the ℓ1 norm of the vector of portfolio positions. Despite the diﬀerent sparse-oriented techniques and diﬀerent strategies for evaluating out-of-sample performance, in both approaches (theirs and ours), sparse portfolios are found overcoming the naive strategy. In our approach one computes sparse portfolios satisfying an eﬃcient or nondomi- nant property and one does it directly and in single run, whereas in [8], there is a need to vary a tunable parameter and select the portfolios according to some criterion to be met (for example, sparsity). It is unclear what sort of eﬃcient or nondominant property their portfolios satisfy. Moreover, we provide results for all cardinality values (from 1 to 48 in FF48 and from 1 to 100 in FF100), while in [8] the authors report results for cardinality values from 4 and 48 (FF48) and from 3 to 60 (FF100). We therefore claim to have a more direct way of dealing Eﬃcient cardinality/mean-variance portfolios 65 with sparsity, which oﬀers a complete determination of an eﬃcient frontier for all cardinalities. A Using direct multisearch to determine eﬃcient cardinality/mean-variance portfolios A few modiﬁcations to problem (5) were required to make it solvable by a mul- tiobjective derivative-free solver, in particular by a direct multisearch one. In practice the ﬁrst modiﬁcation to (5) consisted of approximating the true cardi- nality, by introducing a tolerance ϵ, min w∈RN −µ⊤w w⊤Qw min w∈RN PN i=1 11{\|wi\|>ϵ} subject to e⊤w = 1, subject to e⊤w = 1, Li ≤wi ≤Ui, i = 1, . . . , N. chosen as ϵ = 10−8 (11 represents the indicator function). Secondly, we selected symmetric bounds on the variables Li = −b and Ui = b, min w∈RN −µ⊤w w⊤Qw min w∈RN PN i=1 11{\|wi\|>ϵ} subject to e⊤w = 1, min w∈RN −µ⊤w w⊤Qw min w∈RN PN i=1 11{\|wi\|>ϵ} subject to e⊤w = 1, −b ≤wi ≤b, i = 1, . . . , N, min w∈RN −µ⊤w w⊤Qw min w∈RN PN i=1 11{\|wi\|>ϵ} min w∈RN PN i=1 11{\|wi\|>ϵ} subject to e⊤w = 1, −b ≤wi ≤b, i = 1, . . . , N, setting b = 10. Finally, we eliminated the constraint e⊤w = 1 since direct search methods do not cope well with equality constraints. The version fed to the dms solver was then min w(1:N−1)∈RN−1 −µ⊤w w⊤Qw min w(1:N−1)∈RN−1 PN−1 i=1 11{\|wi\|>ϵ} subject to −b ≤wi ≤b, i = 1, . . . , N −1, −b ≤1 −PN−1 i=1 wi ≤b, (10) (10) where wN in −µ⊤w/w⊤Qw was replaced by 1 −PN−1 i=1 wi. where wN in −µ⊤w/w⊤Qw was replaced by 1 −PN−1 i=1 wi. We used all the default parameters of dms (version 0.2) with the following four exceptions. First, we needed to increase the maximum number of function evaluations allowed (from 20000 to 2000000 for N(= n) up to 50) given the dimension of our portfolios, as well as to require more accuracy by reducing the step size tolerance from 10−3 to 10−7. Then we turned oﬀthe use of the cache of previously evaluated points to make the runs faster (the default version of dms keeps such a list to avoid evaluating points too close to those already evaluated). Lastly, we realized that initializing the list of feasible nondominated points with a singleton led to better results than initializing it with a set of roughly N points as it happens by default. 5 Conclusions and perspectives for future work Also, it implicitly assumes a Gaussian distribution for the return, letting its variance be a natural measure of risk. However, it is known from the analysis of stylized facts that the distribution for the return of securities exhibits tails which are fatter than the Gaussian ones. Practitioners consider other measures of risk and proﬁt better tailored to reality. Our approach to compute the cardinality/mean-variance eﬃcient frontier is ready for application in such general scenarios. R.P. Brito, L.N. Vicente 66 References 1. K. P. Anagnostopoulos and G. Mamanis. A portfolio optimization model with three objectives and discrete variables. Comput. Oper. Res., 37:1285–1297, 2010. 2. K. P. Anagnostopoulos and G. Mamanis. 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The Review of Financial Studies, 22:1915–1953, 2009. 15. J. E. Fieldsend, J. Matatko, and M. Peng. Cardinality constrained portfolio opti- misation. volume 3177 of Lecture Notes in Comput. Sci., pages 788–793. Springer, Berlin, 2004. 16. R. Jagannathan and T. Ma. Risk reduction in large portfolios: Why imposing the wrong constraints hfelps. The Journal of Finance, 58:1651–1684, 2003. 17. D. Lin, S. Wang, and H. Yan. A multiobjective genetic algorithm for portfolio selection. Working Paper. Institute of Systems Science, Academy of Mathematics and Systems Science Chinese Academy of Sciences, Beijing, China, 2001. 18. D. Di Lorenzo, G. Liuzzi, F. Rinaldi, F. Eﬃcient cardinality/mean-variance portfolios 67 A Using direct multisearch to determine eﬃcient cardinality/mean-variance portfolios Thus, we set the option list of dms to zero, which, given the bounds on the variables, assigns the origin to the initial list. Eﬃcient cardinality/mean-variance portfolios References Schoen, and M. Sciandrome. A concave optimization-based approach for sparse portfolio selection. Optim. Methods Softw., 27:983–1000, 2012. 19. H. M. Markowitz. Portfolio selection. The Journal of Finance, 7:77–91, 1952. 20. H. M. Markowitz. Portfolio selection: Eﬃcient diversiﬁcation of investments. Cowles Foundation Monograph, 16, 1959. 68 R.P. Brito, L.N. Vicente 21. M. C. Steinbach. Markowitz revisited: Mean-variance models in ﬁnancial portfolio analysis. SIAM Rev., 43:31–85, 2001. 22. IBMTM. IBM ILOG CPLEX®. 23. IBMTM. IBM SPSS Statistics®. 24. The MathWorksTM. MATLAB®. 25. J. P. Vielma, S. Ahmed, and G. L. Nemhauser. A lifted linear programming branch- and-bound algorithm for mixed-integer conic quadratic programs. INFORMS J. Comput., 20:438–450, 2008. 26. M. Woodside-Oriakhi, C. Lucas, and J. E. Beasley. Heuristic algorithms for the cardinality constrained eﬃcient frontier. European J. Oper. Res., 213:538–550, 2011. Eﬃcient cardinality/mean-variance portfolios 69 Fig. 2. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS2. See the caption of Figure 1 for an explanation of the various symbols. Fig. 2. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS2. See the caption of Figure 1 for an explanation of the various symbols. Fig. 1. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS1. ⋆Naive ▼Markowitz mean per variance ■Markowitz minimum variance ■Markowitz minimum variance • cardinality/mean-variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 3. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS3. See the caption of Figure 1 for an explanation of the various symbols. Fig. 3. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS3. See the caption of Figure 1 for an explanation of the various symbols. Fig. 3. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for DTS3. See the caption of Figure 1 for an explanation of the various symbols. Fig. 4. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF10. ⋆Naive ⋆Naive ▼Markowitz mean per variance ■Markowitz minimum variance ■Markowitz minimum variance • cardinality/mean-variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 5. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF17. See the caption of Figure 4 for an explanation of the various symbols. Fig. 6. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF48. See the caption of Figure 4 for an explanation of the various symbols. Fig. 5. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF17. See the caption of Figure 4 for an explanation of the various symbols. Fig. 6. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF48. See the caption of Figure 4 for an explanation of the various symbols. Fig. 6. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF48. See the caption of Figure 4 for an explanation of the various symbols. Fig. R.P. Brito, L.N. Vicente 5. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF17. See the caption of Figure 4 for an explanation of the various symbols. R.P. Brito, L.N. Vicente 70 Fig. 8. Out-of-sample performance for DTS2 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 7 for an explanation of the various symbols and lines. Fig. 8. Out-of-sample performance for DTS2 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 7 for an explanation of the various symbols and lines. Fig. 8. Out-of-sample performance for DTS2 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 7 for an explanation of the various symbols and lines. Fig. 7. Out-of-sample performance for DTS1 measured by the Sharpe ratio over all the out-of-sample periods. - - Naive - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 10. Out-of-sample performance for FF10 measured by the Sharpe ratio over all the out-of-sample periods. Fig. 9. Out-of-sample performance for DTS3 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 7 for an explanation of the various symbols and lines. - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 11. Out-of-sample performance for FF17 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 11. Out-of-sample performance for FF17 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 12. Out-of-sample performance for FF48 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 12. Out-of-sample performance for FF48 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 11. Out-of-sample performance for FF17 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 12. R.P. Brito, L.N. Vicente Out-of-sample performance for FF48 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Fig. 11. Out-of-sample performance for FF17 measured by the Sharpe ratio over all the out-of-sample periods. See the cap- tion of Figure 10 for an explanation of the various symbols and lines. Eﬃcient cardinality/mean-variance portfolios 71 Fig. 13. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS1. — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance Fig. 14. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2. See the caption of Figure 13 for an explanation of the various symbols and lines. Fig. 14. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2. See the caption of Figure 13 for an explanation of the various symbols and lines. Fig. 13. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS1. Fig. 13. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS1. Fig. 13. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS1. Fig. 14. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2. See the caption of Figure 13 for an explanation of the various symbols and lines. — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance i f h ﬃ Fig. 15. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS3. See the caption of Figure 13 for an explanation of the various symbols and lines. Fig. 16. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF10. Fig. 16. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF10. Fig. 15. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS3. See the caption of Figure 13 for an explanation of the various symbols and lines. — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 17. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF17. See the caption of Figure 16 for an explanation of the various symbols and lines. Fig. 18. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF48. See the caption of Figure 16 for an explanation of the various symbols and lines. Fig. 17. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF17. R.P. Brito, L.N. Vicente See the caption of Figure 16 for an explanation of the various symbols and lines. Fig. 18. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF48. See the caption of Figure 16 for an explanation of the various symbols and lines. Fig. 18. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for FF48. See the caption of Figure 16 for an explanation of the various symbols and lines. R.P. Brito, L.N. Vicente 72 Fig. 20. Out-of-sample performance for DTS2 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 20. Out-of-sample performance for DTS2 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 20. Out-of-sample performance for DTS2 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 20. Out-of-sample performance for DTS2 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 19. Out-of-sample performance for DTS1 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. - - Naive - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 21. Out-of-sample performance for DTS3 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 21. Out-of-sample performance for DTS3 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 19 for an explanation of the various symbols and lines. Fig. 22. Out-of-sample performance for FF10 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. - - Naive - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 23. R.P. Brito, L.N. Vicente Out-of-sample performance for FF17 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 22 for an explanation of the various symbols and lines. Fig. 23. Out-of-sample performance for FF17 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 22 for an explanation of the various symbols and lines. Fig. 24. Out-of-sample performance for FF48 measured by the Sharpe ratio of re- turns net of transaction costs over all the out-of-sample periods. See the caption of Figure 22 for an explanation of the various symbols and lines. Eﬃcient cardinality/mean-variance portfolios 73 Fig. 26. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. See the caption of Figure 25 for an explanation of the various symbols and lines. Fig. 25. Out-of-sample performance for DTS1, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. - - Naive — Markowitz mean per variance -.- Markowitz minimum variance Fig. 26. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. See the caption of Figure 25 for an explanation of the various symbols and lines. Fig. 26. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. See the caption of Figure 25 for an explanation of the various symbols and lines. Fig. 25. Out-of-sample performance for DTS1, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 27. Out-of-sample performance for DTS3, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. See the caption of Figure 25 for an explanation of the various symbols and lines. Fig. 28. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS1, including the ﬁnancial crisis years 2008–2010. Fig. 27. Out-of-sample performance for DTS3, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio over all the out-of-sample periods. See the caption of Figure 25 for an explanation of the various symbols and lines. R.P. Brito, L.N. Vicente — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance Fig. 29. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2, including the ﬁnancial crisis years 2008–2010. See the caption of Figu- re 28 for an explanation of the various sym- bols and lines. Fig. 30. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS3, including the ﬁnancial crisis years 2008–2010. See the caption of Figu- re 28 for an explanation of the various sym- bols and lines. Fig. 29. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2, including the ﬁnancial crisis years 2008–2010. See the caption of Figu- re 28 for an explanation of the various sym- bols and lines. Fig. 29. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS2, including the ﬁnancial crisis years 2008–2010. See the caption of Figu- re 28 for an explanation of the various sym- bols and lines. Fig. 30. Transaction costs of the eﬃ- cient cardinality/mean-variance portfolios for DTS3, including the ﬁnancial crisis years 2008–2010. See the caption of Figu- re 28 for an explanation of the various sym- bols and lines. R.P. Brito, L.N. Vicente 74 Fig. 32. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. See the caption of Figure 31 for an explanation of the vari- ous symbols and lines. Fig. 31. Out-of-sample performance for DTS1, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. - - Naive — Markowitz mean per variance - - Markowitz minimum variance Fig. 32. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. See the caption of Figure 31 for an explanation of the vari- ous symbols and lines. Fig. 32. Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. See the caption of Figure 31 for an explanation of the vari- ous symbols and lines. Fig. 31. Out-of-sample performance for DTS1, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. Fig. 32. R.P. Brito, L.N. Vicente Out-of-sample performance for DTS2, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. See the caption of Figure 31 for an explanation of the vari- ous symbols and lines. - - Naive — Markowitz mean per variance -.- Markowitz minimum variance • cardinality/mean-variance ♦cardinality constrained minimum vari- ance ♦cardinality constrained minimum vari- ance Fig. 34. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF100. Fig. 34. Eﬃcient frontier of the biobjecti- ve cardinality/mean-variance problem for FF100. Fig. 33. Out-of-sample performance for DTS3, including the ﬁnancial crisis years 2008–2010, measured by the Sharpe ratio of returns net of transaction costs over all the out-of-sample periods. See the caption of Figure 31 for an explanation of the vari- ous symbols and lines.
https://openalex.org/W3009741914	https://europepmc.org/articles/pmc7085724?pdf=render	English	null	Blind Quality Assessment of Iris Images Acquired in Visible Light for Biometric Recognition	Sensors	2,020	cc-by	15,823	Mohsen Jenadeleh 1,* , Marius Pedersen 2 and Dietmar Saupe 1 1 Department of Computer and Information Science, University of Konstanz, 78457 Konstanz, Germany; dietmar.saupe@uni-konstanz.de p 2 Department of Computer Science, Norwegian University of Science and Technology, N-2802 Gjøvik, Norway marius.pedersen@ntnu.no d h d l h k d 2 Department of Computer Science, Norwegian University of Science and Technology, N-2802 Gjøvik, Norway; marius.pedersen@ntnu.no * Correspondence: mohsen.jenadeleh@uni-konstanz.de * Correspondence: mohsen.jenadeleh@uni-konstanz.de p j † This paper is an extended version of the conference paper: Jenadeleh, M.; Pedersen, M.; Saupe, D. Realtime quality assessment of iris biometrics in visible light. In Proceedings of the 2018 IEEE/CVF Conference on Computer Vision and Pattern Recognition Workshops (CVPRW), Salt Lake City, UT, USA, 18–22 June 2018. † This paper is an extended version of the conference paper: Jenadeleh, M.; Pedersen, M.; Saupe, D. Realtime quality assessment of iris biometrics in visible light. In Proceedings of the 2018 IEEE/CVF Conference on Computer Vision and Pattern Recognition Workshops (CVPRW), Salt Lake City, UT, USA, 18–22 June 2018. Received: 4 January 2020; Accepted: 25 February 2020; Published: 28 February 2020 Abstract: Image quality is a key issue affecting the performance of biometric systems. Ensuring the quality of iris images acquired in unconstrained imaging conditions in visible light poses many challenges to iris recognition systems. Poor-quality iris images increase the false rejection rate and decrease the performance of the systems by quality ﬁltering. Methods that can accurately predict iris image quality can improve the efﬁciency of quality-control protocols in iris recognition systems. We propose a fast blind/no-reference metric for predicting iris image quality. The proposed metric is based on statistical features of the sign and the magnitude of local image intensities. The experiments, conducted with a reference iris recognition system and three datasets of iris images acquired in visible light, showed that the quality of iris images strongly affects the recognition performance and is highly correlated with the iris matching scores. Rejecting poor-quality iris images improved the performance of the iris recognition system. In addition, we analyzed the effect of iris image quality on the accuracy of the iris segmentation module in the iris recognition system. Keywords: biometric recognition; visible light iris images; image quality assessment; image covariates; quality ﬁltering sensors sensors sensors Blind Quality Assessment of Iris Images Acquired in Visible Light for Biometric Recognition † Mohsen Jenadeleh 1,* , Marius Pedersen 2 and Dietmar Saupe 1 ohsen Jenadeleh 1,* , Marius Pedersen 2 and Dietmar Saupe 1 1. Introduction The stability of iris patterns over the human lifespan and their uniqueness was ﬁrst noticed in 1987 [1]. Since then, biometric iris recognition has been extensively investigated for accurate and automatic personal identiﬁcation and authentication [2]. Most commercial iris recognition systems use near-infrared (NIR) images. However, due to the popularity of smartphones and similar handheld devices with digital cameras, iris recognition systems using images taken in visible light have recently been developed [3–5]. Image quality is a key factor affecting the performance of iris recognition systems [6–8]. In the biometric recognition literature, a biometric quality measure is a covariate that is measurable, inﬂuences performance, and is actionable [9–11]. Quality measurement can include subject and image covariates. Subject covariates are attributes of a person, which may be properties of subjects such as eyelid occlusion, glare, iris deformation, or wearing of glasses. Image covariates depend on sensor and acquisition Sensors 2020, 20, 1308; doi:10.3390/s20051308 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2020, 20, 1308 2 of 22 conditions, such as focus, noise, resolution, compression artifacts, and illumination effects. In this work, we develop a real-time quality measure for image covariates as an actionable quality score, e.g., to decide whether an input iris image sample should be enrolled into a dataset or rejected and a new sample should be captured. The performance of an iris recognition system in visible light suffers from all of the image quality factors mentioned above. To overcome this problem, some researchers have considered image quality in different ways for iris recognition systems [12–17]. However, these systems fall short in two ways: • The considered image covariates and distortions are limited. Only distortions are taken into account that are often seen, such as Gaussian blur, noise, motion blur, and defocus. However, authentic iris images, especially those taken by handheld devices, may additionally suffer from other types of distortion. • Typically, quality assessment is applied to accurately segmented iris images. However, image distortion also affects the performance of the segmentation module of iris recognition systems. Thus, poor image quality can lead to poorly segmented irises and increase in the false rejection rate. In this paper, we propose a general-purpose and fast image quality method that aims to assess the distortion of iris images acquired in unconstrained environments. This method can be used for real-time quality prediction of iris images to rapidly ﬁlter image samples with poor quality. 1. Introduction Iris images with insufﬁcient quality could lead to high dissimilarity scores for matching pairs and increase the false rejection rate of an iris recognition system. We investigate the effect of iris image quality on the recognition performance of a reference iris recognition system for three challenging iris image datasets acquired in visible light. This paper is an extended version of our conference paper [18] and mostly a part of the Ph.D. thesis of the ﬁrst author [19]. The remainder of the paper is organized as follows: Section 2 surveys the literature on iris image quality assessment and iris recognition systems. Section 3 presents the proposed metric for iris image quality assessment. In Section 4, experiments are conducted to study the effect of image quality on the accuracy of iris segmentation. In Section 5, the improvements achieved by ﬁltering poor-quality iris images are discussed using three performance measures on three large iris image datasets acquired in visible light. The paper concludes with suggestions for future research in Section 6. 2. Related Work They focused on the evaluation of iris images after iris segmentation, which allows the systems to process images of poor and good quality in the acquisition phase. They considered only a few image covariates for quality estimation. Proença [3] proposed a metric for the quality assessment of iris images taken in visible light. This metric measures six image quality attributes such as focus score, off-angle score, motion score, occlusion score, iris pigmentation level, and pupil dilation. Then, the impact of image quality on feature matching was analyzed. The results showed a signiﬁcant performance improvement of the iris recognition system by avoiding low-quality images. However, this method requires precisely segmented iris images, and only the motion-blur score is combined with some quality factors related to the subject’s covariates. The authors in [12] proposed an approach that automatically selects the regions of an iris image with the most distinguishably changing patterns between the reference iris image and the distorted version to compute the feature. The measured occlusion and dilation are combined to form a total image quality score to study the correlation between iris image quality and iris recognition accuracy. In the approach of [28], the image quality is assessed locally, based on a fusion schema at the pixel level using a Gaussian mixture model, which gives a probabilistic measure of the quality of local regions of the iris image. The local quality measure is used to detect the poorly segmented pixels and remove them from the fusion process of a sequence of iris images. Recently, many image quality methods have been proposed for perceptual quality assessment of natural images [29–35]. Some of these models use statistics of completed local binary patterns (CLBP) as a part of their feature vectors. In [33], joint statistics of local binary patterns (LBP) and CLBP patterns produced quality-aware features, and a regression function was trained to map the feature space to the perceived quality scores. In [32], features based on several local image descriptors such as CLBP, local conﬁguration patterns (LCP), and local phase quantization (LPQ) were extracted, and then a support vector regressor was used to predict the quality scores. These models are trained to predict the perceptual quality of natural images. Liu et al. [36,37] studied some of these methods for ﬁltering low-quality iris images. 2. Related Work In this section, we review the literature on iris image quality assessment, followed by a brief overview of some state-of-the-art iris recognition systems. Recently, research has been reported to improve the performance of iris recognition systems by considering image quality, but with certain limitations. In some studies, image quality has been examined by considering only certain quality factors, such as sharpness [20], out-of-focus [21], and JPEG compression [22]. These metrics alone cannot be expected to produce reliable quality assessments of authentic in-the-wild iris images. In some other work, iris image quality metrics are applied after segmentation of the iris. In [23], the result of the iris segmentation module is used to form a quality score. Happold et al. [24] proposed a method for predicting the iris matching scores of an iris image pair based on their quality features. They calculated these features for precisely segmented iris images. They labeled a dataset of iris image pairs with the corresponding matching scores. They trained their method for predicting the matching score of an image pair based on their quality features. Therefore, these methods cannot be used to measure iris image quality in the iris recognition system pipeline before segmentation. Several metrics for iris image quality were developed based on a fusion of several quality measures of image and subject covariates. The authors of [25,26] combined quality measures relating to motion blur, 3 of 22 Sensors 2020, 20, 1308 angular deviation, occlusion, and defocus into an overall quality value of an input iris image. These quality metrics were developed for NIR-based images and compared to traditional NIR-controlled iris image acquisition settings. However, images in visible light and under uncontrolled lighting conditions result in notorious differences in the appearance of the acquired images [3]. Therefore, this method may not be used directly to evaluate the quality of iris images in visible light. Li et al. [27] proposed a method for predicting an iris matching score based on iris quality factors such as motion blur, illumination, off-angle, occlusions, and dilation. This method requires segmented irises to compute some of these quality factors (dilation and occlusions). The authors of [10] used combined subject and image covariates, such as the degree of defocusing, occlusion, reﬂection, and illumination, to form an overall quality score. 2. Related Work This study showed inconsistencies for the predicted quality, e.g., removing more low-quality images did not always increase the performance of the iris recognition system. In addition, they removed the low-quality images for each subject separately. Therefore, the ﬁltered images do not have the same range of quality, and there is no global quality-ﬁltering threshold. In summary, some of the methods for iris quality assessment, such as [25,26], are proposed for NIR images, and only a few types of distortion are considered. Some other quality metrics, like those in [3,23,24], require a segmented iris image to calculate their quality features. They also take limited distortion types into account and are not expected to work well for quality assessment of authentic iris images taken in visible light in arbitrary environmental conditions. Iris recognition systems based on authentic images will broaden the scope of iris recognition systems, and require more research to develop robust metrics for quality assessment of authentically distorted iris images. Since we used an iris recognition system as a reference system in this paper, in the following, we brieﬂy review some state-of-the-art iris recognition systems. Sensors 2020, 20, 1308 4 of 22 The fast iris recognition (FIRE) system for images acquired by mobile phones in visible light was proposed by Galdi et al. [38]. It is based on the combination of three classiﬁers by exploiting iris color and texture information. Raja et al. [39] proposed a recognition system for iris images captured in visible light. This method extracts deep sparse features from image blocks and the whole iris image in different color channels to form the feature vector for an input iris image. Minaee et al. [40] proposed an iris feature extraction method based on textural and scattering transform features. The principal component analysis (PCA) technique is used to reduce the extracted feature dimension. Recently, OSIRIS version 4.1, an open-source iris detection system, was proposed by Othman et al. [41]. This system follows the classic Daugman method [42] with some improvements in segmentation, normalization, coding, and matching modules. For iris and pupil segmentation, the Viterbi algorithm is used for optimal contour detection. For normalization, a non-circular iris normalization is performed using the coarse contours detected by the Viterbi algorithm. The coding module is based on 2-D Gabor ﬁlters, which are calculated in different scales and resolutions. 3. Proposed Method In this section, we present our fast and general-purpose method for assessing the quality of iris images acquired in visible light. Earlier works on iris recognition [42,43] employed block-based operations to obtain iris features. Therefore, we can infer that the most distinctive information in the iris pattern comes from the local patterns of an iris image rather than from global features. Local binary patterns (LBP) and their derivatives have been successfully used in many pattern recognition applications, including texture classiﬁcation [44–46], image retrieval [47,48], object recognition [49,50], action recognition [51,52], and biometric recognition [53–56]. Most of the LBP-based biometric recognition methods use statistical analysis of local patterns for their feature extraction. Wu et al. [29] showed that image distortions could change the statistics of LBPs. They then examined the statistics of the LBPs to suggest an index for evaluating natural image quality. However, this index does not accurately predict image quality for some common image distortions, such as Gaussian blur and impulse noise. In the proposed differential sign–magnitude statistics index (DSMI), sign and magnitude patterns are ﬁrst derived. Then, the statistical characteristics of these patterns are analyzed for their sensitivity to iris image distortion. Statistical features of speciﬁc coincidence patterns with high sensitivity to image distortion are identiﬁed. A weighted nonlinear mapping is applied to the features to form the iris image quality score. This metric takes advantage of the observation that low-quality iris images have fewer of these patterns compared with those in high-quality iris images. 2. Related Work Finally, the matching module calculates the global dissimilarity score between two iris codes using the Hamming distance. We used this system as a reference iris recognition system. 3.1. Proposed Quality Metric Our iris image quality metric uses statistical features extracted from patterns of signs and magnitudes of local intensity differences. Then, certain locally weighted statistics of speciﬁc sign–magnitude coincidence patterns are used to deﬁne the quality score. Guo et al. [46] suggested a completed local binary pattern (CLBP) to represent the local difference information that is missed in the LBP representation of an image [57]. We investigate how common distortions in iris images could alter the statistics of the CLBP. Then, a quality metric based on a speciﬁc coincidence of sign and magnitude patterns of the CLBP is proposed. Sensors 2020, 20, 1308 5 of 22 In CLBP, a local grayscale image patch is represented by its central pixel, and the local differences are given by dp = xp −xc, where xc = I(c) is the gray value of the central pixel of the given patch and xp is the gray value of a pixel in the neighborhood. A local difference dp can be decomposed into two components, its sign and its magnitude. These signs and magnitudes of local differences are combined into corresponding patterns, CLBP-S and CLBP-M, as follows. Let C = {(i, j)\|i = 0, · · · , M −1, j = 0, · · · , N −1} be the set of pixels of a normalized grayscale image I of N pixels width by M pixels height. For a given pixel c ∈C, let xc and xp, p = 0, · · · , P −1, denote the gray values of the center pixel c and the P points on a circle of radius R about xc. For example, suppose the coordinates of xc are (0,0); then, the coordinates of xp are (R cos(2πp/P), R sin(2πp/P)). The grayscale value xp is estimated by interpolation if its coordinates do not coincide with the center of a pixel. Then, the CLBP-S patterns are deﬁned by CLBP-SP,R(c) = P−1 ∑ p=0 bp · 2p, bp = ( 1 xp ≥xc 0 otherwise . (1) (1) The CLBP-S operator generates the same code as that of the original LBP operator. The CLBP magnitude patterns are deﬁned similarly by The CLBP-S operator generates the same code as that of the original LBP operator. 3.1. Proposed Quality Metric The patterns in the upper row correspond to CLBP-Sriu2 4,1 , which compares the gray value of the Figure 1. The patterns in the upper row correspond to CLBP-Sriu2 4,1 , which compares the gray value of the central pixel of a patch (xc) with the gray values of its four neighbors (xp). The black and white disks denote smaller and greater values than those of the central pixel value, respectively. In the lower row, CLBP-Mriu2 4,1 compares the absolute values of the differences of the gray values of the central pixel and its neighbors (\|xc −xp\|) with the threshold z from Equation (3). The hatched and white disks denote smaller and greater absolute values than those of the threshold, respectively. Note that the patterns are rotation invariant. Thus, in the case of P = 4 shown here, the patterns for k, l = 1, 2, 3, 5 may be rotated by multiples of 90 degrees without changing the values of CLBP-Sriu2 4,1 and CLBP-Mriu2 4,1 . Finally, the local indices for sign and magnitude have to be combined to give a quality indicator for an iris image as a whole. We ﬁrst join the two types of indices into a set of bitmaps Vk,l(c), indexed by k, l, Finally, the local indices for sign and magnitude have to be combined to give a quality indicator for an iris image as a whole. We ﬁrst join the two types of indices into a set of bitmaps Vk,l(c), indexed by k, l, Vk,l(c) = ( 1 CLBP-Sriu2 P,R (c) = k and CLBP-Mriu2 P,R (c) = l 0 otherwise . (6) (6) For each pair k, l of indices, we form a weighted sum of Vk,l(c) over all pixels c, which is nonlinearly scaled to the unit interval by r(x) = 1 −e−ax as follows: For each pair k, l of indices, we form a weighted sum of Vk,l(c) over all pixels c, which is nonlinearly scaled to the unit interval by r(x) = 1 −e−ax as follows: Qk,l = r 1 \|C\| ∑ c∈C Vk,l(c) ˆσ2(c) + δ2 ! . (7) (7) (7) Here, ˆσ2(c) is the local variance of the P-neighboring pixels of the center pixel c, and δ2 is a small constant value to prevent division by zero. The parameters δ2 and a are empirically set to 0.00025 and 0.01, respectively. 3.1. Proposed Quality Metric The CLBP magnitude patterns are deﬁned similarly by CLBP-MP,R(c) = P−1 ∑ p=0 bp · 2p, bp = ( 1 mp ≥z 0 otherwise, (2) (2) where mp = \|xp −xc\| is the magnitude of the local difference dp. Furthermore, the threshold value z is the average local difference in the P-neighborhoods of all center pixels together, i.e., z = 1 \|C\|P ∑ c∈C P−1 ∑ p=0 \|xp −xc\|. (3) (3) For each pixel c ∈C, we consider the P-bit binary representation of the sums in Equations (1) and (2) as binary codes of CLBP-SP,R and CLBP-MP,R. Using these binary representations, we deﬁne rotation invariant indices or patterns for CLBP-S and CLBP-M in a manner similar to that proposed by Ojala et al. [57] for LBP codes. Equation (4) gives the rotation invariant indices of CLBP-S, CLBP-Sriu2 P,R (c) = G(CLBP-SP,R(c)) = G P−1 ∑ p=0 bp2p ! = ( ∑P−1 p=0 bp U(∑P−1 p=0 bp2p) ≤2 P + 1 otherwise . (4) (4) Here, U gives the number of bit changes (0 to 1 or 1 to 0) of the P-bit binary representation of a number (including circular shift), Here, U gives the number of bit changes (0 to 1 or 1 to 0) of the P-bit binary representation of a number (including circular shift), U P−1 ∑ p=0 bp2p ! = P−1 ∑ p=0 \|bp −b mod (p+1,P)\|. Similarly, Equation (5) gives the uniform rotation invariant patterns of CLBP-M. Similarly, Equation (5) gives the uniform rotation invariant patterns of CLBP-M. CLBP-Mriu2 P,R (c) = G(CLBP-MP,R(c))). (5) CLBP-Mriu2 P,R (c) = G(CLBP-MP,R(c))) (5) Note that these indices, CLBP-Sriu2 P,R and CLBP-Mriu2 P,R , range over the set {0, ..., P + 1}. The ﬁrst indices from 0 up to P correspond to local sign and magnitude patterns with only, at most, two bit changes and, thus, denote uniform local patterns. All non-uniform patterns are assigned to the remaining index P + 1. Note that these indices, CLBP-Sriu2 P,R and CLBP-Mriu2 P,R , range over the set {0, ..., P + 1}. The ﬁrst indices from 0 up to P correspond to local sign and magnitude patterns with only, at most, two bit changes and, thus, denote uniform local patterns. All non-uniform patterns are assigned to the remaining index P + 1. Sensors 2020, 20, 1308 6 of 22 CLBP-Sriu2 P,R generates fewer codes than the basic CLBP-S. 3.1. Proposed Quality Metric It carries less textural information by simplifying the local structure. CLBP-Mriu2 P,R provides a compact representation of textural information derived from local magnitude patterns. For an illustration for the case of P = 4 neighbors at distance R = 1 from the central pixel of a patch, we provide Figure 1. We obtain six indices k and l for sign and magnitude patterns, corresponding to ﬁve rotation invariant uniform patterns (k, l = 0, ..., 4) and one index (k, l = 5) that represents all non-uniform patterns. (a) 𝒌= 0 (b) 𝒌 = 1 (c) 𝒌 = 2 (d) 𝒌 = 3 (e) 𝒌 = 4 (f) 𝒌 = 5 xc xc xc xc xc xc (g) = 0 (h) 𝒍 = 1 (i) 𝒍 = 2 (j) 𝒍 = 3 (m) = 4 (n) = 5 xc xc xc xc xc xc x p≥xc x p<x c CLBP - S4,1 riu 2 \|x p−x c\|<z CLBP- M 4,1 riu 2 \|x p−x c\|≥z Figure 1. The patterns in the upper row correspond to CLBP-Sriu2 4,1 , which compares the gray value of the central pixel of a patch (xc) with the gray values of its four neighbors (xp). The black and white disks denote smaller and greater values than those of the central pixel value, respectively. In the lower row, CLBP-Mriu2 4,1 compares the absolute values of the differences of the gray values of the central pixel and its neighbors (\|xc −xp\|) with the threshold z from Equation (3). The hatched and white disks denote smaller and greater absolute values than those of the threshold, respectively. Note that the patterns are rotation invariant. Thus, in the case of P = 4 shown here, the patterns for k, l = 1, 2, 3, 5 may be rotated by multiples of 90 degrees without changing the values of CLBP-Sriu2 4,1 and CLBP-Mriu2 4,1 . (a) 𝒌= 0 (b) 𝒌 = 1 (c) 𝒌 = 2 (d) 𝒌 = 3 (e) 𝒌 = 4 (f) 𝒌 = 5 xc xc xc xc xc xc (g) = 0 (h) 𝒍 = 1 (i) 𝒍 = 2 (j) 𝒍 = 3 (m) = 4 (n) = 5 xc xc xc xc xc xc x p≥xc x p<x c CLBP - S4,1 riu 2 \|x p−x c\|<z CLBP- M 4,1 riu 2 \|x p−x c\|≥z ure 1. 3.2. Empirical Justiﬁcation Inspired by Wu et al. [29], we examine the distinctiveness of each pattern of CLBP-Sriu2 4,1 , which coincides with patterns of CLBP-Mriu2 4,1 for separating high-quality iris images from distorted versions. To that end, we generated an artiﬁcially distorted iris image dataset from 600 pristine high-quality references taken from the Warsaw-BioBase-Smartphone-Iris v1.0 [4], UTIRIS [58], and GC2 multi-modal [36] datasets. A total of 3 to 12 samples per eye from 75 individuals were selected. This dataset was used only to justify our choice of speciﬁc sign–magnitude patterns and also to investigate how ﬁltering out the low-quality iris images using the DSMI metric could affect the performance of the segmentation module of the reference iris recognition system. The reference iris images have no content-dependent deformations such as eyelid occlusion, and were selected from individuals with high, medium, and low degrees of iris pigmentation. The irises of all of these reference iris images were segmented accurately by the reference iris recognition system. Five common image distortions with different levels and multiple distortions were used to distort the reference iris images. These distortions are Gaussian blur (GB), motion blur (MB), white Gaussian noise (WGN), salt and pepper noise (IN), and overexposure (OE). The parameters of each function and the number of the distorted versions of each reference image are listed in Table 1. In addition to the individual types of distortions, we generated multiple distorted iris images (GB+WGN). First, we distorted the images with GB and then with WGN. Since GB tends to occur during the acquisition phase due to the different working conditions of the image sensors, we applied it ﬁrst. WGN is a noise model that can be used to mimic the effects of random processes, such as sensor noise due to poor illumination and thermal noise in the imaging device. For simplicity, the recommendation of [59] was followed, and WGN was introduced in the end. Table 1. Summary of the artiﬁcially distorted iris image dataset. 3.1. Proposed Quality Metric In Equation (7), the normalization by the local variance emphasizes local minima and maxima, and normalizing the scores to the range [0, 1) is only for ease of interpretation of the quality scores. The value of Qk,l is considered as an image quality score derived from the sign pattern k and the magnitude pattern l. In our experiments, we used four neighbors (P = 4) with unit distance (R = 1) from the central pixel c of a local patch. Sensors 2020, 20, 1308 7 of 22 7 of 22 Our experiments showed that Qk,l with the speciﬁc coincidence of the sign pattern k = 0 and magnitude pattern l = 0 has a high correlation with iris image quality. Therefore, we used Q0,0 as our proposed DSMI quality score. We had summarized the proposed DSMI metric in our conference paper [18], considering, however, only the selected coincidence sign–magnitude patterns. 3.2. Empirical Justiﬁcation Reference Iris Images Degree of Iris Pigmentation Number of Individuals Number of All Iris Images High 25 200 Medium 25 200 Low 25 200 Distorted Iris Images Distortion Type MATLAB Function Parameters Interval Distorted Versions All Distorted Iris Images GB imgaussﬁlt(I, sigma) 0.5–5 10 6000 IN imnoise(I,’salt &pepper’,density) 0.05–0.6 12 7200 OE I+t 10–100 10 6000 MB H=fspecial(’motion’,len, theta); imﬁlter(I,H,’replicate’) 10–60; 10–60 36 21,600 WGN imnoise(I,’gaussian’,0,V) 0.002–0.02 10 6000 GB+WGN imgaussﬁlt(I, sigma); imnoise(I,’gaussian’,0,V) 0.5–5; 0.002–0.02 100 60,000 Table 1. Summary of the artiﬁcially distorted iris image dataset. To analyze the discrimination power of the scores Qk,l for separating the high-quality reference images from their distorted versions, we show the distributions of the corresponding scores Qk,l for some selected combinations of k and l in Figure 2. Visual inspection clearly shows that the coincidence of sign–magnitude patterns with k = 0 and l = 0 gives the greatest discrimination power. The predicted quality scores for the 8 of 22 Sensors 2020, 20, 1308 reference iris images are mostly between 0.8 and 1, and the scores for the distorted versions are mostly less than 0.8. Therefore, we chose this coincidence pattern to form our DSMI quality metric (DSMI = Q0,0). than 0.8. Therefore, we chose this coincidence pattern to form our DSMI quality metric (DSMI = Q0,0). 3.2. Empirical Justiﬁcation 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density GB 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density IN 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density OE 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density MB 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density WGN 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density (a) Qk,l, k = 0, l = 0 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 0.25 0.3 Density (b) Qk,l, k = 0, l ̸= 0 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density (c) Qk,l, k = 0, l = all 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density (d) Qk,l, k = 0, l = 4 GB+WGN Figure 2. The solid red lines show the distributions of the quality scores of the high-quality iris images, and the dotted blue lines show the distributions for the distorted versions with different distortion types, which are shown on the right side of each row. 3.2. Empirical Justiﬁcation The quality scores Qk,l are formed based on four different coincidences of sign (k) and magnitude (l) patterns, shown at the bottom of each column. The ﬁrst column shows the histograms of the quality score Q0,0, and the second, third, and fourth columns show the histograms of the coincidence patterns Q0,l with l ̸= 0, l = all, and l = 4. 3.2. Empirical Justiﬁcation 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density GB 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density IN 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density GB 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density GB 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density IN 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density OE 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.1 0.2 0.3 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density (a) Qk,l, k = 0, l = 0 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density (c) Qk,l, k = 0, l = all 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 Density (d) Qk,l, k = 0, l = 4 GB+WGN 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.2 0.4 0.6 0.8 Density (c) Qk,l, k = 0, l = all 0 0.2 0.4 0.6 0.8 1 Qk,l 0 0.05 0.1 0.15 0.2 0.25 0.3 Density (b) Qk,l, k = 0, l ̸= 0 (b) Qk,l, k = 0, l ̸= 0 (c) Qk,l, k = 0, l = all (d) Qk,l, k = 0, l = 4 (a) Qk,l, k = 0, l = 0 Figure 2. 4. Iris Segmentation Accuracy The performance of iris segmentation in a classical iris recognition system has a signiﬁcant impact on the overall performance. In this section, we analyze how image distortions affect the performance of the segmentation module and how quality ﬁltering could improve the segmentation. Most of the state-of-the-art iris recognition systems for iris imaging acquired in visible light, such as FIRE [38], Raja et al. [39], and OSIRIS, version 4.1 [41], can be used as reference iris recognition systems. We have chosen OSIRIS version 4.1 because (1) OSIRIS is an open-source iris recognition system that facilitates reproducible experiments, (2) it shows high recognition performance [41], and (3) it was used as the reference iris recognition system in some recent biometric recognition studies [4,60–64]. The segmentation module of OSIRIS version 4.1 uses the Viterbi algorithm to detect the iris and pupil contours [65]. The outputs are contours of the iris, which represent the inner boundary between the pupil and iris and the outer boundary between the iris and sclera, resulting in a binary mask for the iris. For our experiments, we used the artiﬁcially distorted dataset from the previous section, which is summarized in Table 1. We segmented all iris images using the OSIRIS segmentation module. The mask of the segmented iris of each reference image was taken as the ground truth for comparison with the segmentation results for the distorted versions. The iris segmentation error is computed by the fraction of mislabeled pixels, 1 e = 1 \|C\| ∑ c∈C T(c) ⊕M(c), where \|C\| is the cardinality of the pixel set C of an iris image, and T and M represent the ground truth and the generated iris masks, respectively. The symbol ⊕represents the exclusive OR operation to identify the segmentation error. If the error e was below the threshold 0.05, the iris segmentation was assumed to be correct. The threshold value was set manually by the authors. where \|C\| is the cardinality of the pixel set C of an iris image, and T and M represent the ground truth and the generated iris masks, respectively. The symbol ⊕represents the exclusive OR operation to identify the segmentation error. If the error e was below the threshold 0.05, the iris segmentation was assumed to be correct. The threshold value was set manually by the authors. 3.2. Empirical Justiﬁcation The solid red lines show the distributions of the quality scores of the high-quality iris images, and the dotted blue lines show the distributions for the distorted versions with different distortion types, which are shown on the right side of each row. The quality scores Qk,l are formed based on four different coincidences of sign (k) and magnitude (l) patterns, shown at the bottom of each column. The ﬁrst column shows the histograms of the quality score Q0,0, and the second, third, and fourth columns show the histograms of the coincidence patterns Q0,l with l ̸= 0, l = all, and l = 4. Sensors 2020, 20, 1308 9 of 22 4. Iris Segmentation Accuracy In Figure 3, we show the fractions of incorrectly segmented irises for the different types of distortion and for low, medium, and high degrees of iris pigmentation. The fractions are given as functions of the percentage of low-quality images that were ﬁltered out using the proposed DSMI quality metric. The results shown indicate a clear correlation between the DSMI quality of iris images and segmentation accuracy. Therefore, ﬁltering out poor-quality images before segmentation will improve the performance by reducing the number of incorrectly segmented images, as indicated by the negative slopes of the plots. In summary, the experiments performed in this section show that the accuracy of the segmentation module varies for iris images with different pigmentations and different distortions. Highly pigmented iris images present a greater challenge for the reference iris recognition system, while the system is more robust for the segmentation of low-pigmented iris images. However, ﬁltering out poor-quality iris images using the proposed DSMI metric increases the accuracy of iris segmentation. Sensors 2020, 20, 1308 10 of 22 10 of 22 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (a) GB 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (b) IN 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (c) OE 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (d) MB 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (e) WGN 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (f) GB+WGN Figure 3. 5. Experimental Results In this section, we investigate to what extent ﬁltering out poor-quality iris images with the proposed quality metric improves the performance of the reference iris recognition system. We also compare our DSMI quality metric with the BRISQUE [66] and WAV1 [67] image quality metrics. BRISQUE uses statistical features extracted from pixel intensities to train a support vector machine for predicting image quality. Pertuz et al. [67] compared 15 metrics to estimate the blur of an image. In their study, WAV1 performed better than the others. WAV1 uses statistical properties of the discrete wavelet transform coefﬁcients. Since blur is a common distortion of iris images taken by handheld imaging devices such as smartphones, we also compare our method with the WAV1 metric. Our experiments were conducted on three large authentic iris image datasets acquired in visible light. 4. Iris Segmentation Accuracy The segmentation performance of the reference iris recognition system is shown for segmenting iris images with high, medium, and low pigmentation, and distorted in different ways. The fraction of incorrectly segmented images is plotted versus the percentage of ﬁltered low-quality images, based on the differential sign–magnitude statistics index (DSMI) metric. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (c) OE 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (a) GB 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (b) IN (c) OE 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (d) MB 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (e) WGN 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Removed low quality iris images 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Incorrectly segmented images high medium low iris pigmentation (f) GB+WGN Incorrectly segmented images (f) GB+WGN Figure 3. The segmentation performance of the reference iris recognition system is shown for segmenting iris images with high, medium, and low pigmentation, and distorted in different ways. The fraction of incorrectly segmented images is plotted versus the percentage of ﬁltered low-quality images, based on the differential sign–magnitude statistics index (DSMI) metric. 5.1. Iris Image Datasets There are many iris image datasets recorded with near-infrared cameras such as CASIA V4 [68], CASIA-Iris-Mobile-V1 [69], IIT Delhi [70], and ND CrossSensor Iris 2013 [71]. However, there are just a few iris image datasets acquired in visible light. Four are widely used in iris recognition research: UTIRIS [58], UBIRIS [72], MICHE [73], and VISOB [74]. An optometric framework in a controlled environment was used for capturing the irises of the UTIRIS dataset, resulting in high-quality iris images. UBIRIS iris images were taken from moving subjects and at different distances, resulting in more heterogeneous images compared to UTIRIS. Nevertheless, the pictures have good quality, better than the expected quality of iris images captured by handheld devices. The MICHE and VISOB datasets are challenging datasets for iris recognition systems, including images with varying degrees of iris pigmentation and eye make-up. In addition, the quality of the images is Sensors 2020, 20, 1308 Sensors 2020, 20, 1308 11 of 22 impaired by lack of focus, gaze deviations, specular reﬂections, eye occlusion, different lighting conditions, and motion blur. impaired by lack of focus, gaze deviations, specular reﬂections, eye occlusion, different lighting conditions, and motion blur. Instead, we chose three datasets of the GC2 multi-modal biometric dataset [36] because they contain authentically distorted iris images typically seen when capturing iris images with handheld devices such as smartphones. In addition, the iris images were taken from many subjects with different handheld cameras in uncontrolled environments at different distances. Iris pigmentation varied, from European subjects with bright iris textures to Asian subjects with very dark iris textures. In addition to the various authentic distortions corresponding to the image covariates, the iris images are subject to a variety of quality losses related to the subject’s covariates, such as gaze deviation, off-angle, reﬂections, eye closure, and make-up. Also, the datasets contain 12–15 iris images of varying quality per eye and person, which is useful for studying the effect of quality ﬁltering. The iris images have more than 30 different resolutions. • The ﬁrst dataset of GC2, REFLEX, was taken with a Canon D700 camera using a Canon EF 100 mm f/2.8 L macro lens (18 megapixels). It contains 1422 irises of 48 subjects. A total of 12 to 15 samples were taken per eye (left and right). • The second dataset, LFC, contains iris images taken by a light ﬁeld camera. 5.1. Iris Image Datasets Normalized histograms of the quality scores according to the DSMI metric on three test iris datasets. Datasets REFLEX LFC PHONE Number of subjects 48 49 50 Total images 1422 1454 1379 Samples per eye 12–15 13–15 12–15 Matching pairs 9457 10,045 9092 Non-matching pairs 975,450 1,056,485 941,039 Camera Canon D700 Light ﬁeld camera Phone Nexus Lowest resolution 1085 × 724 327 × 218 450 × 300 Highest resolution 2813 × 1876 1080 × 1080 1811 × 1208 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (a) REFLEX 0 0.2 0.4 0.6 0.8 1 DSMI 0 1 2 3 4 5 Density (b) LFC 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (c) PHONE Figure 5. Normalized histograms of the quality scores according to the DSMI metric on three test iris datasets. Datasets REFLEX LFC PHONE Number of subjects 48 49 50 Total images 1422 1454 1379 Samples per eye 12–15 13–15 12–15 Matching pairs 9457 10,045 9092 Non-matching pairs 975,450 1,056,485 941,039 Camera Canon D700 Light ﬁeld camera Phone Nexus Lowest resolution 1085 × 724 327 × 218 450 × 300 Highest resolution 2813 × 1876 1080 × 1080 1811 × 1208 0 0.2 0.4 0.6 0.8 1 DSMI 0 1 2 3 4 5 Density (b) LFC 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (a) REFLEX 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (c) PHONE (b) LFC Figure 5. Normalized histograms of the quality scores according to the DSMI metric on three test iris datasets. 5.2. Iris Recognition Performance Analysis To evaluate the performance improvement of iris recognition achieved by quality ﬁltering using an image quality metric, we used three performance methods, namely the Daugman’s decidability index [75], the area under the receiver operating characteristic curves (AUC), and the equal error rates (EER). We compared the performance of three image quality metrics when used for quality ﬁltering. Given a threshold for a metric, we rejected those images that exhibited a quality lower than the threshold. The thresholds for each of the three metrics were chosen such that 1/4, 1/2, and 3/4 of the images were rejected. In our experiments, OSIRIS version 4.1 was used as a reference iris recognition system. 5.1. Iris Image Datasets The LFC dataset contains 1454 iris images from the right and left eyes of 49 subjects. For each eye, 13 to 15 samples were taken. 1454 iris images from the right and left eyes of 49 subjects. For each eye, 13 to 15 samples were taken. • The third dataset, PHONE, was taken by a smartphone (Google Nexus 5, 8 megapixel camera). It contains 1379 iris images from the right and left eyes of 50 subjects, and 12 to 15 samples were taken per eye. • The third dataset, PHONE, was taken by a smartphone (Google Nexus 5, 8 megapixel camera). It contains 1379 iris images from the right and left eyes of 50 subjects, and 12 to 15 samples were taken per eye. We compare an iris image with all iris images from the same dataset. Table 2 summarizes these datasets and shows the number of matching and non-matching iris pairs. Figure 4 shows some samples from these datasets, and Figure 5 shows the histograms of the quality scores of the datasets, estimated by the proposed DSMI metric. Figure 4. Some iris image samples with high, medium, and low pigmentation from the multi-modal biometric dataset GC2 [36]. The ﬁrst, second, and third rows show some images from the REFLEX, LFC, and PHONE datasets, respectively. Figure 4. Some iris image samples with high, medium, and low pigmentation from the multi-modal biometric dataset GC2 [36]. The ﬁrst, second, and third rows show some images from the REFLEX, LFC, and PHONE datasets, respectively. 12 of 22 Sensors 2020, 20, 1308 Table 2. Summary of the GC2 dataset. Table 2. Summary of the GC2 dataset. Table 2. Summary of the GC dataset. Datasets REFLEX LFC PHONE Number of subjects 48 49 50 Total images 1422 1454 1379 Samples per eye 12–15 13–15 12–15 Matching pairs 9457 10,045 9092 Non-matching pairs 975,450 1,056,485 941,039 Camera Canon D700 Light ﬁeld camera Phone Nexus Lowest resolution 1085 × 724 327 × 218 450 × 300 Highest resolution 2813 × 1876 1080 × 1080 1811 × 1208 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (a) REFLEX 0 0.2 0.4 0.6 0.8 1 DSMI 0 1 2 3 4 5 Density (b) LFC 0 0.2 0.4 0.6 0.8 1 DSMI 0 0.5 1 1.5 2 2.5 3 3.5 Density (c) PHONE Figure 5. 5.2.1. Daugman’s Decidability Index We can now study the effect of quality ﬁltering on the performance of the iris recognition system. In Figure 7, we show Daugman’s decidability index as a function of the fraction of removed poor-quality images. DSMI, BRISQUE, and WAV1 image quality metrics were used for quality ﬁltering. Filtering out low-quality iris images using the DSMI metric leads to the largest performance improvement in the REFLEX dataset, while quality ﬁltering in the PHONE dataset leads only to small improvements. This could be due to the DSMI metric performing better in quality assessment on iris images in the REFLEX dataset or to the PHONE dataset posing a greater challenge to the reference iris recognition system. The Daugman index for the PHONE dataset is only 1.36, compared to 2.02 and 1.90 for REFLEX and LFC, respectively (see Figure 6). 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (a) REFLEX 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (b) LFC 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (c) PHONE Figure 7. Daugman’s decidability index for all iris images, after ﬁltering different parts of the iris images with the poorest quality using three image quality metrics on three test datasets. 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (b) LFC 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (c) PHONE 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 1 1.5 2 2.5 3 3.5 d ' DSMI BRISQUE WAV1 (a) REFLEX (b) LFC Figure 7. Daugman’s decidability index for all iris images, after ﬁltering different parts of the iris images with the poorest quality using three image quality metrics on three test datasets. From the Daugman’s decidability index values in the three test datasets, as shown in Figure 7, we can conclude that ﬁltering out the iris images with the poorest quality using the proposed DSMI metric improves the recognition accuracy of the reference iris recognition system. 5.2.1. Daugman’s Decidability Index Daugman’s decidability index [75] is a widely used method for assessing the performance of iris recognition systems [3,36,75]. In an iris recognition system like OSIRIS, a binary phase code is derived for each presented iris image. Then, the fractional Hamming distance to the phase code of a reference iris image is computed. The distributions of these Hamming distances are compared between a set of matching and a set of non-matching iris image pairs from a test dataset. The larger the overlap between the distributions, the more likely recognition errors become. The Daugman index (d′) measures the separation of these distributions by d′ = \|µE −µI\| q 1 2(σ2 E + σ2 I ) , d′ = \|µE −µI\| q 1 2(σ2 E + σ2 I ) , where µE and µI are the means and σE and σI are the standard deviations of the distributions. Larger values correspond to better discrimination. We follow this procedure using the GC2 multi-modal biometric where µE and µI are the means and σE and σI are the standard deviations of the distributions. Larger values correspond to better discrimination. We follow this procedure using the GC2 multi-modal biometric 13 of 22 Sensors 2020, 20, 1308 dataset and plot the histograms of the Hamming distances for the matching and the non-matching iris pairs in Figure 6. For visualization, normal distributions were ﬁtted to the histograms. 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 20 Density d' = 2.0246 (a) REFLEX 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 Density d' = 1.8952 (b) LFC 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 Density d' = 1.3617 (c) PHONE Figure 6. Normal distributions ﬁtted to the normalized histograms of Hamming distances of matching (solid lines) and non-matching (dash lines) iris pairs are shown for three test image datasets. 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 20 Density d' = 2.0246 (a) REFLEX 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 Density d' = 1.8952 (b) LFC 0 0.1 0.2 0.3 0.4 0.5 0.6 Distance 0 5 10 15 Density d' = 1.3617 (c) PHONE (b) LFC Figure 6. Normal distributions ﬁtted to the normalized histograms of Hamming distances of matching (solid lines) and non-matching (dash lines) iris pairs are shown for three test image datasets. 5.2.2. Receiver Operating Characteristic Curve The area under the curve (AUC) of the receiver operating characteristic (ROC) is a widely used performance metric for comparing the accuracy of iris recognition systems. The iris recognition system with the larger AUC is considered to be a more accurate system. To visualize and measure the improvements of the performance of the reference iris recognition system by ﬁltering out the poor quality iris images, the ROC curves were generated for each dataset by plotting the true positive rate against the false positive rate at various fractional Hamming distances (see Figure 8). Figure 8 shows the ROC curves for the three test datasets with different quality ﬁltering thresholds using our DSMI metric, BRISQUE, and WAV1 metrics. The solid red lines in Figure 8 show the performance of the reference iris recognition system without quality ﬁltering. Without quality ﬁltering, the corresponding AUC value for the REFLEX dataset is 0.9065, for the LFC dataset it is 0.8861, and for the PHONE dataset it is 0.8226. The AUC values show again that the PHONE dataset is the most challenging one for the reference iris recognition system. 5.2.2. Receiver Operating Characteristic Curve 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 DSMI = 0.55, AUC = 0.9471 DSMI = 0.76, AUC = 0.9533 DSMI = 0.85, AUC = 0.9869 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 BRISQUE = 32.88, AUC = 0.9292 BRISQUE = 23.11, AUC = 0.9457 BRISQUE = 12.75, AUC = 0.9742 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 WAV1 = 4.34, AUC = 0.8853 WAV1 = 5.40, AUC = 0.8617 WAV1 = 6.31, AUC = 0.8120 REFLEX 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 DSMI = 0.57, AUC = 0.9139 DSMI = 0.68, AUC = 0.9263 DSMI = 0.73, AUC = 0.9367 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 BRISQUE = 41.05, AUC = 0.8985 BRISQUE = 34.85, AUC = 0.9091 BRISQUE = 29.4, AUC = 0.8993 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 WAV1 = 0.9177, AUC = 0.9051 WAV1 = 1.0956, AUC = 0.8973 WAV1 = 1.2914, AUC = 0.8745 LFC 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 DSMI = 0.51, AUC = 0.8275 DSMI = 0.63, AUC = 0.8402 DSMI = 0.72, AUC = 0.8815 (a) DSMI 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 BRISQUE = 33.21, AUC = 0.8304 BRISQUE = 22.61, AUC = 0.8435 BRISQUE = 12.73, AUC = 0.8293 (b) BRISQUE 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 WAV1 = 7.1, AUC = 0.8095 WAV1 = 7.98, AUC = 0.8092 WAV1 = 8.79, AUC = 0.8257 (c) WAV1 PHONE Figure 8. The receiver operating characteristic (ROC) curves for the three test datasets (REFLEX, LFC, and PHONE) with different quality ﬁltering thresholds using our DSMI metric, BRISQUE, and WAV1. 5.2.2. Receiver Operating Characteristic Curve The solid red, dashed blue, dot-dashed green, and dotted black lines were plotted without quality ﬁltering, after ﬁltering out one-quarter, half, and three-quarters of the poorest-quality images, respectively. 5.2.2. Receiver Operating Characteristic Curve 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 WAV1 = 4.34, AUC = 0.8853 WAV1 = 5.40, AUC = 0.8617 WAV1 = 6.31, AUC = 0.8120 REFLEX 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 BRISQUE = 32.88, AUC = 0.9292 BRISQUE = 23.11, AUC = 0.9457 BRISQUE = 12.75, AUC = 0.9742 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.9065 DSMI = 0.55, AUC = 0.9471 DSMI = 0.76, AUC = 0.9533 DSMI = 0.85, AUC = 0.9869 REFLEX 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 DSMI = 0.57, AUC = 0.9139 DSMI = 0.68, AUC = 0.9263 DSMI = 0.73, AUC = 0.9367 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 WAV1 = 0.9177, AUC = 0.9051 WAV1 = 1.0956, AUC = 0.8973 WAV1 = 1.2914, AUC = 0.8745 LFC 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8861 BRISQUE = 41.05, AUC = 0.8985 BRISQUE = 34.85, AUC = 0.9091 BRISQUE = 29.4, AUC = 0.8993 LFC 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 WAV1 = 7.1, AUC = 0.8095 WAV1 = 7.98, AUC = 0.8092 WAV1 = 8.79, AUC = 0.8257 (c) WAV1 PHONE 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 DSMI = 0.51, AUC = 0.8275 DSMI = 0.63, AUC = 0.8402 DSMI = 0.72, AUC = 0.8815 (a) DSMI 0 0.2 0.4 0.6 0.8 1 False positive rate 0.5 0.6 0.7 0.8 0.9 1 True positive rate all, AUC = 0.8226 BRISQUE = 33.21, AUC = 0.8304 BRISQUE = 22.61, AUC = 0.8435 BRISQUE = 12.73, AUC = 0.8293 (b) BRISQUE PHONE Figure 8. 5.2.1. Daugman’s Decidability Index The BRISQUE metric also performs well in the REFLEX dataset, but it is not consistent for quality ﬁltering in the LFC and PHONE datasets. WAV1 is not consistent with quality ﬁltering on all three test datasets. Sensors 2020, 20, 1308 14 of 22 14 of 22 5.2.2. Receiver Operating Characteristic Curve The receiver operating characteristic (ROC) curves for the three test datasets (REFLEX, LFC, and PHONE) with different quality ﬁltering thresholds using our DSMI metric, BRISQUE, and WAV1. The solid red, dashed blue, dot-dashed green, and dotted black lines were plotted without quality ﬁltering, after ﬁltering out one-quarter, half, and three-quarters of the poorest-quality images, respectively. Sensors 2020, 20, 1308 15 of 22 15 of 22 We also computed the AUC values after removing 1/4, 1/2, and 3/4 of the iris images with the poorest quality from each test dataset. The AUC values are listed in the ﬁgure legends for all of the test datasets. Using the proposed DSMI metric for quality ﬁltering increased the AUC value in all test datasets. In the REFLEX dataset, ﬁltering out a quarter of the iris images with the poorest quality using the DSMI metric greatly improves the performance of the reference iris recognition system in terms of AUC by 0.0406 (4.5%). However, ﬁltering out the second quarter only increases AUC by 0.0062 (0.65%). This indicates that the middle two quarters of the iris images have a small quality deviation, and ﬁltering a part of these images does not result in a considerable improvement in the performance of the iris recognition system. However, ﬁltering the third quarter of the iris images with the poorest quality improves the AUC signiﬁcantly by 0.0336 (3.5%). g y y The performance improvements for the LFC dataset after ﬁltering out the ﬁrst, second, and third quarters of the iris images with the poorest quality using the DSMI metric are 0.0278 (3.1%), 0.0124 (1.4%), and 0.0104 (1.1%), respectively. The values for performance improvement on the PHONE dataset are 0.0049 (0.6%), 0.0127 (1.5%), and 0.0413 (4.9%). Filtering out the ﬁrst quarter of the iris images with the poorest quality using the DSMI metric only slightly improves the AUC value, but ﬁltering out three quarters of the iris images with the poorest quality improves the performance signiﬁcantly by 7.2%. We visualized these performance improvements in Figure 9. 5.2.2. Receiver Operating Characteristic Curve 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (a) REFLEX 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (b) LFC 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (c) PHONE Figure 9. Area under the curve (AUC) values for all iris images after removing different parts of the iris images with the poorest quality. 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (c) PHONE 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (b) LFC 0% 12.5% 25% 37.5% 50% 62.5% 75% Removed low quality iris images 0.8 0.85 0.9 0.95 1 AUC DSMI BRISQUE WAV1 (a) REFLEX (b) LFC (b) LFC (a) REFLEX Figure 9. Area under the curve (AUC) values for all iris images after removing different parts of the iris images with the poorest quality. The analysis of the AUC values shows that the performance of the reference iris recognition system has improved by quality ﬁltering in all test datasets when using the DSMI metric for quality assessment. In contrast, BRISQUE is consistent for quality ﬁltering for the REFLEX dataset, but not for the other two test datasets. WAV1 shows inconsistent performance in all test datasets. The reason for this could be that the DSMI metric is optimized for assessing the image quality of iris images and BRISQUE for the perceptual quality of natural images. Both, however, can assess image quality for different image distortions. The WAV1 metric is optimized for blur assessment. Since blur is common in iris images taken with handheld devices, we compare our method with the WAV1 metric. However, the iris images in test datasets have more complicated authentic in-the-wild image distortions, and these distortions degrade the performance of WAV1 in all test datasets. 5.2.3. Equal Error Rate The equal error rate (EER) is the rate at which both accept and reject errors are equal. The EER is used for comparing the accuracy of classiﬁcation systems with different receiver operating characteristic (ROC) curves. With the EER approach, the system with the lowest EER is considered the most accurate. In Table 3, we calculated the EER values when three image quality metrics were used to ﬁlter out the poor-quality iris images from the test datasets. The greatest performance improvement is achieved by Sensors 2020, 20, 1308 16 of 22 16 of 22 ﬁltering out poor-quality iris images using the DSMI metric on the REFLEX dataset. The PHONE dataset is the more challenging dataset for the reference iris recognition system, resulting in higher EER values. The results conﬁrm that rejecting poor-quality images using the proposed DSMI metric improves the iris recognition performance consistently, while this observation does not hold for BRISQUE and WAV1 metrics. The results conﬁrm that rejecting poor-quality images using the proposed DSMI metric improves the iris recognition performance consistently, while this observation does not hold for BRISQUE and WAV1 metrics. Table 3. The equal error rate (EER) values are calculated after ﬁltering different parts of the iris images with the poorest quality from each test dataset. This table shows the EER values when all iris images are passed to the iris recognition system and after ﬁltering out one quarter, half, and three quarters of the iris images with the poorest quality from the REFLEX, LFC, and PHONE datasets using the DSMI, BRISQUE, and WAV1 quality metrics. Removed Part REFLEX LFC PHONE DSMI BRISQUE WAV1 DSMI BRISQUE WAV1 DSMI BRISQUE WAV1 0% 0.1469 0.1469 0.1469 0.1770 0.1770 0.1770 0.2466 0.2466 0.2466 25% 0.0987 0.1202 0.1714 0.1500 0.1604 0.1562 0.2418 0.2374 0.2594 50% 0.0878 0.978 0.1963 0.1376 0.1528 0.1692 0.2293 0.2276 0.2595 75% 0.0382 0.0520 0.2443 0.1287 0.1724 0.1955 0.1845 0.2412 0.2434 In summary, for all of the test iris image datasets (REFLEX, LFC, PHONE) and all of the performance evaluation methods (Daugman’s decidability index, AUC, EER), the performance of the reference iris recognition system (OSIRIS, Version 4.1) increased consistently by ﬁltering out iris images with the poor quality using the proposed DSMI quality metric. In contrast, for the other two image quality metrics (BRISQUE, WAV1), the experiments showed inconsistencies, i.e., removing more low-quality images did not always increase the performance of the reference iris recognition system. 5.2.3. Equal Error Rate (a) 0.35 (b) 0.27 (c) 0.09 (d) 0.33 (e) 0.39 (f) 0.35 Figure 10. The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed as low-quality samples by our DSMI metric. All of these samples would be falsely rejected with high dissimilarity scores (>0.47) by the reference iris detection system. However, if we ﬁlter out a quarter of the iris images with the poorest quality from each test dataset, these samples will be removed and not passed to the iris recognition system. The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the iris samples. (a) 0.53 (b) 0.54 (c) 0.57 (d) 0.59 (e) 0.43 (f) 0.55 Figure 11. The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed by our DSMI metric as iris samples of sufﬁcient quality if only one quarter of the iris images with the poorest quality are ﬁltered out. Therefore, these images are passed to the iris recognition pipeline for further processing. However, all of these samples would be falsely rejected by the reference iris recognition system with high dissimilarity values (>0.47). The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the (a) 0.35 (f) 0.35 (e) 0.39 (d) 0.33 (c) 0.09 (b) 0.27 (a) 0.35 (c) 0.09 (a) 0.35 (d) 0.33 (d) 0.33 (f) 0.35 (c) 0.09 (e) 0.39 (b) 0.27 Figure 10. The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed as low-quality samples by our DSMI metric. All of these samples would be falsely rejected with high dissimilarity scores (>0.47) by the reference iris detection system. However, if we ﬁlter out a quarter of the iris images with the poorest quality from each test dataset, these samples will be removed and not passed to the iris recognition system. The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the iris samples. (a) 0.53 (b) 0.54 (c) 0.57 (d) 0.59 (e) 0.43 (f) 0.55 Figure 11. 5.2.3. Equal Error Rate The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed by our DSMI metric as iris samples of sufﬁcient quality if only one quarter of the iris images with the poorest quality are ﬁltered out. Therefore, these images are passed to the iris recognition pipeline for further processing. However, all of these samples would be falsely rejected by the reference iris recognition system with high dissimilarity values (>0.47). The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the iris samples. (b) 0.54 (f) 0.55 (c) 0.57 (e) 0.43 (d) 0.59 (b) 0.54 (f) 0.55 (a) 0.53 (d) 0.59 (c) 0.57 (a) 0.53 (e) 0.43 Figure 11. The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed by our DSMI metric as iris samples of sufﬁcient quality if only one quarter of the iris images with the poorest quality are ﬁltered out. Therefore, these images are passed to the iris recognition pipeline for further processing. However, all of these samples would be falsely rejected by the reference iris recognition system with high dissimilarity values (>0.47). The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the iris samples. The iris samples that are shown in Figure 11 have fewer image distortions compared to the sample shown in Figure 10. Therefore, our quality metric predicts higher quality scores for these iris images. Some of these images have quality degradations related to subject covariates, such as eyelashes obscuring the iris or closed eyes. If we ﬁlter out half of the iris samples with the poorest quality, these samples will be ﬁltered. However, by setting a higher quality ﬁltering threshold, some iris samples may be rejected unnecessarily. 5.2.3. Equal Error Rate Figure 10 shows some iris samples from the test datasets with poor quality scores predicted by the proposed DSMI metric. These samples will be ﬁltered out when we remove a quarter of the iris images with the poorest quality from each test dataset. If we pass these samples to the reference iris detection system for iris recognition, all of them will be falsely rejected. Thus, the proposed DSMI metric can be used to decide whether an input iris sample should be enrolled in a dataset or rejected, and a new sample should be captured based on the quality score. Although our method is designed to consider only image covariates, some subject covariates, such as eyelid occlusion due to blinking, may also result in motion blur or other image quality distortions that can be measured by our proposed quality metric, as shown in Figure 10c. All iris samples shown in Figure 10 suffer from authentic image distortion and other quality degradation due to subject covariates. g j Figure 11 shows some iris samples with DSMI scores that are higher than the threshold for ﬁltering out one quarter of the iris samples with the poorest quality from each test dataset. Our proposed framework passes these images for iris segmentation and identiﬁcation when only a quarter of the iris images with the poorest quality are ﬁltered out from the test datasets. However, all of these samples will be falsely rejected by the reference iris recognition system. Some of these images have quality degradation related to subject covariates, such as eyelashes obscuring the iris or closed eyes. 17 of 22 17 of 22 Sensors 2020, 20, 1308 (a) 0.35 (b) 0.27 (c) 0.09 (d) 0.33 (e) 0.39 (f) 0.35 Figure 10. The ﬁrst row shows some iris samples from the multi-modal biometric dataset GC2 [36], which are classiﬁed as low-quality samples by our DSMI metric. All of these samples would be falsely rejected with high dissimilarity scores (>0.47) by the reference iris detection system. However, if we ﬁlter out a quarter of the iris images with the poorest quality from each test dataset, these samples will be removed and not passed to the iris recognition system. The second row shows the segmentation result of the segmentation module of the reference iris recognition system. The DSMI scores are listed below the iris samples. 5.3. Computational Complexity It is straightforward to assess the computational complexity of the DSMI quality metric by checking the algorithmic steps, outlined in Section 3.1, one by one. The result is a time complexity, linear in the size of the input image. More precisely, it is O(N × M × P), where N × M is the image size in pixels, and P is the number of points checked in the neighborhood of each pixel for deriving the sign and magnitude patterns. We also recorded the actual speed of the quality metric using our implementation, running on an MSI GP60 laptop with an Intel Core i7 processor and 16GB RAM with MATLAB version 2018b in Ubuntu 18.04.3 LTS. We computed the DSMI quality scores on four parts of the test datasets, each containing iris 18 of 22 18 of 22 Sensors 2020, 20, 1308 images of the same size in pixels, ranging from 596 × 397 up to 2036 × 1358 (see Table 4). The table conﬁrms the linear time complexity, amounting to roughly 0.06 × 10−6 seconds per pixel. At that processing speed, a throughput of 66 frames per second (FPS) can be achieved at resolution 596 × 397. For the higher resolutions, 625 × 537, 1233 × 810, and 2036 × 1358, the speed is 40, 16, and 6 FPS, respectively. Therefore, the proposed method can be used to assess the quality of iris images in interactive applications, such as iris recognition systems based on handheld imaging devices. Table 4. Comparison of the average running time (seconds) on four sets of iris images with different resolutions. Image Resolutions 596 × 397 625 × 537 1233 × 810 2036 × 1358 Average running time per image (seconds) 0.015 0.026 0.061 0.181 Average running time per pixel (microseconds) 0.065 0.062 0.062 0.064 Frames per second (FPS) 66 40 16 6 Comparison of the average running time (seconds) on four sets of iris images with different resolutions. 6. Conclusions and Future Work In this paper, we presented a fast image quality metric, based on statistical features of the sign–magnitude transform to estimate the quality of iris images acquired by handheld devices in visible light. We suggest that this method can be used to decide whether an input iris sample should be enrolled in a dataset or rejected, and a new sample should be captured based on the quality score to improve the speed and the recognition rate of the reference iris recognition system. We conducted extensive experiments to demonstrate these improvements using three performance methods for measuring the iris recognition accuracy on three large datasets acquired in unconstrained environments in visible light. The experiments showed that the proposed approach improved the accuracy of the reference iris recognition system. However, we would like to point out that the inclusion of quality ﬁltering in an iris recognition system can increase the computational costs of iris image recognition, and some iris images may be rejected unnecessarily. This could be caused by an error in the quality metric, by too conservative of a setting of the quality threshold, or by quality factors related to the subject covariates. In our future work, we will propose a metric for iris image quality assessment that takes into account all of these factors. Furthermore, another future work is to develop an algorithm to monitor criteria, such as recognition performance, time and number of photos required per person, and customer satisfaction, in order to dynamically adapt the threshold for quality ﬁltering to achieve optimal performance. It may also be promising to examine the use of the proposed quality metric to assess the quality of other biometric images, such as facial image, and NIR biometric images. Author Contributions: Conceptualization, M.J. and M.P.; Investigation, M.J.; methodology M.J., M.P., D.S.; validation M.J. and D.S.; Writing—original draft preparation, M.J.; Writing—review and editing, M.J., M.P., D.S.; Visualization, M.J., M.P., D.S.; Supervision, M.P. and D.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by the Exzellenzstrategie des Bundes und der Länder (the Excellence Strategy of the German Federal and State Governments), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project-ID 251654672—TRR 161 and the Research Council of Norway within project no. 221073 HyPerCept–Color and quality in higher dimensions. Acknowledgments: The authors thank Jon Yngve Hardeberg, Katrin Franke, and Sokratis Katsikas for their helpful discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: The authors thank Jon Yngve Hardeberg, Katrin Franke, and Sokratis Katsikas for their helpful discussions. Author Contributions: Conceptualization, M.J. and M.P.; Investigation, M.J.; methodology M.J., M.P., D.S.; validation M.J. and D.S.; Writing—original draft preparation, M.J.; Writing—review and editing, M.J., M.P., D.S.; Visualization, M.J., M.P., D.S.; Supervision, M.P. and D.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by the Exzellenzstrategie des Bundes und der Länder (the Excellence Strategy of the German Federal and State Governments), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project-ID 251654672—TRR 161 and the Research Council of Norway within project no. 221073 HyPerCept–Color and quality in higher dimensions. 6. Conclusions and Future Work Acknowledgments: The authors thank Jon Yngve Hardeberg, Katrin Franke, and Sokratis Katsikas for their helpful discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 19 of 22 Sensors 2020, 20, 1308 References 1. Flom, L.; Saﬁr, A. Iris Recognition System. U.S. Patent 4,641,349, 3 February 1987. 2. Daugman, J. New methods in iris recognition. IEEE Trans. Syst. Man Cybern. Part B Cybern. 2007, 37, 1167–1175. 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https://openalex.org/W3026007273	https://europepmc.org/articles/pmc7277918?pdf=render	English	null	Using Life History Calendars to Estimate in Utero and Early Life Pesticide Exposure of Latinx Children in Farmworker Families	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	7,194	Received: 22 March 2020; Accepted: 14 May 2020; Published: 16 May 2020 Abstract: (1) Background: Early life exposure to neurotoxic chemicals can have later impacts on child health. Most research designs must assume that current exposure is similar to past. Life history calendar methods can help to provide data on early life exposure. (2) Methods: Life history calendars were completed by mothers of 8-year-old children from Latinx farmworker and non-farmworker families (n = 73 and 65, respectively). Measures were created of months exposure through living adjacent to farm ﬁelds and having household members who worked in jobs exposing them to toxic chemicals. Data were divided into time periods of in utero, early childhood (birth-35 months) and later childhood (36–96 months). Cluster analysis compared the measures for children from farmworker and non-farmworker parents. (3) Results: Although, as a group, children from farmworker families have greater lifetime months of probable exposure to pesticides than children in non-farmworker families, cluster analysis reveals groups of children who do not follow that pattern. (4) Conclusions: The life history calendar is a technique for obtaining data on early life toxic chemical exposure that may help assign children to proper exposure groups. Conducting secondary analyses using such information can help to clarify the association of exposures to health outcomes. Keywords: Latino/Hispanic; agricultural health; health disparities; migrant and seasonal farmworkers; exposure assessment International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Using Life History Calendars to Estimate in Utero and Early Life Pesticide Exposure of Latinx Children in Farmworker Families Sara A. Quandt 1,* , Dana C. Mora 2, Theresa L. Seering 3, Haiying Chen 4, Thomas A. Arcury 2 and Paul J. Laurienti 5 Sara A. Quandt 1,* , Dana C. Mora 2, Theresa L. Seering 3, Haiying Chen 4, Thomas A. Arcury and Paul J. Laurienti 5 1 Department of Epidemiology and Prevention, Division of Public Health Sciences, Wake Forest School o Medicine, Winston-Salem, NC 27157, USA 1 Department of Epidemiology and Prevention, Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Department of Family and Community Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA; dmora@wakehealth.edu (D.C.M.); tarcury@wakehealth.edu (T.A.A.) 3 Department of Anthropology, Lawrence University, Appleton, WI 54911, USA; Theresa.l.seering@lawrence.edu 2 Department of Family and Community Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA; dmora@wakehealth.edu (D.C.M.); tarcury@wakehealth.edu (T.A.A.) 3 Department of Anthropology, Lawrence University, Appleton, WI 54911, USA; Theresa.l.seering@lawrence.edu 4 Department of Biostatistics and Data Science, Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA; hchen@wakehealth.edu 5 Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA; plaurien@wakehealth.edu p * Correspondence: squandt@wakehealth.edu; Tel.: +1-336-716-6015 1. Introduction Pesticides used in the United States include substances that are potent neurotoxicants. Agricultural workers are exposed directly to multiple classes of pesticides and pesticide residues in their work [1–4], and these workers are often the vector by which pesticides enter the home and result in indirect exposure of family members [5,6], including pregnant women and children [7]. Family members are also subject to direct exposure by drift from nearby ﬁelds and from using pesticides for the control of pests in housing, which is often in poor repair, gardens and yards [8,9]. Int. J. Environ. Res. Public Health 2020, 17, 3478; doi:10.3390/ijerph17103478 www.mdpi.com/journal/ijerph 2 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 Children in farmworker families are particularly susceptible to pesticide exposure [10]. Children have greater surface area relative to body volume, so absorb a relatively greater amount of pesticides through the skin than adults [11]. Their metabolic systems are less mature, so their bodies are unable to detoxify some chemicals, such as the organophosphorus pesticides commonly used in agriculture and, thus, they are more vulnerable to their eﬀects than adults [12]. Behaviorally, children come in contact with pesticides through hand-to-mouth activity and playing on contaminated ﬂoors [13–15]. Compared to adults, children are particularly susceptible to eﬀects of early life pesticide exposure. The developing neurological system is characterized by critical periods, with early in utero exposures likely to result in structural malformations, and later in utero and early life exposures expected to result in permanent functional and behavioral alterations [16]. In prospective studies of child cognitive development, children exposed to pesticides prenatally and during early life demonstrate lower IQ by seven years of age [17–19]. However, prospective studies are diﬃcult in many vulnerable populations, because of the expense of following children over years, as well as the diﬃculty of tracking members of a mobile population. Farmworkers, many of whom migrate to follow crops and resist eﬀorts at tracking because of immigration concerns, constitute such a population that is both subject to pesticide exposure, but diﬃcult to follow. Therefore, determining methods of ascertaining prenatal and postnatal exposure without the time, expense and diﬃculty of a prospective research design is of value. Life history or life event calendars have been used to aid respondent recall of events occurring long prior to the data collection [20]. 2. Materials and Methods The study reported here is part of a larger two-group, prospective, longitudinal study, examining the health and cognitive eﬀects of pesticide exposure in children in farmworker families. The larger study uses a comparative design, recruiting a sample of children in families of Latinx farmworkers (the exposed sample) and a sample of children in similar families, but without a member who was a farmworker (the non-exposed sample). 1. Introduction This method uses life event cues of signiﬁcant milestones to encourage respondents to remember less memorable events or circumstances. In environmental health, life history calendars have been used to elicit occupational histories from migrant farmworkers in the US [21], agricultural exposures among minority adult farmers [22], and prenatal and early life pesticide exposures among school children in Nicaragua [23]. In a comparison with a traditional questionnaire method, calendar-based questions were found to be more eﬀective in eliciting the details of complex life histories [24]. In a test-retest assessment of reliability of calendar-based work histories, the calendar was found to be eﬀective, particularly if data were categorized rather than used as direct counts [25]. p y g The goal of this paper is to explore the use of life history calendars, to characterize and compare pesticide exposure in children in Latinx farmworker and non-farmworker families in the US. The speciﬁc aims are: (1) to describe the lifetime pesticide exposures of Latinx children whose families either do or do not include farmworkers; (2) to identify the typical proﬁles of exposure in these children; and (3) to compare the lifetime exposure and typical proﬁles of exposure in children in farmworker families with those of children in non-farmworker families. 2.1. Inclusion Criteria and Participant Recruitment Inclusion criteria for the children were similar in both samples. Children were aged 8 years at baseline and had to have completed the ﬁrst grade in the United States. Children had to be able to cooperate with and complete an assessment battery. All children had to be from families that self-identiﬁed as Latino or Hispanic, and with household incomes below 200% of the US federal poverty line. In the farmworker sample, the mother/guardian or her partner must have been employed in farm work on non-organic farms during the past three years. In the non-farmworker sample, adults could not have been employed in industry that involves routine exposure to pesticides (e.g., farm work, landscaping, pest control) in the previous three years. Families in the non-farmworker sample could not have lived adjacent to agricultural ﬁelds in the previous three years. 3 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 Exclusion criteria for both samples included children having life threatening illnesses, prior history of neurological conditions, physical condition or development disorder that would not allow them to complete or would interfere with the results of neurobehavioral tests or MRIs (used in the larger main study), primary language other than Spanish or English spoken in the home or refusal of mother/guardian to complete the questionnaires. Children and their mother/guardians were recruited from March 2018, to December 2019. A total of 76 children were recruited for the farm work sample and 66 for the non-farm work sample. Of these, life history calendars were completed by mothers for 73 children in the farm work sample and 65 children in the non-farm work sample. This sample of 138 is used in this paper. For children in farmworker families, the community partner North Carolina Farmworkers Project developed a list of families with an 8-year-old child, and the locations where they lived. This included families who had participated in a previous study of child growth and nutrition. In addition, other community organizations that served farmworker families in the recruitment area were contacted. Study personnel contacted the mothers. Similarly, for the non-farmworker sample, local recruiters in Winston-Salem and community members developed a list. For both samples, mothers were contacted by a bilingual staﬀmember who explained the overall study procedures, answered questions and, if the mother agreed to participate, obtained signed informed consent from the mother and assent from the child. 2.1. Inclusion Criteria and Participant Recruitment As recruitment progressed, community partners worked with the study team to balance the two samples on socioeconomic status. All procedures were approved by the Wake Forest University Institutional Review Board. The study received a Certiﬁcate of Conﬁdentiality from the National Institutes of Health. 2.3. Variable and Measures Variables from the baseline questionnaire were used to create measures to describe the sample. These included marital status of the mother (married or living as married versus not married); age at enrollment for the child, mother and spouse/partner; sex of the child; educational attainment of mother and spouse/partner; and current occupation of mother and spouse/partner. From the life history calendar interview, the following measures were compiled for the period pregnancy (possible values 1–9 months) and each year of life of the child (1–12 months): months living in housing near agricultural ﬁelds, months mother worked in job exposing her to toxic chemicals, months mother’s spouse/partner worked in a job exposing him to toxic chemicals, months up to two other co-resident individuals worked in jobs exposing them to toxic chemicals. Measures were also created for each of these individuals for months worked mixing or applying toxic chemicals. Jobs encountered in the calendars that were considered exposing workers to toxic chemicals included agricultural jobs, including packing and work with livestock, cleaning hotels and restaurants, landscaping, carpet installation and welding. The jobs, including farm work, that were deemed to expose individuals to any form of toxic chemicals are referred to in this paper as “toxic jobs”. Two staﬀmembers independently calculated measures from each calendar; their data were compared, and any discrepancies were resolved. The ﬁnal measures data were entered into a REDcap ﬁle. After these variables were created directly as counts from the calendars, the following cumulative measures were created for three time periods: pregnancy; early childhood, deﬁned as birth month through age 35 months; and later childhood, deﬁned as age 36 months through age 96 months. The measures were: total months the parents (mother plus spouse/partner) worked in toxic jobs; total months the other co-resident adults (up to two) worked in toxic jobs; total months all the adults (mother, spouse/partner, up to two co-resident adults) worked in toxic jobs; total months the parents (mother plus spouse/partner) worked mixing or applying toxic chemicals; total months the other co-resident adults (up to two) worked mixing or applying toxic chemicals; total months all the adults (mother, spouse/partner, up to two co-resident adults) worked mixing or applying toxic chemicals. No measures were created for home pesticide use. The information provided by the respondents was quite vague. 2.3. Variable and Measures Virtually all respondents in both samples stated that they used pesticides when there were pests in the dwelling, but that use was not routine and not continuous. Therefore, the data appeared to be diﬃcult to quantify or to assign to diﬀerent life periods. 2.2. Data Collection Public Health 2020, 17, 3478 stressed the need to probe employment of mothers, their spouse or partners, and up to two co-resident adults for interactions with pesticides, cleaning products, paint and other substances that could be neurotoxic. Once data collection began, life history calendars were reviewed upon completion by project staﬀ, and interviewers were asked to re-contact the mother if clarifying information was needed. 2.2. Data Collection The data in this paper were gathered at the baseline visit for the study. Mothers completed a standardized interviewer-administered questionnaire. This questionnaire included demographic and background data on the family and child. This part of the interview was collected using tablets. Data were entered in real time during the interviews using Research Electronic Data Capture (REDCap). REDCap is hosted at Wake Forest School of Medicine through the Clinical and Translational Science Institute. The REDCap system provides secure, web-based applications for a variety of types of research [26]. To collect data on the possible exposures the child might have experienced to pesticides, the mother completed a life history calendar for the child, beginning with prenatal life and ending at the time of the interview. The interviewer used a long paper form, representing the life span of the child, and she wrote information directly onto this form with the mother watching. The mother was queried to identify the child’s birth date (month and year), which was marked on the top row of the calendar with a sticker. Birth month anniversaries up to the eighth birthday were then marked with stickers going forward in the calendar, and 9 months were counted back from the birth month to indicate the approximate date of conception. Other notable family events (e.g., the birth of other children) were added to this ﬁrst row to help the mother remember context for the child’s life. Data for other rows on the calendar were then queried, in this order: residence (whether the family lived in rural or urban areas, classed as rural if they lived next to ﬁelds; type of housing; source of household water), maternal employment (where employed, tasks performed, crops worked if farm work, probing for potential exposures), paternal employment, and employment of other co-resident individuals. Finally, the mother was queried across the calendar for whether pesticides were used within the family dwelling and whether she, the father or any co-resident workers mixed or applied pesticides or other toxic substances. Interviewers were instructed to write notes throughout the calendar form to provide detail that might be important in detecting exposures. All interviewers were native Spanish speakers; all spoke English, but with varying degrees of proﬁciency. Interviewers participated in training, including practice interviews (both questionnaire and life history calendars) that were recorded and critiqued, before any data collection began. Training 4 of 11 Int. J. Environ. Res. 3.2. In Utero Exposure The two samples generally conformed to expectations that more months of in utero pesticide exposure would be reported in the farmworker sample (Table 1). This is clearest for living in housing close to ﬁelds, where 60 of 73 farmworker cases lived adjacent to ﬁelds for 7 to 9 months of the pregnancy, and 57 of 58 non-farmworker cases never lived adjacent to ﬁelds. Farmworker cases reported, on average, 7.42 months living close to ﬁelds, compared to 0.16 reported by non-farmworker cases (p < 0.001). Comparing the number of person-months household residents worked in toxic jobs, the results were less clear. Nineteen of 73 in the farmworker sample reported 0 person-months, and 16 of the 62 in the non-farmworker sample reported at least seven person-months of work in toxic jobs. Nevertheless, the diﬀerences were signiﬁcant, with farmworker cases reporting an average 8.44 months, compared to 2.32 months in non-farmworker cases, working in toxic jobs (p < 0.001). Few of either sample reported household residents mixing or applying chemicals, but more of the farmworker sample reported more than 0 months in these tasks (1.27 months versus 0.52 months, p = 0.33). Table 1. In utero exposure to pesticides and other toxic chemicals, comparing number and percent for month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Table 1. In utero exposure to pesticides and other toxic chemicals, comparing number and percent for month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Table 1. In utero exposure to pesticides and other toxic chemicals, comparing number and percent for month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Table 1. In utero exposure to pesticides and other toxic chemicals, comparing number and percent for month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. 3.1. Personal Characteristics The sample consisted of 69 boys and 69 girls, ages 8 to 9 years of age at recruitment. Most children were born in the US (128, 92.75%). The remainder were born in Mexico (seven, 5.07%) and other Latin American countries (three, 2.17%). Mothers reported almost no use of alcohol (two, 1.45%), tobacco (one, 0.72%), and marijuana or other substances (0%) during the pregnancy. Mothers reported that most of the children were ﬂuent in English (126, 91.30%) and Spanish (108, 78.26%), though only 25 (18.12%) reported that they themselves were ﬂuent in English. 2.4. Data Analysis Counts/percentages were calculated for data describing the child and mother. We display data for each sample on exposure by month or person-month to assist in visualizing the within and between sample diﬀerences. We used negative binomial models to compare the diﬀerences in the average number of months of exposures between farmworkers and non-farmworkers. We used a K-means clustering approach to classify children into distinct groups based on the exposures summarized in Section 2.3. The ﬁrst cluster analysis used the housing and toxic job variables for all three time periods. Pseudo-F statistics and cubic clustering criterion were used to help determine the optimal number of clusters. Similar cluster analysis was performed using the housing and toxic job variables from the in utero and early childhood time periods. All analyses were conducted with SAS 9.4 (Cary, NC, USA). 5 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 3.2. In Utero Exposure Months Variable Missing 0 1–6 7–9 10–17 18–27 n (%) n (%) n (%) n (%) n (%) Mother lived adjacent to ﬁelds, months Farmworker 0 12 (17) 1 (1) 60 (82) na na Non-farmworker 7 57 (98) 0 1 (2) na na Household residents worked in toxic jobs, months Farmworker 0 19 (26) 4 (5) 30 (41) 11 (15) 9 Non-farmworker 3 46 (74) 0 15 1 0 Household residents mixed or applied chemicals, months Farmworker 0 61 (84) 2 (3) 10 (14) 0 0 Non-farmworker 3 59 (95) 0 2 (3) 1 (2) 0 3.3. Early Childhood Exposure Person-Months Variable Missing 0 1–12 13–36 37–60 61–84 85–108 n (%) n (%) n (%) n (%) n (%) n (%) Child lived adjacent to ﬁelds, person-months Farmworker 0 6 (8) 3 (4) 64 (88) na na na Non-farmworker 4 57 (93) 1 (2) 3 (5) na na na Household residents worked in toxic jobs, person-months Farmworker 0 7 (10) 13 (18) 22 (30) 22 (30) 7 (10) 2 (3) Non-farmworker 0 39 (60) 4 (6) 18 (28) 3 (5) 1 (2) 0 Household residents mixed or applied chemicals, person-months Farmworker 0 56 (78) 6 (8) 11 (15) 0 0 0 Non-farmworker 0 58 (89) 1 (2) 5 (8) 1 (2) 0 0 3.4. Later Childhood Exposure 3.3. Early Childhood Exposure Again, the two samples generally conformed to the expectation that the farmworker sample would have greater exposure than the non-farmworker sample (Table 2). Farmworker cases reported, on average, 31.10 person-months living adjacent to ﬁelds, compared to 1.85 person-months among the non-farmworker cases (p < 0.0001). More in both samples reported household adults working at least some person-months in toxic jobs than was the case for in utero exposure. However, the farmworker sample had more reports of adults in toxic jobs and had the most extreme reports up to 108 person-months in toxic jobs over the 36 months. For farmworker cases, the mean was 35.18 person-months, versus 12.48 person-months for non-farmworker cases (p = 0.0005). Few mothers in 6 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 either sample reported household residents mixing or applying chemicals (farmworker cases average was 5.42 person-months, compared to 3.02 person-months in non-farmworker cases; p = 0.48). either sample reported household residents mixing or applying chemicals (farmworker cases average was 5.42 person-months, compared to 3.02 person-months in non-farmworker cases; p = 0.48). Table 2. Early childhood (birth-35 months) exposure to pesticides and other toxic chemicals, comparing number and non-missing percentages for person-month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Person-Months Variable Missing 0 1–12 13–36 37–60 61–84 85–108 n (%) n (%) n (%) n (%) n (%) n (%) Child lived adjacent to ﬁelds, person-months Farmworker 0 6 (8) 3 (4) 64 (88) na na na Non-farmworker 4 57 (93) 1 (2) 3 (5) na na na Household residents worked in toxic jobs, person-months Farmworker 0 7 (10) 13 (18) 22 (30) 22 (30) 7 (10) 2 (3) Non-farmworker 0 39 (60) 4 (6) 18 (28) 3 (5) 1 (2) 0 Household residents mixed or applied chemicals, person-months Farmworker 0 56 (78) 6 (8) 11 (15) 0 0 0 Non-farmworker 0 58 (89) 1 (2) 5 (8) 1 (2) 0 0 3.4. Later Childhood Exposure Table 2. Early childhood (birth-35 months) exposure to pesticides and other toxic chemicals, comparing number and non-missing percentages for person-month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Table 2. Early childhood (birth-35 months) exposure to pesticides and other toxic chemicals, comparing number and non-missing percentages for person-month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. 3.4. Later Childhood Exposure As would be expected from the study inclusion criteria, there are clear diﬀerences between the samples in later childhood in living adjacent to ﬁelds, and in having household members working in toxic jobs (Table 3). Nonetheless, some children in the farmworker sample did not live adjacent to ﬁelds and household adults worked limited months in toxic jobs. Similarly, there are children in the non-farmworker sample whose proﬁle looks more like the farmworkers. However, the overall diﬀerences were signiﬁcant. Among the farmworker cases, a mean of 60.68 person-months living adjacent to ﬁelds was reported, compared to only 3.32 person-months among the non-farmworker cases (p < 0.0001). Similar diﬀerences are apparent for adult household residents working in toxic jobs (71.92 person-months, versus 23.43 person-months; p < 0.0001). As in the other time periods, few household members in either sample participated in mixing or applying chemicals (14.75 person-months versus 5.95 person-months; p = 0.21). Table 3. Later childhood (36–96 months) exposure to pesticides and other toxic chemicals, comparing number and non-missing percentages for person-month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Person-Months Variable Missing 0 1–12 13–36 37–60 61–96 97–121 122–204 n (%) n (%) n (%) n (%) n (%) n (%) Child lived adjacent to ﬁelds, person-months (%) Farmworker 0 3 (4) 0 2 (3) 4 (5) 64 (88) na na Non-farmworker 3 56 (90) 1 (2) 1 (2) 4 (6) 0 na na Household residents worked in toxic jobs, person-months (%) Farmworker 0 0 1 (1) 14 (19) 15 (21) 27 (37) 9 (12) 7 (10) Non-farmworker 0 36 (55) 4 (6) 4 (6) 7 (11) 12 (18) 1 (2) 1 (2) Household residents mixed or applied chemicals, person- months (%) Farmworker 0 49 (67) 2 (3) 8 (11) 1 (1) 13 (18) 0 0 Non-farmworker 0 55 (85) 2 (3) 3 (5) 1 (2) 4 (6) 0 0 Table 3. Later childhood (36–96 months) exposure to pesticides and other toxic chemicals, comparing number and non-missing percentages for person-month ranges, by children in farmworker (n = 73) and non-farmworker (n = 65) families. Int. J. Environ. Res. Public Health 2020, 17, 3478 7 of 11 7 of 11 3.5. Cluster Analysis The ﬁrst cluster analysis using the housing and toxic job variables for all three time periods produced two clusters (Table 4). Cluster 1 included 65 of 73 children in the farmworker sample; cluster 2 included 63 of 65 children in the non-farmworker sample. Examination of the data points for the two non-farmworker children classiﬁed into cluster 1 showed that one had lived adjacent to ﬁelds consistently through the child’s life, and the other appears to have had exposure due to living near ﬁelds and household member toxic jobs in both early and later childhood. For the eight farmworker children who were classiﬁed into cluster 2, none lived adjacent to ﬁelds in utero and most did not have adults working in toxic jobs in that same period. Table 4. Results of cluster analyses using two variables (months living adjacent to ﬁelds and person-months household residents worked in toxic jobs) for three time periods (in utero and early childhood, later childhood) and two time periods (in utero and early childhood), comparing farmworker (n = 73) and non-farmworker (n = 65) samples. Counts and percentages. Cluster Analysis Including in Utero, Early Childhood, and Later Childhood Variables Cluster 1 n (%) Cluster 2 n (%) Total Farmworker 65 (89) 8 (11) 73 Non-farmworker 2 (3) 63 (97) 65 Total 67 (49) 71 (51) 138 Cluster Analysis Including in Utero, and Early Childhood Cluster 1 Cluster 2 Total Farmworker 60 (82) 13 (18) 73 Non-farmworker 1 (2) 64 (98) 65 Total 61 (44) 77 (56) 138 Cluster Analysis Including in Utero, Early Childhood, and Later Childhood Variables The second cluster analysis using data only from the in utero and early childhood time periods also produced two clusters. Cluster 1 included 60 of 73 children in the farmworker sample; cluster 2 included 64 of 65 children in the non-farmworker sample. Examination of the data points for the one non-farmworker child who was classiﬁed in cluster 1 showed that the family had lived adjacent to ﬁelds consistently through the child’s life. For the 13 farmworker children who were classiﬁed into cluster 2, it appears that some did not live adjacent to ﬁelds until later childhood, and there was less household member engagement in toxic jobs in early life. 4. Discussion This study used a life history calendar approach to develop indices of exposure of children from time in utero through the ﬁrst 96 months of life. Using the life history calendar allowed construction of quantitative work and residential histories using proxies for exposure (residence near ﬁelds, adult occupational activity) known to be key to the pesticide take-home exposure pathway. We have characterized exposure in three periods: prenatal, early childhood and later childhood. These periods are of value, because the exact critical period for the eﬀects of pesticides on the developing child is not known [27,28]. Furthermore, it is likely that the critical period will vary, depending on the particular class of pesticide, the neurological system in question and the particular outcome examined. The parent study will examine brain anatomy and function, as well as developmental and behavior outcomes, so being able to characterize exposure into at least these three time periods is useful. Life history calendar studies address the problem of environmental exposures distant in time from their outcomes [21]. While prospective studies would be the optimal means of documenting early life exposures and later outcomes, these are impractical when the population is mobile, when funding is not inﬁnite and when answers to questions concerning the negative consequences of toxic exposures 8 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 are needed sooner rather than later. Even those important prospective studies of early life pesticide exposure and child outcomes, which have documented the association of pesticide exposure and IQ, have no data before late pregnancy [17–19]. Assisting respondents in recalling past events through the use of a calendar helps to provide much needed and targeted early life information. Other methods, like job-exposure matrices, are also diﬃcult to use when the respondent is not necessarily a worker, and when exposures stretch across 9 years, often in two countries, for workers whose tasks are often quite varied, depending on the demands of the employer. It is not clear what the best metrics are to describe early life exposure to chemicals such as pesticides. Prior studies have used the single variable proxy of living in communities close to ﬁelds for early life pesticide exposure [29] or biomarkers from birth (meconium or umbilical cord blood), which reﬂect only recent exposures or persistent pesticides [17]. Life history calendars have received only limited attention. 4. Discussion This approach was reviewed in a special issue of the American Journal of Industrial Medicine (volume 63, number 4) in 2001, as a means of obtaining prior pesticide exposure data among farmers and farmworkers. Since then, the only reports of the use of such calendars for estimating pesticide exposure are from Costa Rica [30,31] and Nicaragua [23], where they have been used to estimate prenatal and early life exposure of children, as in the current study. In those cases, parents were asked to name speciﬁc pesticides to which children were exposed. While knowing the exact pesticide, or at least the class of pesticides, to which a child was exposed could help reﬁne metrics, that is not always possible, and may not even be accurate when provided by the parents. Based on our previous experience with farmworkers in the US, we chose not to focus on speciﬁc pesticides, as farmworkers, much less family members who do not participate in farm work, often have no way of knowing the names of pesticides being used. One expects children in current farm-working families to have a greater lifetime exposure than those in current non-farm working families. From the data collected in this study, that does appear to be the case. Our data show that their lifetime exposure, from time in utero on, is likely high, relative to the children in non-farmworker families. Nevertheless, there is important variability within both groups of children. Within the two earlier time periods, a group of the children in current farmworker families did not live adjacent to ﬁelds and had relatively little co-resident adult involvement in toxic jobs. Similarly, there are children in the current non-farmworker families who have a pattern of early exposure similar to children in farmworker families. This paper has a number of limitations that should be taken into account. The research was restricted to families in North Carolina. It may be that families elsewhere in the US have diﬀerent patterns of exposure. While the proportions of children falling (according to the cluster analyses) outside the groups of original assignment is small, those proportions could be quite diﬀerent in another population. Home use of pesticides was excluded. There is always a risk of fabrication or faulty memory in collecting early life data, and veriﬁcation is diﬃcult. 4. Discussion While one could research vital statistics and other records to ﬁnd places of birth or dates of immigration, such research could be costly and invade the privacy of these vulnerable participants. Nonetheless, the methods used have been undertaken with success with similar types of populations (e.g., low literacy and low educational attainment) elsewhere. 5. Conclusions The life history calendar can be used by researchers with Latinx parents in the US to obtain detailed information on factors such as places of residence during pregnancy and the early life of children, which may be key to understanding their exposure to pesticides and other toxic substances. While the method is somewhat time consuming, mothers of children are willing to participate and provide detailed data. Children classiﬁed as coming from current farmworker families include a subset that data from the life history calendar method reveals have had lower than expected early life exposure. Similarly, a subset of children in the families of non-farmworkers have likely had higher exposures than would be 9 of 11 Int. J. Environ. Res. Public Health 2020, 17, 3478 expected. Researchers should be aware of this and conduct analyses taking into account these ﬁndings. In the case of the parent study, conducting secondary analyses using a cluster assignment, rather than a recruitment assignment, is warranted. expected. Researchers should be aware of this and conduct analyses taking into account these ﬁndings. In the case of the parent study, conducting secondary analyses using a cluster assignment, rather than a recruitment assignment, is warranted. Author Contributions: S.A.Q. participated in all aspects of the study conceptualization and design, analysis, and manuscript writing. D.C.M. participated in the participant recruitment, data collection, data management, and manuscript writing. T.L.S. participated in background research, data coding, and manuscript writing. H.C. participated in the study design, data analysis, and manuscript writing. T.A.A. participated in all aspects of the study conceptualization and design, analysis, and manuscript writing. P.J.L. participated in all aspects of the study conceptualization and design, analysis, and manuscript writing. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Institute of Environmental Health Science, Grant/Aw Number R01 ES08739. Acknowledgments: The authors appreciate the support and participation of their community partner, the NC Farmworkers Project, and of Student Action with Farmworkers. They also appreciate the valuable contributions of our community ﬁeld interviewers in carrying out participant recruitment and data collection. They especially thank the mothers who participated in this study. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. 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https://openalex.org/W4307167122	https://e-journal.usd.ac.id/index.php/IJHS/article/download/5147/2918	English	null	USE OF THE WORD ‘ANAK’ (CHILD) IN INDONESIAN FROM ECOLINGUISTIC PERSPECTIVE	International Journal of Humanity Studies (IJHS)	2,022	cc-by-sa	5,198	Abstract This article presents the results of research on the use of the word ‘anak’ (child) in Indonesian compound words from an ecolinguistic perspective. The problem studied is whether the use of the word ‘anak’ in Indonesian compound words has a relationship with the environment. When it shows a relationship with the environment, what type of environment will the word ‘anak’ in Indonesian compound words refer to? Because the problem is studied from an ecolinguistic perspective, the theory used in this research is the theory of language concerning the environment. A language whose manifestation is in the form of grammatical units is assumed to designate a certain type of environment as its reference. The data of this research are in the form of compound words which contain the word ‘anak’ in Indonesian. The data were taken from a primary source, namely the online version of the Kamus Besar Bahasa Indonesia (2015), and from a secondary source, namely the researcher's introspective data as a native Indonesian speaker. The data were collected by the referential method and analyzed by the referential equivalent method. The results of data analysis were presented with informal methods and formal methods. The results of this study indicate that the use of the word ‘anak’ in Indonesian compound words refers to ten types of environment, namely (1) the family environment, (2) the educational environment, (3) the work environment, (4) the place environment, (5) the socio-economic environment, (6) the socio-ethical environment, (7) the animal environment, (8) the plant environment, (9) the natural environment, and (10) the artificial object environment. This finding is one of the pieces of evidence that language has a relationship with the environment. Keywords: ‘anak’ word, compound words, ecolinguistics, environment, Indonesian JHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 JHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 International Journal of Humanity Studies http://e-journal.usd.ac.id/index.php/IJHS Sanata Dharma University, Yogyakarta, Indonesia USE OF THE WORD ‘ANAK’ (CHILD) IN INDONESIAN FROM ECOLINGUISTIC PERSPECTIVE Praptomo Baryadi Isodarus Sanata Dharma University, Indonesia correspondence: praptomo@usd.ac.id https://doi.org/10.24071/ijhs.v6i2.5147 received 9 September 2022; accepted 13 October 202 Praptomo Baryadi Isodarus Sanata Dharma University, Indonesia correspondence: praptomo@usd.ac.id https://doi.org/10.24071/ijhs.v6i2.5147 received 9 September 2022; accepted 13 October 2022 Introduction This article presents the results of research on the use of the word ‘anak’ in Indonesian from an ecolinguistic perspective. What is meant by the use of the word ‘anak’ is the combination of the word ‘anak’ with other words to form a compound 68 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 word. For example, the word ‘anak’ joins the word ‘kandung’ (biological) to form ‘anak kandung’ (biological child), the word ‘anak’ joins the phrase ‘sekolah dasar’ (elementary school) to form ‘anak sekolah dasar’ (elementary school pupil/student), the word ‘anak’ joins the word ‘ayam’ (chicken) to form ‘anak ayam’ (chick), the word ‘anak’ joins the word ‘sungai’ (river) to form ‘anak sungai’ (tributary). Thus, the object of this research is the use of the word ‘anak’ in Indonesian compound words. The word ‘anak’ that combines with other words to form compound words can generate certain meanings. The word ‘anak’ in ‘anak kandung’ (biological child) contains the meaning of 'his own child'. The word ‘anak’ in ‘anak sekolah dasar (elementary school pupil/student) expresses the meaning of 'pupil' or 'student'. The word ‘anak’ in anak ayam (chick) expresses the meaning of 'small chicken'. The word ‘anak’ in ‘anak sungai’ (tributary) means 'branches of the river'. This study aims to prove that language has a relationship with the environment. The relationship between language and the environment is the object of studies in ecolinguistics (Halliday, 2001) or language ecology (Haugen, 1972). Therefore, the use of the word ‘anak’ in Indonesian compound words is studied from the perspective of ecolinguistics or language ecology. In this study, the object of the study is part of the language, namely the use of the word ‘anak’ in Indonesian compound words. In this case, the use of the word ‘anak’ in Indonesian compound words is examined concerning the environment. The problem studied is whether the use of the word ‘anak’ in Indonesian compound words has a relationship with the environment. When it shows a relationship with the environment, what type of environment will the word ‘anak’ in Indonesian compound words refer to? Introduction For example, the word ‘anak’ in ‘anak kandung’ (biological child) has a relationship with the family environment, the word ‘anak’ in ‘anak sekolah dasar’ (elementary school pupil/student) has a relationship with the educational environment, the word ‘anak’ in ‘anak ayam’ (chick) has a relationship with the animal environment, and the word ‘anak’ in ‘anak sungai’ (tributary) has a relationship with the natural environment. Discussions on the use of the word ‘anak’ in Indonesian compound words are found, among others, in the book by Kridalaksana (1988; 1993), Chaer's book (1984), Echols’ and Shadily’s book (1994), Badudu (1981), and the online version of the Indonesian Dictionary (2015). Kridalaksana (1988; 1993) discusses the use of the word ‘anak’ to study the combination of lexemes in Indonesian. The combinations of lexemes containing the lexeme of ‘anak’ are listed and classified according to their grammatical meaning. In his work of an Indonesian dictionary of idioms, Chaer (1984) has also listed idioms containing the word ‘anak’ and explained their meaning. In the Kamus Besar Bahasa Indonesia (2015), the online version also has a list of compound words containing the word ‘anak’ and their respective meanings. In these books, the use of the word ‘anak’ in Indonesian compound words, of course, has not been discussed from an ecolinguistic perspective. There have been articles that present research results on the use of words or lexicon from an ecolinguistic perspective, namely articles by Sibarani (2014) and Sinungharjo (2020). Sibarani (2014) examines ‘bamboo’ vocabulary in Batak Toba 69 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 language from an ecolinguistic perspective. Based on the results of his research, Sibarani (2014) shows a list of vocabulary related to bamboo that is almost extinct in Batak Toba language. Sinungharjo (2020) examines the biotic lexicon in music from an ecolinguistic perspective. The results of his research, among others, show that there is a relationship between musical genres and the combination of biotic lexemes. Biotic lexemes are more dominant in dangdut music. The use of biotic elements as the central element is found in popular music and rock music genres and the use of biotic elements as attributes is found in reggae, punk, and hardcore music. Introduction In addition to the articles, there is also a thesis by Tarigan (2018) which describes the Karo language lexicon of nouns and verbs related to the rice environment in Rumah Pil-Pil Village and describes a picture of people's understanding of the noun and verb lexicon of rice. The results showed that the rice lexicon in Karo in Rumah Pil-Pil Village consisted of 5 groups, namely (1) the pre-planting stage lexicon, (2) the planting stage lexicon, (3) the post-planting stage lexicon, (4) the animal and plant lexicon related to rice, (5) the lexicon of processed rice in Rumah Pil-Pil Village. From the five lexicon groups, 118 noun lexicon and 50 verb lexicon were obtained. The number of the rice lexicon found in Rumah Pil-Pil Village is 168 lexicon. From the results of testing the understanding of the people of Rumah Pil-Pil Village on the rice lexicon, the results show that there has been a decreasing understanding of the lexicon in each age group of respondents, especially the adolescent age group. Respondents' understanding of noun lexicon at the age of 45 years was 97.7%, aged 21-45 years was 84.6%, and aged 15-20 years was 60.5%. Respondents' understanding of the verb lexicon at the age of 45 years was 98.6%, the age of 21-45 years was 82.6%, and the age of 15-21 years was 39.8%. g y The use of the word ‘anak’ in Indonesian compound words is studied from an ecolinguistic perspective, so the theory used in this study is the theory of language concerning the environment. The ecolinguistic paradigm includes a functional paradigm (Isodarus, 2022), namely the study of language in terms of language relations and other aspects of life. Ecolinguistics is the study of language concerning the environment (Wenjuan, 2017, Chen 2016). Language concerning the environment contains three concepts, namely language, environment, and the relationship of language to the environment. Language is a sign or symbol consisting of form and meaning. A language which consists of form and meaning is manifested in grammatical units, namely words, sentences, and discourse. In this study, the grammatical unit studied is the word, namely the word ‘anak’ which is combined with other words to form a compound word. The environment, in this case, includes the metaphorical environment, namely the language user, as proposed by Haugen (1972) and the non-metaphorical environment as stated by Halliday (2001). The environment is everything that surrounds human life. Introduction The environment includes the human environment, animal environment, plant environment, natural environment, and the environment of artificial objects. Each type of environment can still be broken down into more specialized types. The human environment, for example, can be detailed into the individual environment, 70 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 family, school environment, work environment, place/location environment, socio- economic environment, socio-ethical environment, and so on. The relationship between language and the environment can be explained through the semantic triangle proposed by Ogden and Richards (1989: 11). Ogden and Richards (1989) explain the relationship between symbols, thoughts, and referents. The symbol serves to symbolize the mind and therefore there is a direct relationship between the two. The mind points to the referent and therefore there is a direct relationship between the two. The relationship between symbols and referents must go through the mind, namely through references or designations, and therefore between symbols and referents there is an indirect relationship. The indirect relationship is shown by a dotted line. The following presents the semantic triangle of Ogden and Richards (1989: 11). Figure 1. The semantic triangle by Ogden and Richards (1989:11) Figure 1. The semantic triangle by Ogden and Richards (1989:11) The semantic triangle theory from Ogden and Richards (1989: 11) is adapted in this study to explain the relationship between language and the environment. In this study, what is meant by a symbol is a verbal symbol or language and the referent in question is the environment. The human mind is the axis of the relationship between language and the environment. It is the mind that connects language with the environment. Language and environment do not have a direct relationship. The indirect relationship is shown by a dotted line. The relationship between language and the environment must go through the mind. This is following the view that language is in the human mind (Subiyanto, 2013). The relationship between language, thought, and environment can be described in the following ecolinguistic triangle. Figure 2 The ecolinguistic triangle Figure 2 The ecolinguistic triangle 71 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 The relationship between language and the environment can be explained from two starting points, namely starting from the environment as a referent and starting from language as a symbol. Introduction If it departs from the environment, the environment is perceived by the mind through the senses to form a concept and then the concept is symbolized to form language. The symbolized concept is called meaning. Thus, a language, which consists of form and meaning, is formed. If it departs from language, the language element consisting of form and meaning refers to a certain environment. In this study, the relationship between language and the environment starts from the elements of language that have been formed. The language element that has been formed is the word ‘anak’, which combines with other words to form a compound word. The word ‘anak’ in the compound word expresses a particular meaning. The word ‘anak’ is a compound word that expresses a particular meaning and has a relationship with a certain type of environment. Findings and Discussion Based on the environment it refers to, the word ‘anak’ in Indonesian compound words designates ten types of environment, namely (1) the family environment, (2) the educational environment, (3) the work environment, (4) the place/location environment, (5) the socio-economic environment, (6) the socio-ethical environment, (7) the animal environment, (8) the plant environment, (9) the natural environment, and (10) the artificial object environment. The word ‘anak’ which refers to the family environment generally means 'descendants'. The data for the word ‘anak’ which refers to the family environment are presented in table 1 below. Table 1. The word ‘anak’ referring to the family environment No Compound Words Makna 1 anak kandung his/her own child 2 anak sulung the first child 3 anak bungsu the last child 4 anak tungal the only child 5 anak kembar children (two, three, or more) born from the same womb in a pregnancy 6 anak sapihan (newly) weaned child 7 anak istri children and wife or family 8 anak bini children and wife or family 9 anak cucu children and grandchildren or descendants 10 anak yatim a child who no longer has a father 11 anak yatim piatu a child who no longer has a father and a mother 12 anak tiri husband's or wife's inborn child who is not the result of marriage to the current wife or husband 13 anak angkat a child who is given the cost of education by someone, but still lives with his parents 14 anak asuh a child who is given the cost of education by someone, but still lives with his parents 15 anak haram a child born from a relationship outside the marriage bond 16 anak jadah children born from relationships outside of marriage bond 17 anak emas the most loved child Table 1. The word ‘anak’ referring to the family environment o Compound Words Makna The word ‘anak’ which refers to the educational environment is found in the combination of the word ‘anak’ and the name of the education unit, the combination of the word ‘anak’ with the name of the level in education, the combination of the word ‘anak’ with the type of education, the combination of the word ‘anak’ and the word student. The word ‘anak’ that refers to the educational environment generally contains the meaning of 'pupil' or 'student'. Method The object of this research is the word ‘anak’ in Indonesian. The research data are compound words that contain the word ‘anak’. The data were obtained from two sources, namely (1) from a book containing a list of compound words containing the word ‘anak’ in Indonesian and (2) from a creative generation of the Indonesian language that researchers use daily. The data from the first source were taken from the online version of the Kamus Besar Bahasa Indonesia (2015). The data were obtained from a second source, which Botha (1981: 67) calls introspective data. This introspective data are compound words containing the word ‘anak’ which is not found in the online version of the Kamus Besar Bahasa Indonesia (2015). Data collection from the first source was carried out using the method of listening or observation (Sudaryanto, 2015: 203), namely by listening to a list of compound words containing the word ‘anak’ found in the first data source. The results of the listening are then recorded on the data card. Data collection from the second source was carried out using an intuitive method (Labov, 1972: 9), namely by generating the language competence of the researcher as an Indonesian speaker. In this case, according to Robins (1992: 9), the researcher is an internalist, that is, the researcher is also a speaker of the language being studied. The data that have been collected are then classified based on the types of environment it refers to. The classified data are then analyzed using the referential equivalent method, which is a method that uses referents as a tool to prove certain lingual facts (Sudaryanto, 2015). This referential equivalent method is used to prove that the word ‘anak’ in a certain compound refers to a certain type of environment. The results of the data analysis are presented using informal methods and formal methods. The informal presentation method is the formulation of the results of data analysis in ordinary words, even with technical terminology. The formal presentation method is the formulation of the results of data analysis with what is commonly known as signs and symbols (Sudaryanto, 2015: 241). With the formal method, the results of data analysis in this study are presented in a table. 72 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 Findings and Discussion The data for the word ‘anak’ which refers to the educational environment are presented in Table 2 below. 73 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 Table 2. The word ‘anak’ referring to the educational environment No Combined Words Meaning 1 anak taman kanak-kanak (TK) kindergarten pupil or student 2 anak sekolah dasar (SD) elementary school pupil or student 3 anak sekolah menengah pertama (SMP) junior high school pupil or student 4 anak sekolah menengah atas (SMA) high school pupil or student 5 anak sekolah menengah kejuruan (SMK) vocational high school student 6 anak sekolah pupil or student 7 anak kursus student taking a course 8 anak madrasah 1. madrasa (a college for Islamic instruction) student 9 anak seminari seminary student 10 anak kelas I student of the first grade 11 anak kelas II student of the second grade 12 anak kelas III student of the third grade 13 anak didik pupil or student In table 2, data numbers 1 to 5 is a combination of the word ‘anak’ and the name of the education unit. Data number 6 to 9 includes a combination of the word ‘anak’ and the name of the type of education. Data number 10 to 12 is a combination of the word ‘anak’ and the name of the level in the education unit. Data 13 is a combination of the word ‘anak’ and the word ‘didik’ (educate). The word ‘anak’ that refers to the place/location environment is found in the combination of the word ‘anak’ and the name of the place. The word ‘anak’ followed by a place name usually means 'a child who comes from a particular area' or 'a child who lives in a particular area'. The data for the word ‘anak’ that refers to the environment in which it is located are presented in Table 3 below. Table 3. Findings and Discussion The word ‘anak’ pointing to the place/location environment No Combined Words Meaning 1 anak Aceh a person coming from Aceh 2 anak Bali a person coming from Bali 3 anak Yogyakarta a person coming from or Yogyakarta 4 anak Papua a person coming from Papua 5 anak kota a person coming from a city 6 anak desa a person coming from a village 7 anak asrama a person living in dormitories 8 anak kampung a person living in a kampong or village 9 anak kos a person living in a boarding house 10 anak jalanan a person living on the street Table 3. The word ‘anak’ pointing to the place/location environment No Combined Words Meaning In table 3, data 1 to 5 are a combination of the word ‘anak’ with the name of the place of origin. The combination of words 1 to 6 means 'children who come from….' The combination of words 7 to 10 means 'children who live in ….' 74 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 The word ‘anak’ that refers to the work environment means 'people who work in', 'players', or 'subordinates'. The word ‘anak’ that refers to the work environment is contained in the compound words presented in table 4 below. Table 4. The word ‘anak’ referring to the work environment No Compound Words Meaning 1 anak kapal officer on duty on the ship 2 anak perahu ship's crew 3 anak panggung performers in plays, drama, comedies, and so on 4 anak komidi comedian, play performer, theatrical performer 5 anak dabus player of dabus (Sundanese martial arts showing human abilities that are immune to sharp weapons, hard water, and others) 6 anak dayung rower 7 anak buah subordinate Table 4. The word ‘anak’ referring to the work environment N C d W d M i The word ‘anak’ that refers to the socio-economic environment is found in the combination of the word ‘anak’ and the name of the parent's occupational background and the combination of the word ‘anak’ with the name of the level of the parents' economic ability. The word ‘anak’ that refers to the socio-economic environment generally means 'a child whose parents’ status is …' or 'a child whose family's economic condition is capable or not.' Table 5. Findings and Discussion The word ‘anak’ referring to the socio-economic environment No Compound Words Meaning 1 anak pejabat A person whose parents are officials 2 anak petani A person whose parents are farmers 3 anak pedagang A person whose parents are traders 4 anak buruh A person whose parents are laborers 5 anak pengusaha A person whose parents are businessmen 7 anak kaya A person whose parents are rich 8 anak miskin children of poor parents 7 anak mampu children of rich parents 8 anak tidak mampu children from poor families Table 5. The word ‘anak’ referring to the socio-economic environment No Compound Words Meaning The word ‘anak’ referring to the socio-ethical environment is found in the combination of the word ‘anak’ and words that state the good or bad behaviour of a person. The compound word containing the word ‘anak’ that refers to a socio-ethical environment means 'a child who behaves according to or not following societal norms.' Table 6 The Word ‘Anak’ Referring to the Socio-ethical Environment No Compound Words Meaning 1 anak baik a good child 2 anak saleh a religiously obedient child 3 anak nakal a child who behaves deviantly from social norms 75 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 4 anak durhaka a child behaving against parental orders or God's orders 5 anak brandal a child who behaves not in accordance with the norms of society 6 anak kurang ajar a child behaving impolitely 7 anak santun a well-behaved child 8 anak penurut a child who obeys parental orders The word ‘anak’ that refers to the animal's environment is found in the combination of the word ‘anak’ and the name of the animal. The word ‘anak’ followed by the name of an animal usually means 'a small animal'. The data for the word ‘anak’ that refer to the animal environment are in the combination of words presented in table 7 below. Findings and Discussion Table 7 The Word ‘Anak’ Pointing to Animal Environment No Kata Majemuk Meaning 1 anak kelinci a little bunny 2 anak kucing a little cat 3 anak harimau a little tiger 4 anak sapi a little cow 5 anak burung a little bird 6 anak gajah a little elephant 7 anak ular a little snake Table 7 The Word ‘Anak’ Pointing to Animal Environment The word ‘anak’ that designates the plant environment is found in a combination of the word ‘anak’ and the name of the plant or the name of the plant part. Data for the word ‘anak’ that refers to the plant environment are presented in table 8 below. Table 8 The Word ‘Anak’ Pointing to the Plant Environment No Compound Words Meaning 1 anak daun leaf blade 2 anak pisang small banana tree 3 anak cabang twig The word ‘anak’ that refers to the natural environment is found in the combination of the word ‘anak’ and the name of the type of natural environment. The word ‘anak’ that refers to the natural environment generally means 'section' or 'branch'. Data for the word ‘anak’ that refers to the natural environment are presented in table 9 below. 76 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 Table 9 The Word ‘Anak’ Pointing to the Natural Environment No Compound Words Meaning 1 anak sungai river branch 2 anak bukit smaller hill 3 anak air gutter or ditch 4 anak gunung a smaller mountain that appears near a larger mountain 5 anak angin softer wind 6 anak laut smaller sea The word ‘anak’ that designates the environment of artificial objects is found in the combination of the word ‘anak’ and the names of artificial objects. Findings and Discussion Table 10 The Word ‘Anak’ Pointing to the Environment of Artificial Objects No Compound Words Meaning 1 anak kunci a key to open/unlock a lock 2 anak tangga the part of the ladder that is used to stand on 3 anak panah an arrow released with the bow 4 anak meja drawer 5 anak busur arrow 6 anak timbangan weighing device as a measure of the weight of goods 7 anak dacin stone (iron and so on) on a scale with a certain weight 8 anak batu tulis stationery to write on slate 9 anak batu cylinder shaped stone for crushing pepper (chili, spices, etc.) 10 anak bedil bullet 10 anak kalimat part of a sentence (clause) that cannot stand alone as a sentence' or 'bound clause' 11 anak judul addition to the title of an essay or a writing 12 anak bab part of the chapter in a writing 13 anak baju underwear 14 anak perusahaan a company part of a larger company le 10 The Word ‘Anak’ Pointing to the Environment of Artificial Objects The word ‘anak’ in the combination of these words means 'part'. The data for the word ‘anak’ that refers to the environment of artificial objects are presented in table 10 above. Conclusion The conclusion from the data analysis in this study is that the word ‘anak’ in Indonesian compound words refers to ten types of environment, namely (1) family environment, (2) educational environment, (3) work environment, (4) place/location environment, (5) socio-economic environment, (6) socio-ethical environment, (7) animal environment, (8) plant environment, (9) natural environment, and (10) artificial object environment. The results of this study have answered the research problem, 77 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 namely whether the use of the word ‘anak’ in a combination of Indonesian words has a relationship with the environment. The word ‘anak’ that designates certain environment contains certain meanings. The word ‘anak’ which refers to the family environment means 'descendants'. The word ‘anak’ which refers to the educational environment means 'pupil' or 'student'. The word ‘anak’ that refers to the work environment means 'people who work in', 'players', or 'subordinates'. The word ‘anak’ which refers to the environment in which it is located means 'person who comes from' or 'person who lives in'. The word ‘anak’ which refers to the socio-economic environment means 'children whose parents are' or 'children whose socio-economic status is able/poor'. The word ‘anak’ which refers to a socio- ethical environment means 'a child who behaves well or not well'. The word ‘anak’ which refers to an animal environment means 'a small animal'. The child's word that refers to the plant environment means 'part'. The word ‘anak’ which refers to the natural environment means 'part'. The word ‘anak’ that refers to the environment of artificial objects also means 'part'. The results of this study prove that the use of the word ‘anak’ in Indonesian compound words can refer to various types of environments. The type of environment that the word ‘anak’ denotes depends on the word it combines. The words they combine also affect the meaning expressed by the word ‘anak’. Furthermore, the results of this study further confirm the basic assumption in ecolinguistics that language has a relationship with the environment. The theory of the relationship between language and the environment will be stronger when more research is carried out on the elements of language from an ecolinguistic perspective. References Badan Pengembangan Bahasa dan Perbukuan. (2015). Kamus besar Bahasa Indonesia (Edisi V). Jakarta: Kementerian Pendidikan dan Kebudayaan Republik Indonesia. Badudu, J.S. (1981). Kamus ungkapan Bahasa Indonesia. Bandung: CV Pustaka Prima dan PT Harapan Botha, R. P. (1981). The Conduct of Linguistics Inquiry: A Systemic Introduction to the Methodology of Generative Grammar. ITL – International Journal of Applied Linguistics, 62, 84-87. https://doi.org/10.1075/itl.62.07swi g p g aer, A. (1984). Kamus idiom Bahasa Indonesia. Ende-Flores: Penerbit Nusa Indah. Chaer, A. (1984). Kamus idiom Bahasa Indonesia. Ende-Flores: Penerbit Nusa Indah. Chen, S. (2016). Language and ecology: A content analysis of ecolinguistics as an emerging research field. Apersand, 3, 108-116. https://doi.org/10.1016/j.amper.2016.06.002 Echols, J. M., & Shadily, H. (1994). Kamus Indonesia-Inggris (Edisi Ketiga). Jakarta: Penerbit PT Gramedia. Halliday, M. A. K. (2001). New ways of meaning: The challenge to applied linguistics. Paper presented at the World Congress of Applied Linguistics sponsored by the International Association of Applied Linguistics (9th, Thessaloniki, Greece, April 15-21, 1990). Haugen, E. (1972). The ecology of language. Stanford, CA: Stanford University Press. 78 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 6, No. 2, September 2022, pp. 68-79 Isodarus, P. B. (2021). Perubahan paradigma dalam kajian bahasa. Jurnal Sintesis, 15(1), 67-79. https://doi.org/10.24071/sin.v15i1.3273 Isodarus, P. B. (2021). Perubahan paradigma dalam kajian bahasa. Jurnal Sintesis, 15(1), 67-79. https://doi.org/10.24071/sin.v15i1.3273 ( ), p g Kridalaksana, H. (1993). Kamus linguistik (Edisi Ketiga). Jakarta: PT Gramedia Pustaka Utama Kridalaksana, H. (1993). Kamus linguistik (Edisi Ketiga). Jakarta: PT Gramedia Pustaka Utama Kridalaksana, H., & Harimurti. (1988). Beberapa prinsip perpaduan leksem dalam Bahasa Indonesia. Yogyakarta: Penerbit Kanisius. Labov, W. (1972). Some principles of linguistic methodology. Language and Society 1, 97-120. Ogden, C. K., & Richards, I. A. (1989). The meaning of meaning. San Diego: Harcourt Brace Javanovich, Inc. Robins, R. H. (1992). Linguistik umum: Sebuah pengantar (Soenarjati Djajanegara, Trans.). Yogyakarta: Penerbit Kanisius. Sibarani, T. (2014). Ekolinguistik kebambuan dalam masyarakat bahasa batak toba. Medan Makna, 12(2), 95-104. https://doi.org/10.26499/mm.v12i2.1033 Sinungharjo, F. X. (2020). Leksikon biotik di panggung musik: Perspektif ekolinguistik. Sintesis, 14(2), 109-130. https://doi.org/10.24071/sin.v14i2.2675 Subiyanto, A. (2013). Ekolinguistik: Model analisis dan penerapannya. Humanika, 18(2), https://doi.org/10.14710/humanika.18.2. Sudaryanto. (2015). Metode dan aneka teknik analisis bahasa: Pengantar penelitian wahana kebudayaan secara linguistis. Yogyakarta: Sanata Dharma University Press. Tarigan, G. E. (2018). Leksikon perpadian dalam bahasa karo kajian ekolinguistik (Undergraduate thesis, Universitas Sumatra Utara Medan). Retrieved from http://repositori.usu.ac.id/handle/123456789/10789 Wenjuan, Z. (2017). Ecolinguistics: Towards a new harmony. Language & Communication, 62, 124-138. https://doi.org/10.1016/j.langsci.2017.04.004 Wenjuan, Z. (2017). References Ecolinguistics: Towards a new harmony. Language & Communication, 62, 124-138. https://doi.org/10.1016/j.langsci.2017.04.004 the Indonesian version, this article was presented online at the 29th Anniversary minar of the Faculty of Letters, Sanata Dharma University, on April 4, 2022. Notes: Notes: Notes: In the Indonesian version, this article was presented online at the 29th Anniversary Seminar of the Faculty of Letters Sanata Dharma University on April 4 2022 In the Indonesian version, this article was presented online at the 29th Anniversary Seminar of the Faculty of Letters, Sanata Dharma University, on April 4, 2022. In the Indonesian version, this article was presented online at the 29th Anniversary Seminar of the Faculty of Letters, Sanata Dharma University, on April 4, 2022. 79 79
https://openalex.org/W2412668035	https://zenodo.org/records/1693824/files/article.pdf	English	null	The Penetration of Electrolytes into Gels	Biochemical journal. Cellular aspects	1,921	public-domain	2,816	LXXVI. THE PENETRATION OF ELECTRO- LYTES INTO GELS. IV. THE DIFFUSION OF SULPHATES IN GELS. BY WALTER STILES. From the Department of Botany, University College, Reading. (Received August 18th, 192-1.) INTRODUCTION. IT is a curious fact relating to the nutrition of plants that plant tissue appears to absorb chlorides and nitrates to a considerably greater extent than sulphates. This phenomenon appears to be very general. Thus Wodehouse [1917] found chloride abundant in the marine alga Valonia, whereas sulphate was absent from it, although abundant in the sea water in which the alga was growing. Stiles and Kidd [1919] found that sulphates were absorbed to a much less extent than chlorides and nitrates by potato tuber and carrot root. The investigations of Pantanelli [1915, 1918] on a.large number of plants point to the same conclusion with regard to root absorption. This state of affairs is the more surprising as sulphur is an essential element for the plant, whereas many plants are able to thrive if unprovided with chlorine in any form. It would appear to be significant in regard to the apparent difficulty of absorption of sulphates by plants as compared with the absorption of chlorides, that a similar difference has been observed in the matter of the penetration of these salts through precipitation memiabranes [Traube, 1867; Tammann, 1892; Walden, 1892] and membranes of-collodion [Brown, 1915]. It thus would seem possible that the small quantity of sulphate that enters living plant tissue as compared with the quantity of chloride and nitrate, might be due to the different degrees of difficulty of sulphates on the one hand, and chlorides and nitrates on the other, in passing through colloidal membranes, of the protoplast [see Stiles, 1921]. It becomes of interest, there- fore, to determine whether the diffusion of sulphates in gels presents any anomalous features similar to those presented in their diffusion through membranes, which might help to explain the unexpected phenomenon of their comparatively slight absorption by plant tissue. The only recorded observations on the diffusion of sulphates in gels appear to be those of von Fiirth and Bubanovic [1918] who used agar-agar containing barium nitrate as an indicator. They did not calculate coefficients of diffusion 40 2 W. STILES 630 from their results, nor do they provide the data to enable this to be done absolutely, but instead they calculate a quantity called by them the " invasion factor," which should be approximately proportional to the square root of the coefficient of diffusion. INTRODUCTION. From the data of earlier workers von Fiirth and Bubanovic have calculated the coefficients of diffusion in water at 180 of the substances with which they worked. In Table I are collected the numbers they quote for sulphates together with the invasion factors they found for the same substances and the coefficients of diffusion obtained by taking the squares of the invasion factors. Table I. Coefficients of diffusion at 18° of some sulphates in water and in 2 % agar-agar containing barium nitrate. (Calculated from the data of von Fiirth and Bubanovic, the coefficient of diffusion of sodium chloride being taken as unity.) Table I. Coefficients of diffusion at 18° of some sulphates in water and in 2 % agar-agar containing barium nitrate. (Calculated from the data of von Fiirth and Bubanovic, the coefficient of diffusion of sodium chloride being taken as unity.) y Coefficient of Coefficient of Invasion factor diffusion diffusion Sulphate in gel in gel in water Hydrogen sulphate 098 096 1-33 Potassium sulphate 1-06 1-12 0 74 Zinc sulphate 074 0 55 0-48 Magnesium sulphate 067 045 0 50 Ammonium sulphate 0'67 045 0-76 These results exhibit no sort of relation between the coefficients of diffusion in the gel and in water. It has, however, already been pointed out in the first paper in this series [Stiles, 1920], that von Fiirth and Bubanovic neglect the influence of the concentration of the indicator in their experiments, although this influences very considerably the invasion factor, and consequently the coefficient of diffusion calculated from it. A fresh series of determinations of the coefficients of diffusion of a number of sulphates in gels of gelatin and agar-agar has therefore been made; the values obtained are recorded in a subsequent section of this paper. METHOD. The method employed was that outlined in the third paper in this series [Stiles and Adair, 1921]. Gels of 0 5 % agar-agar and 10 % gelatin were employed, and barium chloride in 0 01 N concentration by weight was used as the indicator in the gel. The solutions of sulphates employed were in normal concentration. The experiments were carried out in thermostats main- tained at 00 and about 20.5°. In calculating the coefficients of diffusion from the experimental data, the more exact of the two formulae obtained by Adair [1920] was employed. For this purpose the coefficient of diffusion of barium chloride was assumed to be 0-76 x 10-5 C.g.S. units under the experimental conditions. Although exact data of this constant do not exist, such values for the coefficient of DIFFUSION OF SULPHATES IN GELS 631 diffusion of barium chloride as have been obtained indicate the value used as approximately correct. Even a 50 % error in the value of the constant introduces only a very slight error in the calculated coefficient of diffusion, and it is safe to conclude that any error arising from an assumption of an incorrect value for the coefficient of diffusion of barium chloride is negligible. EXPERIMENTAL RESULTS. The sulphates employed were those of ammonium, potassium, sodium, magnesium and copper. The experimental data are summarised in Table II. Table II. Penetration of various sulphates from a N solution into gels of agar-agar and gelatin containing barium chloride as indicator. (Concentra- tion of barium chloride in the gel, 0.01 N; time in hours; penetration in centimetres of precipitate; diffusion coefficient k in c.g.s. units x 105.) Diffusing salt (NH4)2S04 K2SO4 ,, ,, ,, Na2SO4 S,, MgS04 ,,9 ,.9 ,, ,, cus04j Gel 0 5 % agar 10 % gelatin 0 5 % agar 10 % gelatin ,, 05 % agar ,, 10 % gelatin 0.5 % agar 1 l, n 10 % gelatin 0 5 % agar 10 % gelatin 9,, Temperature 0 . 0 20-3 20-5 0 203 20-5 ,, 0 20-3 ,, 205 0 20-0 ,, 20-5 20-0 9, 20-5 9, Time 22-36 2345 4768 2266 46-87 22-28 23-44 2267 t690 22-45 23-69 47-60 22-70 46-90 22-12 20-39 45 30 22-60 46-80 22-20 20-25 45-23 22-74 4695 Penetration 2-220 3-202 4.599 2-748 3.955 2-218 3-200 2745 3924 1-920 2937 4-213 2-515 3-615 1-657 2-192 3316 2002 2-872 1-793 2276 3.439 1-796 2592 k 0 474 0920 0-932 0-706 0-706 0475 0-918 0704 0-695 0-355 0-770 0-787 0 593 0593 0-269 0-502 0-517 0-384 0382 0-314 0 545 0 557 0 3I1 0-314 A first glance at these results as they stand shows that the coefficient of diffusion is considerably raised with increase of temperature from 00 to 200; that at the same temperature diffusion is much more rapid in 0-5 % agar than in 10 % gelatin, and that the coefficients of diffusion of different sub- stances in the gels are roughly parallel to the coefficients of diffusion in water. Each one of these qaestions will now be considered more in detail. W. STILES 632 2. Influence of gel concentration. In the previous paper of this series the influence of the concentration of the gel on the diffusion of sodium chloride was investigated over a wide range of gel concentrations. In order to determine whether the effect is similar in the case of sulphates it was considered sufficient to carry out experiments with a thin gel of low concentration (0.5 % agar) and a stiff gel of moderately high concentration (10 % gelatin). The values previously obtained with sodium chloride indicate that with that salt the coefficient of diffusion in 0 5 per cent. agar is about 1-3 times that in 10 % gelatin at the same temperature. The ratios of the coefficients-of diffusion of the sulphates examined in 0 5 % agar and 10 % gelatin at 20.50 are as follows: g Ammonium sulphate ... 131 Magnesium sulphate ... 1P34 Potassium sulphate ... 1-31 Copper sulphate ... 1-78 Sodium sulphate ... 1P32 With the exception of copper sulphate, tfhe relation between the coefficients of diffusion in 0 5 % agar and in 10 % gelatin is much the same in all the sulphates examined, and in sodium chloride. Copper sulphate forms a very conspicuous exception to the rule in that the coefficient of diffusion is relatively much greater in 05 % agar than would be expected from the rate, of diffusion in 10 % gelatin. 1. Influence of temperature. If we assume, as is usually done, that the coefficient of diffusion increases with rise of temperature according to a linear relation between diffusivity and temperature, we have the following equations for the coefficient of diffusion of the various salts examined at any temperature 0 lying between 00 and 20°. Salt Coefficient of diffusion at 6' C. Ammonium sulphate 0 474 (1+0-04630) Potassium sulphate 0475 (1 +0.0463 0) Sodium sulphate 0355 (1+00592 0) Magnesium sulphate 0269 (1+00446 0) Copper sulphate 0-314 (1 +00382 0) The increase in the rate of diffusion for rise of -temperature of 10 relative to the coefficient of diffusion at 00 thus varies somewhat with different salts, being least for copper sulphate and greatest for sodium sulphate. The numbers are however all of the same order. The value of this quantity in the case of the diffusion of sodium chloride in 0-5 % agar at temperatures between 00 and 200 is however much the same, namely 00385, as that for copper sulphate. It is clear that the influence of temperature on the diffusion of sulphates in agar gels presents no unusual features. 3. Comparison of the coefficients of diffusion in gels and in water. The observations that have been made in the past on the diffusion of sul- phates in pure water have been made at a number of differing temperatures DIFFUSION OF SULPHATES IN GELS 633 and with very varied concentrations of diffusing salts. A comparison between the actually observed rates of diffusion in gels and water cannot therefore be made. However from the results obtained for the coefficient of diffusion of sulphates in 05 % agar at 0° and 200, it is at once possible to obtain by interpolation the values for the coefficient of diffusion in this gel at the temperatures used in earlier investigations on diffusion in pure water. In the following Table are shown, for the sulphates examined in this investigation, the values of the coefficients of diffusion in pure water and in 0-5 % agar- agar at the same temperature. The concentrations of the diffusing salts are not always the same, but the coefficients of diffusion in pure water tabulated are those for the concentration of salt nearest to normal, that used in the experiments with gels. Observations made by Oholm [1905] on other salts suggest that generally decrease in concentration results in an increase in the coefficient of diffusion, but the influence of concentration is not very great over small ranges of concentration. Table III. Comparison of the coefficients of diffusion of various sulphates in water and in 0-5 %, agar-agar. (Coefficient of diffusion, k, in c.g.s. units x 105; concentration of salt in diffusion in gels N in all cases; concentration of salt in diffusion in water given in normalities in fifth column.) Concentration in normalities of Diffusing Temperature salt in diffusion Observer of k salt 0 C. k in gel k in water in water in water (Lang-Hedin- (NH4)2SO4 18 0-869 0-880 2-0 von Fiirth and ( Bubanovic K2SO4 19 6 0 903 {0914 905} Thovert Na2SO4 10-4 0 574 0-567 2-58 Graham-Stefan M904 r5.5 0-335 0-37 1.08 Shfe MgSO4 1 10 0-389 0 394 0°80 Scheffer CuS04 17 0-518 0-336 1.00 Thovert The numbers presented in this Table show conclusively that, with the exception of copper sulphate, the values for the coefficient of diffusion of the sulphates in 0-5 % agar are very close to those for diffusion of the same salts in pure water. 3. Comparison of the coefficients of diffusion in gels and in water. The evidence is that the coefficient in agar is slightly less than in pure water, as would be expected from the increased viscosity in the gel. CONCLUSION. From the results of this study of the diffusion of a number of sulphates in gelatin and agar-agar gels, it appears evident that, with the single exception of copper sulphate, the diffusion of sulphates in gels presents the same features as that of sodium chloride. Diffusion proceeds almost as rapidly in 0 5 % agar- agar as in pure water, while in 10 % gelatin the diffusion coefficient is reduced about 25 %. The influence of temperature is similar to that on the coefficient of diffusion of sodium chloride. Copper sulphate, on the other hand, shows W. STILES 634 an abnormally large coefficient of diffusion in 0-5 % agar-agar, although the coefficient of diffusion of this salt in 10 % gelatin bears more nearly, though not exactly, the same relation to the coefficient of diffusion in water as the other salts examined. This is a very curious phenomenon for which at present no explanation can be offered, but which certainly deserves further in- vestigation. A reference to Table I of this paper shows that the results of von Fiirth and Bubanovic indicate that the coefficients of diffusion of potassium sulphate and zinc sulphate are greater, and that of ammonium sulphate considerably less, in a 2 % agar-agar gel than in pure water. The more exact determinations recorded in this paper show clearly that such a divergence between diffusion of these sulphates in water and in gels does not exist. g The comparatively small intake of sulphates by plant tissue is therefore not to be explained on account of any abnormal behaviour of these salts in diffusing through colloidal systems. Further evidence in this direction is forth- coming from the observations of Stiles and Kidd [1919] in which it was shown that the plant tissue examined absorbs sulphates initially faster than either chlorides or nitrates, but that the rate of absorption of sulphate falls off very much more rapidly than that of chloride or nitrate. It would thus appear that it is a question of equilibrium in a chemical combination, or in adsorption, that limits the intake of sulphates, rather than any retardation owing to a particularly slow rate of diffusion, or to a change brought about by the sulphate on the colloidal system as such which renders it much more resistant to the passage of the ions. SUMMARY. The coefficients of diffusion of a number of sulphates in 0-5 % agar-agar and 10 % gelatin have been determined by the indicator method. In the cases of the sulphates of ammonium, potassium, sodium and magnesium the coefficient of diffusion in 0-5 % agar-agar is very close to, though slightly less than, that in pure water at the same temperature and for the same concentration of diffusing salt, while the coefficient of diffusion in 10 % gelatin is about 25 % less than in 0-5 % agar-agar. The diffusion coefficient of copper sulphate in -0-5 % agar-agar is considerably greater than in water, but in 10 % gelatin it is smaller than in water but yet somewhat higher than the values obtained for other sulphates would indicate. Increase of temperature from 00 to 200 increases the diffusion coefficient about 1-76 to 2-2 times according to the salt. These effects are of the same order as in the case of sodium chloride. Except in the case of copper sulphate the diffusion of sulphates in the gels examined proceeds normally and the results of von Fiirth and Bubanovic indicating the contrary are not confirmed. The comparatively small uptake of sulphates by plant tissue is not to be explained on the basis of retarded diffusion through a gel. DIFFUSION OF SULPHATES IN GELS 635 REFERENCES. Adair (1920). Biochem. J. 14, 762. Brown (1915). Biochem. J. 9, 591. v. Fiirth and Bubanovi6 (1918). Biochem. Zeitsch. 90, 265; 92, 139. Oholm (1905). Zeit8ch. phy8ikal. Chem. 50, 309. ( ) p y , Pantanelli (1915). Jahrb. Wi88. Bot. 56, 689; Bull. Orto Bot., R. Univ. Napoli, 5, 1. p y Pantanelli (1915). Jahrb. Wi88. Bot. 56, 689; Bull. Orto Bot., R. Univ. Napoli, 5, 1. (1918). Bull. Orto Bot., R. Univ. Napoli, 6, 1. Stiles (1920). Biochem. J. 14, 58. - (1921). New Phyt. 20, 48. Stiles and Adair (1921). Biochem. J. 15, 620. Stiles and Kidd (1919). Proc. Roy. Soc. B, -90, 487. Tammann (1892). Zeitsch. phy8ikal. Chem. 10, 255. Traube (1867). Arch. Anat. Phy8iol. Wi8s. Med. 87. Walden (1892). Zeit8ch. phy8ikal. Chem. 10, 699. Wodehouse (1917). J. Biol. Chem. 29, 453.
https://openalex.org/W2997785597	https://nottingham-repository.worktribe.com/file/3694335/1/s42005-020-0290-x	English	null	Resonant tunnelling into the two-dimensional subbands of InSe layers	Communications physics	2,020	cc-by	7,337	1 School of Physics and Astronomy, University of Nottingham, Nottingham NG7 2RD, UK. 2 Department of Physics, Loughborough University, Loughborough, LE11 3TU, UK. 3 Institute of Microelectronics Technology and High Purity Materials, RAS, Chernogolovka 142432, Russia. 4 Institute for Problems of Materials Science, The National Academy of Sciences of Ukraine, Chernivtsi Branch, Chernivtsi 58001, Ukraine. email: amalia.patane@nottingham.ac.uk COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys Resonant tunnelling into the two-dimensional subbands of InSe layers Furthermore, Esaki-type tunnel diodes10 have demonstrated the potential of 2D materials for transistors with a low subthreshold swing and NDC by band-to-band tun- nelling of carriers through the forbidden energy gap of a vdW heterojunction11–13. H the current–voltage I–VSD curve at a measurement temperature, T = 2.0 K, and Vg = 0. For \|VSD\| ≲0.5 V, the tunnel current remains small (<10 pA) for both positive and negative applied voltages. However, for VSD > 0.5 V, the current increases rapidly and the I–VSD curve reveals a sequence of resonant features. Pronounced peaks in the I–VSD can be seen over an extended range of VSD and are followed at higher bias (\|VSD\| > 1.1 V) by weaker resonances. These resonances, labelled S1, S2… in Fig. 1b can be seen more clearly in the differential conductance, G = dI/ dVSD, plot shown in Fig. 1c. We attribute each resonance to the threshold of resonant tunnelling through a quantized 2D subband of the InSe layer when the energy of the subband minimum aligns with the chemical potential of the MLG-source electrode (inset of Fig. 1c, d). The asymmetry of the I–VSD curve with respect to positive and negative voltages suggests that the two interfaces of the InSe with the two MLG-electrodes are different. The reso- nances can be observed more clearly when electrons tunnel from the bottom to the top MLG (VSD > 0), suggesting that the InSe layer forms a cleaner interface with the bottom MLG. This asymmetry is likely to arise from the lower quality of the top surface of InSe, which tends to be more contaminated than the bottom surface due to the use of polymer stamps for the exfo- liation of InSe. The resonances are observed over a wide temperature range (Fig. 2a–b): on increasing the temperature from T = 2 K to 144 K, the resonances shift to lower values of VSD and tend to broaden; they cannot be resolved clearly at T = 300 K (inset of Fig. 2a). We assign this behavior to two main effects: the thermal broadening of the electron distribution in the MLG-source; secondly, the resonant subband states of the InSe layer are effectively broadened due to scattering of charge carriers by thermally excited lattice vibrations. This phenomenon is further examined in the Discussion section. Resonant tunnelling into the two-dimensional subbands of InSe layers j Here, we report on a different type of TFET, a resonant tun- neling transistor (RTT), which exhibits multiple regions of NDC. These device structures exploit the tunnelling of charge carriers from a source of electrons into the distinct 2D subband states of an InSe vdW crystal a few atomic layers thick. We use important features of the RTT: due to strong quantum conﬁnement of the light effective mass electrons14, the energy of the 2D subbands can be tuned by varying the thickness of the InSe layer. By applying a voltage, VSD, across two multilayer graphene (MLG) electrodes, which serve as the source (S) and drain (D) electrodes to the InSe layer, the chemical potential in the source can be tuned in and out of resonance with a given 2D subband, leading to a peak in the current–voltage (I–VSD) characteristics and a region of NDC. Multiple regions of NDC can be thus induced by tunnelling into consecutive 2D subbands of InSe, whose number increases with the number of InSe layers. Our ﬁndings suggest prospects for further research avenues and applications. InSe- based RTTs could aid the development of multivalued logic and frequency multiplication. Furthermore, intersubband transitions of individual InSe layers or repeated multilayered stacks could be exploited in photon sources and detectors. InSe offers exciting possibilities for these technological developments due to its high room temperature electron mobility and its tuneable band gap and energy subbands14–16. Tunnelling into the 2D subbands of InSe. InSe-based devices can be designed and fabricated with different thickness, t, of the InSe layer and hence with different subband energies. Figure 3a shows the t-dependence of the energy minima of the InSe sub- bands. Here, we use the simplifying assumption that the electrons are conﬁned by a square quantum well (QW) potential of inﬁnite height, i.e., En ¼ n2h2=8t2me c, where n is an integer, me c= 0.08me is the electron effective mass for motion along the c-axis in bulk InSe18, and me is the free electron mass. The calculated energy separation, ΔE2–1, between the lowest two subbands decreases from hundreds of meV to a few meV with increasing t from 2 to 20 layers (Fig. 3a). Resonant tunnelling into the two-dimensional subbands of InSe layers Zakhar R. Kudrynskyi 1, James Kerfoot1, Debarati Mazumder1, Mark T. Greenaway 1,2, Evgeni E. Vdovin3, Oleg Makarovsky1, Zakhar D. Kovalyuk4, Laurence Eaves1, Peter H. Beton 1 & Amalia Patanè1 Two-dimensional (2D) van der Waals (vdW) crystals have attracted considerable interest for digital electronics beyond Si-based complementary metal oxide semiconductor technologies. Despite the transformative success of Si-based devices, there are limits to their miniatur- ization and functionalities. Here we realize a resonant tunnelling transistor (RTT) based on a 2D InSe layer sandwiched between two multilayered graphene (MLG) electrodes. In the RTT the energy of the quantum-conﬁned 2D subbands of InSe can be tuned by the thickness of the InSe layer. By applying a voltage across the two MLG electrodes, which serve as the source and drain electrodes to the InSe, the chemical potential in the source can be tuned in and out of resonance with a given 2D subband, leading to multiple regions of negative differential conductance that can be additionally tuned by electrostatic gating. This work demonstrates the potential of InSe and InSe-based RTTs for applications in quantum electronics. 1 ARTICLE COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x H eterostructures based on two-dimensional (2D) van der Waals (vdW) crystals1–3, such as graphene, hexagonal boron nitride (hBN), and metal mono- (e.g., InSe, GaSe…) and di-chalcogenide (e.g., SnSe2, MoS2…) layers have physical properties that may overcome some of the technological limitations of traditional covalent semiconductors4. Beyond their versatility for a wide range of band structure-engineered devices by stacking and/or twisting the layers, these structures can be also readily transferred onto substrates that are compatible with the well-established complementary metal–oxide–semiconductor (CMOS) device fabrication processes used in modern integrated circuits for digital logic, image sensors and transceivers. In recent years, different types of tunnel ﬁeld effect transistors (TFET) based on vdW crystals have emerged, stimulating research of fundamental and applied interest5–13. These devices include the graphene/insulator/graphene TFET with a region of negative differential conductance (NDC) in the current–voltage char- acteristics due to resonant tunnelling8. Similar TFET structures based on transition metal di-chalcogenides (TMDs) rather than graphene layers have been also suggested and studied theoreti- cally, revealing improved transport characteristics due to quali- tative differences in the band structure of these materials compared to graphene9. Resonant tunnelling into the two-dimensional subbands of InSe layers Figure 3b shows the corresponding t-depen- dence of the band-edge exciton recombination energy, i.e., EX ¼ E3D X þ h2=8t2μex c , where E3D X = 1.25 eV is the value of EX for bulk InSe at T = 300 K, μex c ¼ 1=½1=me c þ 1=mh c is the reduced exciton mass, and mh c= 0.17me is the hole effective mass19. Similar dependences of EX and ΔE2–1 on t were predicted by density functional theory20,21, thought with larger deviations from the simple QW model at smaller t21. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x The device is constructed by dry transfer of individual layers (e.g., InSe, hexagonal boron nitride (hBN), and multilayer graphene (MLG)) onto a SiO2/Si substrate and is encapsulated by hBN. The top insets show the crystal structure of single-layer InSe and the in-plane energy dispersion of the two- dimensional (2D) InSe subbands. b Current-voltage, I–VSD, characteristics of a device with ten-layers (10 L) InSe at a gate voltage Vg = 0 V and temperature T = 2 K. Inset: zoom in of the I–VSD curve in forward bias. c Differential conductance, G–VSD, curve at Vg = 0 V and T = 2 K. Inset: sketch of the tunnelling of electrons from a MLG-electrode (source) into the 2D subbands of InSe. d Schematic of tunnelling of electrons from a MLG-electrode (source) into the 2D subbands of InSe and into MLG (drain) for an increasing source-to-drain voltage, VSD. Bottom inset: Brillouin zone of InSe and MLG. Fig. 2 Temperature dependence of the transport characteristics. a Current–voltage characteristics, I−VSD, of a device with ten-layers InSe at a gate voltage Vg = 0 V and at different temperatures T. The inset compares the I−VSD curves at T = 300 K and 2 K. b I−VSD curves from part (a) in the low voltage region. Fig. 2 Temperature dependence of the transport characteristics. a Current–voltage characteristics, I−VSD, of a device with ten-layers InSe at a gate voltage Vg = 0 V and at different temperatures T. The inset compares the I−VSD curves at T = 300 K and 2 K. b I−VSD curves from part (a) in the low voltage region. layer, the I–VSD also reveal a distinct temperature dependence: the conductivity exhibits a thermally activated behavior, sugges- tive of thermal emission of electrons from shallow donor and defect states in InSe (Supplementary Fig. 2). exhibit a similar shift with the applied gate voltage (e.g., dVSD=dVg ~ −0.005). The spacing between the ﬁrst two reso- nances S1 and S2 is ΔVSD ~ 0.2 V at Vg = +30 V. This is in good agreement with the calculated energy spacing ΔE1–2 between the lowest electron energy subbands of InSe shown in Fig. 3a. Decreasing the layer thickness from 10 to 8 layers increases the measured value of ΔVSD from ~0.2 V to ~0.3 V (Fig. 3c). As can be seen in Fig. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x -1 0 1 0 10 S5 S4 S3 S2 G (nS) VSD (V) S1 -1 0 1 10 -11 10 -10 10 -9 10 -8 S5 S4 S3 S2 S1 I (A ) VSD (V) T = 2 K Vg= 0 V t = 10 L a b c k// k// d MLG MLG InSe eVSD eVSD VSD = 0 e e e- e- 0.8 1.2 0 2 VSD(V) I (nA) Au MLG Au MLG VSD Vg Fig. 1 InSe-based resonant tunnelling transistors with NDC. a Optical image (scale bar: 20 μm) and schematic of an InSe-based resonant tunnelling transistor. The device is constructed by dry transfer of individual layers (e.g., InSe, hexagonal boron nitride (hBN), and multilayer graphene (MLG)) onto a SiO2/Si substrate and is encapsulated by hBN. The top insets show the crystal structure of single-layer InSe and the in-plane energy dispersion of the two- dimensional (2D) InSe subbands. b Current-voltage, I–VSD, characteristics of a device with ten-layers (10 L) InSe at a gate voltage Vg = 0 V and temperature T = 2 K. Inset: zoom in of the I–VSD curve in forward bias. c Differential conductance, G–VSD, curve at Vg = 0 V and T = 2 K. Inset: sketch of the tunnelling of electrons from a MLG-electrode (source) into the 2D subbands of InSe. d Schematic of tunnelling of electrons from a MLG-electrode (source) into the 2D subbands of InSe and into MLG (drain) for an increasing source-to-drain voltage, VSD. Bottom inset: Brillouin zone of InSe and MLG. a k// k// Au MLG Au MLG VSD Vg d MLG MLG InSe eVSD eVSD VSD = 0 e e -1 0 1 10 -11 10 -10 10 -9 10 -8 S5 S4 S3 S2 S1 I (A ) VSD (V) T = 2 K Vg= 0 V t = 10 L b 0.8 1.2 0 2 VSD(V) I (nA) d a c -1 0 1 0 10 S5 S4 S3 S2 G (nS) VSD (V) S1 c e- e- -1 0 1 0 10 S5 S4 S3 S2 ( ) VSD (V) S1 eVSD e e- e- Fig. 1 InSe-based resonant tunnelling transistors with NDC. a Optical image (scale bar: 20 μm) and schematic of an InSe-based resonant tunnelling transistor. Results p We now consider the measured I–VSD characteristics of three devices with different InSe layer thicknesses t = 8, 10, and 19 layers, as estimated from the photoluminescence (PL) spectra of these three samples at T = 300 K (inset of Fig. 3b). The PL peak energy corresponds to that of the band-edge exciton21,22 and is very sensitive to the layer thickness: it increases from EX~1.28 eV for t = 19 layers to EX ~ 1.39 eV for t = 8 layers. Figure 3c shows the I–VSD and G–VSD curves of these three devices, revealing that the current and voltage spacing between consecutive resonances increases with decreasing t. We note that no resonances were observed in the device with t = 19 layers or in other devices with thicker layers. For these devices with a relatively thicker InSe Resonant tunnelling transistors with NDC. Figure 1a illustrates the schematic layered structure and an optical image of a typical device formed by dry transfer17 of exfoliated MLG, hBN, and InSe on a SiO2/Si substrate (details of the sample preparation and measurement techniques are described in the Methods section and Supplementary Note 1). In this device, the InSe layer has an in-plane area of ~100 μm2 and thickness t ~ 8 nm (ten layers of InSe). A voltage (VSD) is applied to the top MLG-electrode relative to the bottom to align different 2D subbands of InSe to the chemical potential in the source. The Si-substrate serves as a gate electrode and the gate voltage, Vg, is used to adjust the chemical potential in the adjacent/bottom MLG. Figure 1b shows COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys 2 COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x ARTICLE COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x a Differential conductance, G–VSD, curves at applied magnetic ﬁelds B = 0 and 14 T for devices with eight-layers (main panel) and ten-layers (inset) InSe. In both cases the gate voltage is Vg = 0 V, the temperature is T = 2 K and the magnetic ﬁeld is applied perpendicular to the direction of the current. b G(B, VSD) color map for the device with eight- layers InSe (T = 2 K). The inset is a schematic of the injection of electrons f th l t d i t th bb d f I S Th b l ill t t th i Fig. 3 2D subbands in InSe. a, b Calculated dependence of the subbands a and exciton recombination b energies on the number of InSe layers The Fig. 3 2D subbands in InSe. a, b Calculated dependence of the subbands a and exciton recombination b energies on the number of InSe layers. The arrows in part a show the energy spacing between the lowest two subbands for three speciﬁc layer thicknesses. The inset in part b shows the normalized photoluminescence spectra of devices with 8, 10, and 19 layers (L) of InSe at T = 300 K. c Current–voltage, I–VSD, characteristics of devices with different InSe layer thickness (t = 8, 10, and 19 layers) at the gate voltage Vg = 0 V and T = 2 K. Inset: differential conductance, G–VSD, plots for devices with thickness t = 8, 10, and 19 layers. The multiplication factor indicates the number by which each curve has been multiplied to facilitate a comparison of different samples. Fig. 3 2D subbands in InSe. a, b Calculated dependence of the subbands a Fig. 3 2D subbands in InSe. a, b Calculated dependence of the subbands a and exciton recombination b energies on the number of InSe layers. The arrows in part a show the energy spacing between the lowest two subbands for three speciﬁc layer thicknesses. The inset in part b shows the normalized photoluminescence spectra of devices with 8, 10, and 19 layers (L) of InSe at T = 300 K. c Current–voltage, I–VSD, characteristics of devices with different InSe layer thickness (t = 8, 10, and 19 layers) at the gate voltage Vg = 0 V and T = 2 K. Inset: differential conductance, G–VSD, plots for devices with thickness t = 8, 10, and 19 layers. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x The emergence of this additional narrow feature in the transport characteristics of our devices is similar to recent observations of resonant electron tunneling through localized states due to defects in hBN23. The stronger dependence of R on Vg (dVSD=dVg ~ −0.05) compared to that of Sn (dVSD=dVg ~ −0.005) suggests that R arises from carrier tunnel- ing into the states of a localized defect close to the bottom gate electrode. Similar narrow resonances can be seen in the same device under negative applied gate voltages and VSD ~ +1.0 V (Fig. 4b). Also, close inspection of the G–VSD plots of different devices reveals that the main Sn-resonances can comprise additional weak features closely spaced in energy (Fig. 5a). This quasi-regular modulation of the current is weakly affected by temperature, although the increasing noise and broadening of the incremental change of the applied voltage corresponding to a change in energy dE of a particular subband. A value of f ≈1 is in line with the observation of electroluminescence from our devices when the chemical potentials in the source and drain differs by an energy ΔE = eVSD that is approximately equal to the exciton recombination energy in the InSe layer, i.e., eVSD ~ EX (Supple- mentary Note 2). This is also consistent with a simple electrostatic model of our devices that gives f-values between 1 and 2, depending on the applied gate voltage (Supplementary Note 3). Fig. 4 Electrostatic gating of the NDC. a Differential conductance, G–VSD, curves at different gate voltages, Vg, at a temperature T = 2 K for a device with ten-layers InSe (curves are offset for clarity). b Color map of G versus VSD and Vg at T = 2 K. Black and white lines identify different resonances. depending on the applied gate voltage (Supplementary Note 3). We now examine additional characteristic features of these RTTs. As shown in Fig. 4a for the device with t = 10 layers, at Vg ~ +20 V, a narrow resonance, R, emerges at VSD ~ +0.5 V. This can be seen more clearly in the G(VSD, Vg) color map of Fig. 4b. Here, the white lines highlight the shift and splitting of the narrow R-resonance. The emergence of this additional narrow feature in the transport characteristics of our devices is similar to recent observations of resonant electron tunneling through localized states due to defects in hBN23. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x The multiplication factor indicates the number by which each curve has been multiplied to facilitate a comparison of different samples. Fig. 5 Magneto-tunnelling studies. a Differential conductance, G–VSD, curves at applied magnetic ﬁelds B = 0 and 14 T for devices with eight-layers (main panel) and ten-layers (inset) InSe. In both cases the gate voltage is Vg = 0 V, the temperature is T = 2 K and the magnetic ﬁeld is applied perpendicular to the direction of the current. b G(B, VSD) color map for the device with eight- layers InSe (T = 2 K). The inset is a schematic of the injection of electrons from the electrode into the subbands of InSe. The parabolas illustrate the in- plane energy dispersion of the subbands. incremental change of the applied voltage corresponding to a change in energy dE of a particular subband. A value of f ≈1 is in line with the observation of electroluminescence from our devices when the chemical potentials in the source and drain differs by an energy ΔE = eVSD that is approximately equal to the exciton recombination energy in the InSe layer, i.e., eVSD ~ EX (Supple- mentary Note 2). This is also consistent with a simple electrostatic model of our devices that gives f-values between 1 and 2, depending on the applied gate voltage (Supplementary Note 3). dd l h f f h incremental change of the applied voltage corresponding to a change in energy dE of a particular subband. A value of f ≈1 is in line with the observation of electroluminescence from our devices when the chemical potentials in the source and drain differs by an energy ΔE = eVSD that is approximately equal to the exciton recombination energy in the InSe layer, i.e., eVSD ~ EX (Supple- mentary Note 2). This is also consistent with a simple electrostatic model of our devices that gives f-values between 1 and 2, depending on the applied gate voltage (Supplementary Note 3). We now examine additional characteristic features of these RTTs. As shown in Fig. 4a for the device with t = 10 layers, at Vg ~ +20 V, a narrow resonance, R, emerges at VSD ~ +0.5 V. This can be seen more clearly in the G(VSD, Vg) color map of Fig. 4b. Here, the white lines highlight the shift and splitting of the narrow R-resonance. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x 3a, this value of ΔVSD is in agreement with the calculated energy spacing ΔE2–1, which increases from ΔE2–1 = 0.20–0.32 eV with decreasing t from 10 to 8 layers. Thus our data suggest a leverage factor f = e(dVSD/dE) ≈1, where dVSD is the To estimate the energy spacing between the 2D subbands, we examine the Vg-dependence of the main conductance peaks S1, S2, S3… in the device with ten-layers InSe. As shown in Fig. 4a, an increase of Vg decreases the VSD-position of these peaks. This behavior can be seen more clearly in the G(VSD, Vg) color map of Fig. 4b. Here, the dashed black lines illustrate that for a sufﬁciently large applied gate voltage, all the Sn-resonances COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys 3 COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x ARTICLE incremental change of the applied voltage corresponding to a Fig. 3 2D subbands in InSe. a, b Calculated dependence of the subbands a and exciton recombination b energies on the number of InSe layers. The arrows in part a show the energy spacing between the lowest two subbands for three speciﬁc layer thicknesses. The inset in part b shows the normalized photoluminescence spectra of devices with 8, 10, and 19 layers (L) of InSe at T = 300 K. c Current–voltage, I–VSD, characteristics of devices with different InSe layer thickness (t = 8, 10, and 19 layers) at the gate voltage Vg = 0 V and T = 2 K. Inset: differential conductance, G–VSD, plots for devices with thickness t = 8, 10, and 19 layers. The multiplication factor indicates the number by which each curve has been multiplied to facilitate a comparison f diff l Fig. 5 Magneto-tunnelling studies. a Differential conductance, G–VSD, curves at applied magnetic ﬁelds B = 0 and 14 T for devices with eight-layers (main panel) and ten-layers (inset) InSe. In both cases the gate voltage is Vg = 0 V, the temperature is T = 2 K and the magnetic ﬁeld is applied perpendicular to the direction of the current. b G(B, VSD) color map for the device with eight- layers InSe (T = 2 K). The inset is a schematic of the injection of electrons from the electrode into the subbands of InSe. The parabolas illustrate the in- plane energy dispersion of the subbands. Fig. 5 Magneto-tunnelling studies. Discussion d This weak dependence on B is consistent with the strong quan- tum conﬁnement of electrons along the c-axis: at B = 14 T, the magnetic length l ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ h=eB p is equal to 6.8 nm, comparable to or smaller than the thickness of the InSe layer (~6 and 8 nm in the devices with eight and ten-layers InSe, respectively). Furthermore, for this orientation of magnetic ﬁeld, when an electron tunnels from the source into a 2D subband, it acquires an additional in- plane wave vector Δk ¼ eBs=h24 due to the Lorentz force exerted on the electrons as they tunnel. Here, s is the effective tunneling length from the source to the centre of the InSe layer. Electrons that tunnel from states close to the K-points of the MLG into states near the Γ-point of the InSe layer require a much larger change of in-plane wave vector than that due to the Lorentz force. As discussed before, this can be provided by the scattering potential at the InSe–graphene interface. For s = t/2 = 3 nm and B = 14 T, we estimate k ¼ eB?s=h = 6.4 × 107 m−1, correspond- ing to a change in energy Δε = h2k2=2me == ~ 1 meV. This weak dispersion is due to the relatively large effective mass of the conduction electrons in the layer plane (me === 0.14me)21. Thus, even with magnetic ﬁelds of up to 14 T, the change in the in-plane wave vector k (and corresponding kinetic energy Δε) of the tunnelling electron is too small to detect a voltage shift ΔVSD = ε (Δk)/e or change in amplitude of the resonances in the I–VSD characteristics. p As \|VSD\| increases, electrons tunnel through the states of dis- tinct InSe subbands or states with increasing in-plane momentum within the same subband. When, with increasing \|VSD\|, an additional subband becomes resonantly aligned with the chemical potential in the source, an additional resonant feature appears in I–VSD, along with a further increase of the current. Although each 2D subband should produce a distinct peak in I–VSD, the over- lapping contributions from different subbands acts to increase the valley current between adjacent resonances, giving rise to a steadily increasing monotonic background current. Furthermore, as can be seen in the I–VSD plots of Fig. 1b, the current increases by two orders of magnitude going from S1 to S5. Discussion d Our devices based on a few layers of InSe sandwiched between two MLG electrodes differ in several important aspects from the well-established resonant tunnelling diodes (RTDs) based on heterostructures, in which the crystal lattices of the component layers are aligned and lattice matched. A typical example is the RTD in which two (AlGa)As tunnel barriers enclose a GaAs QW with the source and drain contacts composed of doped GaAs24. The GaAs-AlGaAs RTDs are ideally suited to conserve both the energy and momentum of the tunnelling carriers so that strong resonant peaks and extended regions of NDC with high peak-to- valley ratios are observed. In our devices, the hexagonal lattices of InSe (a = 4.002 Å) and graphene (a = 2.46 Å) are strongly mismatched (~60%) and are not intentionally aligned. Furthermore, in undoped graphene and MLG the occupied states are located around the K and K′ points of the hexagonal Brillouin zone, whereas the conduction band edge of InSe is at the Γ-point (Fig. 1d). For electron tunneling from MLG to InSe, an electron must overcome the large momentum difference between the K-(K′-) and Γ-points. This can be provided by the scattering potential at the InSe–graphene interface due to the large lattice mismatch of the InSe and gra- phene layers, and/or disorder in the layers. This is analogous to the situation encountered in previous studies of almost lattice- matched graphene–hBN–graphene tunnel transistors, in which the lattices of the graphene electrodes were misaligned. This misalignment gives rise to a difference between the in-plane momenta of the initial and ﬁnal states of electrons tunnelling between the two graphene layers. In this case, the in-plane scat- tering allows energy-conserving resonant tunnelling to occur even when the wave vectors of the tunnelling electrons in the collector and emitter are different8. As shown in the Supplementary Note 4, our measured region of NDC in the MLG–InSe–MLG heterostructures can be explained in terms of a similar model. pp y g ) As shown in Fig. 5a, a magnetic ﬁeld of up to B = 14 T applied perpendicular to the direction of the current (e.g., parallel to the c-axis) has almost no effect on the amplitude and voltage position of the Sn-resonances in the devices with eight and ten-layers InSe. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x The stronger dependence of R on Vg (dVSD=dVg ~ −0.05) compared to that of Sn (dVSD=dVg ~ −0.005) suggests that R arises from carrier tunnel- ing into the states of a localized defect close to the bottom gate electrode. Similar narrow resonances can be seen in the same device under negative applied gate voltages and VSD ~ +1.0 V (Fig. 4b). Also, close inspection of the G–VSD plots of different devices reveals that the main Sn-resonances can comprise additional weak features closely spaced in energy (Fig. 5a). This quasi-regular modulation of the current is weakly affected by temperature, although the increasing noise and broadening of the Fig. 4 Electrostatic gating of the NDC. a Differential conductance, G–VSD, curves at different gate voltages, Vg, at a temperature T = 2 K for a device with ten-layers InSe (curves are offset for clarity). b Color map of G versus VSD and Vg at T = 2 K. Black and white lines identify different resonances. 4 COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys 4 ARTICLE COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x a band alignment that takes the form shown schematically in Fig. 1d. By reducing the thickness of the InSe layer the energy gap of InSe can be increased due to quantum conﬁnement, increasing the effective height of the InSe tunnel barrier. This also changes the number of 2D subbands and their energy spacing. Thus, in these RTTs, the InSe layer thickness can control both the tun- nelling rate of the charge carriers and the resonant enhancement of the current through 2D subbands whose energy position is very sensitive to quantum conﬁnement. An additional feature of interest is that, despite the absence of a real energy barrier between the MLG and InSe layers, the subband states of the InSe layer are sufﬁciently well deﬁned to produce prominent resonant features in the current–voltage characteristics. The difference between the I–VSD characteristics of the eight-layer and ten-layer devices suggests that aside the transmission of electrons through a barrier of ﬁnite length and height, additional mechanisms con- tribute to the transport characteristics of these devices. For example, impurities and/or interface disorder are likely to affect both the magnitude of the current and the broadening of the resonances, with a larger current and/or broader resonances in layers that are more disordered due to scattering (Supplementary Note 4 and Supplementary Fig. 8). COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-020-0290-x resonances prevent its clear observation above T = 100 K (Supplementary Fig. 6). The positions of the main resonances Sn and that of the additional features are only weakly affected by a magnetic ﬁeld (B) applied perpendicular to the direction of the current (Fig. 5a–b). These dependences are further discussed below. COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys Methods M t i l O 13. Liu, X. et al. Modulation of quantum tunneling via a vertical two-dimensional black phosphorus and molybdenum disulﬁde p–n junction. ACS Nano 11, 9143–9150 (2017). Materials. Our InSe crystals were grown using the Bridgman method from a polycrystalline melt of In1.03Se0.97. The primitive unit cell contains three InSe layers, each of which has a thickness of 8.320 Å and consists of four covalently bonded monoatomic sheets in the sequence Se–In–In–Se (Fig. 1a); along the c-axis, the primitive unit cell has a lattice constant of c = 24.961 Å and, within each a–b plane, atoms form hexagons with lattice parameter a = 4.002 Å. In its bulk form, InSe contains native donors due to In-interstitial atoms. From Hall effect mea- surements on bulk InSe at T = 300 K, we estimate an electron density n ~ 1021 m−3, an electron mobility μ = 0.1 m2V−1s−1, and a Fermi energy EF ~ 0.21 eV below the conduction band minimum. 14. Mudd, G. W. et al. Tuning the bandgap of exfoliated InSe nanosheets by quantum conﬁnement. Adv. Mater. 25, 5714–5718 (2013). 15. Bandurin, D. A. et al. High electron mobility, quantum Hall effect and anomalous optical response in atomically thin InSe. Nat. Nanotechnol. 12, 223–227 (2016). 16. Brotons-Gisbert, M. et al. Nanotexturing to enhance photoluminescent response of atomically thin indium selenide with highly tunable band gap. Nano Lett. 16, 3221–3229 (2016). 17. Castellanos-Gomez, A. et al. Deterministic transfer of two-dimensional materials by all-dry viscoelastic stamping. 2D Mater. 1, 011002 (2014). Device fabrication. Our TFETs are assembled on a conventional SiO2/Si substrate (SiO2 layer thickness of 300 nm) using mechanically exfoliated MLG, hBN, and InSe ﬂakes. These are deposited sequentially onto preprepared metal contacts, Au/ Cr (30 nm/10 nm), using a dry transfer method under ambient conditions15. Flakes of hBN, MLG, and InSe are ﬁrst exfoliated onto polydimethylsiloxane (PDMS) from a low-tack, low-residue tape and identiﬁed using transmission and reﬂection mode optical microscopy. Then, using a micromanipulator stage, exfoliated ﬂakes on PDMS are aligned to the metal contacts/predeposited ﬂakes and brought into contact. The PDMS is then slowly retracted in order to sequentially deposit a bottom hBN ﬂake, a MLG electrode, few layer InSe, a top MLG electrode, and an encapsulating hBN ﬂake. The latter protects the whole heterostructure from con- tamination during long-term exposure to atmospheric conditions. The thickness of the MLG electrodes in different devices is between 10 and 18 layers. 18. Methods M t i l O Kress-Rogers, E., Nicholas, R., Portal, J. C. & Chevy, A. Cyclotron resonance studies on bulk and two-dimensional conduction electrons in InSe. Solid State Commun. 44, 379–383 (1982). 19. Ferrer-Roca, Ch., Segura, A., Andrés, M. V., Pellicer, J. & Muñoz, V. Investigation of nitrogen-related acceptor centers in indium selenide by means of photoluminescence: determination of the hole effective mass. Phys. Rev. B. 55, 6981–6987 (1997). 20. Magorrian, S. J., Ceferino, A., Zólyomi, V. & Fal’ko, V. I. Hybrid k · p tight- binding model for intersubband optics in atomically thin InSe ﬁlms. Phys. Rev. B 97, 165304 (2018). 21. Mudd, G. W. et al. The direct-to-indirect band gap crossover in two- dimensional van der Waals indium selenide crystals. Sci. Rep. 6, 39619–39629 (2016). Techniques. Magneto-transport measurements are conducted with the device mounted inside a superconducting magneto-cryostat at magnetic ﬁelds B of up to 14 T applied perpendicular to the direction of the current. The low-temperature transport characteristics reveal an electrical noise of ~1 pA. This is mainly limited by the measurement equipment. As the temperature increases, the source of noise is from both the device (e.g., thermal noise) and equipment. The experimental setup for the μPL spectroscopy studies at room temperature comprises lasers (λ = 633 nm and 405 nm), a x–y–z motorized stage and an optical confocal microscope system equipped with a 0.5-m long monochromator with 150 and 1200 g/mm gratings. The laser beam is focused to a diameter d ~ 1 μm using a 100× objective. The signal is detected by a Si-charge-coupled device camera. The optical studies are performed at low excitation power (P < 10−4 W) to avoid excessive heating. Images of the layers’ topography were acquired by atomic force microscopy (AFM, MFP- 3D) in non-contact mode under ambient conditions. 22. Shubina, T. V. et al. InSe as a case between 3D and 2D layered crystals for excitons. Nat. Commun. 10, 3479 (2019). 23. Greenaway, M. et al. Tunnel spectroscopy of localised electronic states in hexagonal boron nitride. Commun. Phys. 1, 94 (2018). 24. Hayden, R. K. et al. Probing the hole dispersion curves of a quantum well using resonant magneto-tunneling spectroscopy. Phys. Rev. Lett. 66, 1749–1752 (1991). 25. Mudd, G. W. et al. High broad‐band photoresponsivity of mechanically formed InSe–graphene van der Waals heterostructures. Adv. Mater. 27, 3760–3766 (2015). 26. Kuroda, N. & Nishina, Y. Methods M t i l O Resonant Raman scattering at higher M0 exciton edge in layer compound InSe. Solid State Commun. 28, 439–443 (1978). 27. Seabaugh, A. C. et al. In Ultrafast Electronics and Optoelectronics (eds J. Shah & U. Mishra) (OSA Proceedings Series, Optical Society of America, 1993). 28. Capasso, F., Sen, S., Cho, A. Y. & Sivco, D. Resonant tunneling devices with multiple negative differential resistance and demonstration of three-state memory cell for multiple-valued memory applications. IEEE Electron Device Lett. 8, 297–299 (1987). References 1. Geim, A. K. & Grigorieva, I. V. Van der Waals heterostructures. Nature 499, 419–425 (2013). 2. Novoselov, K. S., Mishchenko, A., Carvalho, A. & Castro Neto, A. H. 2D materials and van der Waals heterostructures. Science 353, 461–473 (2016). 3. Mounet, N. et al. Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds. Nat. Nanotechnol. 13, 246–252 (2018). 4. Fiori, G. et al. Electronics based on two-dimensional materials. Nat. Nanotechnol. 9, 768–779 (2014). 5. Britnell, L. et al. Field-effect tunneling transistor based on vertical graphene heterostructures. Science 335, 947–950 (2012). 6. Feenstra, R. M., Jena, D. & Gu, G. Single-particle tunneling in doped graphene-insulator-graphene junctions. J. Appl. Phys. 111, 043711 (2012). 7. Zhao, P., Feenstra, R. M., Gu, G. & Jena, D. SymFET: a proposed symmetric graphene tunneling ﬁeld-effect transistor. IEEE Trans. Electron Devices 60, 951–957 (2013). 8. Britnell, L. et al. Resonant tunnelling and negative differential conductance in graphene transistors. Nat. Commun. 4, 1794 (2013). 9. Campbell, P. M., Tarasov, A., Joiner, C. A., Ready, W. J. & Vogel, E. M. Enhanced resonant tunneling in symmetric 2D semiconductor vertical heterostructure transistors. ACS Nano 9, 5000–5008 (2015). 10. Esaki, L. New phenomenon in narrow germanium p−n junctions. Phys. Rev. 109, 603 (1958). 11. Yan, R. et al. Esaki diodes in van der Waals heterojunctions with broken-gap energy band alignment. Nano Lett. 15, 5791–5798 (2015). 12. Roy, T. et al. Dual-gated MoS2/WSe2 van der Waals tunnel diodes and transistors. ACS Nano 9, 2071–2079 (2015). Received: 10 September 2019; Accepted: 7 January 2020; Received: 10 September 2019; Accepted: 7 January 2020; Received: 10 September 2019; Accepted: 7 January 2020; control of these factors are required in order to optimize the performance of the RTTs. p In conclusion, we have realized vertical tunnel transistors with an InSe layer between two MLG contacts. Within the wide family of vdW crystals, InSe provides a very promising conduction channel due to the strong dependence of its 2D energy subbands on quantum conﬁnement, offering a route to the modulation of electrical properties. Electron tunneling into consecutive 2D subbands of InSe leads to multiple regions of NDC that can be tuned by electrostatic gating. Our devices could provide the foundation for further technologies, such as low-power con- sumption transistors and broad-band sources and detectors compatible with existing photonic and CMOS technologies. RTTs could offer opportunities to perform more logic operations per transistor than conventional transistors, providing a foundation for multivalued logic and frequency multiplication27–29. Recent observations of intersubband transitions by near-ﬁeld optics in TMDs30 have demonstrated the potential of 2D intersubband transitions for optoelectronics. The InSe-based RTTs demon- strated in this work has the potential for a broad spectral range with intraband transitions tuneable from the near infrared in a bilayer InSe to the far-infrared/terahertz range in thicker InSe ﬁlms. In particular, by suitable design of the layer thicknesses, it may be possible to engineer population inversion between the subbands and tune the photon emission, as done in quantum cascade lasers based on traditional semiconductors31. ARTICLE ARTICLE Discussion d This increase can be understood by reference to the schematic diagram in Fig. 1d. With increasing voltage, there is an increasing number of ﬁlled and empty states in the source and drain electrodes, respectively. Thus, the current and the amplitude of the differential con- ductance increase with increasing VSD. Finally, we attribute the temperature dependence of the dif- ferential conductance peaks (Fig. 2) to the thermal broadening of the electron distribution around the Fermi energy in the MLG- source and to phonon-assisted tunneling. The energy of the optical phonon modes in InSe is in the range 14–30 meV26. When these lattice vibrations are thermally activated, they inelastically scatter the tunneling electrons. In turn, this broadens the resonances and shifts their position to lower voltages with increasing T (Fig. 2). Elastic scattering mechanisms also inﬂuence the tunnel current. In particular, ﬂuctuations of the electrical conductance characteristics can be seen in our devices (Fig. 5a). This can vary from sample to sample due to scattering sites at the interfaces, local electric ﬁelds arising from surface dipoles due to the local atomic structure and charge distribution at the surface of the layers (Supplementary Fig. 6). A better understanding and We note that InSe has a much smaller band gap energy than that of hBN, which was previously used to realize TFETs5,8,23. The band alignment of InSe and hBN with respect to the Dirac points of graphene (or the conduction band of MLG) is also quite different; whereas the Dirac point lies almost in the middle of the band gap of hBN, it is positioned closer to the conduction band edge of InSe25. A MLG–InSe–MLG heterostructure therefore has 5 Correspondence and requests for materials should be addressed to A.P. 31. Faist, J. et al. Quantum cascade laser. Science 264, 553–556 (1994). 31. Faist, J. et al. Quantum cascade laser. Science 264, 553–556 (1994). Reprints and permission information is available at http://www.nature.com/reprints This work was supported by the Engineering and Physical Sciences Research Council [grant numbers EP/M012700/1 and EP/N033906/1]; the European Union’s Horizon 2020 research and innovation programme Graphene Flagship Core 2 under grant agreement number 785219; the Defence Science and Technology Laboratory (Dstl); the University of Nottingham; The National Academy of Sciences of Ukraine; The Lever- hulme Trust [RF-2017–224]; and The Royal Society [IE160395]. E.E.V. acknowledges the support from the Russian Science Foundation (17–12–01393). A.P. acknowledges the Chinese Academy of Sciences (CAS) for the Award of a “President’s International Fel- lowship for Visiting Scientists”. 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/. Author contributions J.K. fabricated the devices; Z.R.K., E.E.V. and O.M. conducted the transport measure- ments; Z.R.K., J.K. and D.M. conducted the optical studies; J.K. conducted the AFM studies; Z.R.K. and Z.D.K. grew the bulk InSe crystals; Z.R.K., E.E.V., O.M., L.E., P.H.B. and A.P. analysed and interpreted the experimental data; M.T.G. developed a model to describe the transport properties of the devices. A.P. wrote the paper; all authors took part in the discussion and analysis of the data. Data availability All relevant data that support the ﬁndings of this study are available in Nottingham Research Data Management Repository (https://doi.org/10.17639/nott.7030). COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys 6 ARTICLE Additional information 29. Wei, S. J. & Lin, H. C. Multivalued SRAM CEll Using Resonant Tunneling Diodes. IEEE J. Solid-State Circuits 27, 212–216 (1992). Supplementary information is available for this paper at https://doi.org/10.1038/s42005- 020-0290-x. 30. Schmidt, P. et al. Nano-imaging of intersubband transitions in van der Waals quantum wells. Nat. Nanotechnol. 13, 1035–1041 (2018). 30. Schmidt, P. et al. Nano-imaging of intersubband transitions in van der Waals quantum wells. Nat. Nanotechnol. 13, 1035–1041 (2018). Competing interests © The Author(s) 2020 p g The authors declare no competing interests. 7 COMMUNICATIONS PHYSICS \| (2020) 3:16 \| https://doi.org/10.1038/s42005-020-0290-x \| www.nature.com/commsphys
https://openalex.org/W2765326749	https://repository.nwu.ac.za/bitstream/10394/27644/1/2017The_impact_of_maintenance.pdf	English	null	The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile	Banks and bank systems	2,017	cc-by	9,356	“The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile” AUTHORS Ketsia Lorraine Motlhabane ARTICLE INFO Ketsia Lorraine Motlhabane (2017). The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile. Banks and Bank Systems, 12(3), 283-297. doi:10.21511/bbs.12(3-1).2017.12 DOI http://dx.doi.org/10.21511/bbs.12(3-1).2017.12 RELEASED ON Tuesday, 31 October 2017 RECEIVED ON Friday, 24 March 2017 ACCEPTED ON Friday, 28 July 2017 LICENSE This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License JOURNAL "Banks and Bank Systems" ISSN PRINT 1816-7403 ISSN ONLINE 1991-7074 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 38 NUMBER OF FIGURES 0 NUMBER OF TABLES 2 © The author(s) 2018. This publication is an open access article. “The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile” AUTHORS Ketsia Lorraine Motlhabane ARTICLE INFO Ketsia Lorraine Motlhabane (2017). The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile. Banks and Bank Systems, 12(3), 283-297. doi:10.21511/bbs.12(3-1).2017.12 DOI http://dx.doi.org/10.21511/bbs.12(3-1).2017.12 RELEASED ON Tuesday, 31 October 2017 RECEIVED ON Friday, 24 March 2017 ACCEPTED ON Friday, 28 July 2017 LICENSE This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License JOURNAL "Banks and Bank Systems" ISSN PRINT 1816-7403 ISSN ONLINE 1991-7074 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 38 NUMBER OF FIGURES 0 NUMBER OF TABLES 2 © The author(s) 2018. This publication is an open access article. “The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile” Ketsia Lorraine Motlhabane, Ph.D. Candidate; MBA; Post- Graduate Diplomas in Taxation; Entrepreneurship, Lecturer in Personal/Business Finance, North-West University, School of Management Sciences, South Africa. JEL Classification G21, L84 JEL Classification G21, L84 This is an Open Access article, distributed under the terms of the Creative Commons Attribution-Non- Commercial 4.0 International license, which permits re-use, distribution, and reproduction, provided the materials aren’t used for commercial purposes and the original work is properly cited. BUSINESS PERSPECTIVES LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine Abstract credit profile, maintenance fees, cardless services, university students credit profile, maintenance fees, cardless services, university students Received on: 24th of March, 2017 Accepted on: 28th of July, 2017 The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile BUSINESS PERSPECTIVES © Ketsia Lorraine Motlhabane, 2017 This is an Open Access article, distributed under the terms of the Creative Commons Attribution-Non- Commercial 4.0 International license, which permits re-use, distribution, and reproduction, provided the materials aren’t used for commercial purposes and the original work is properly cited. LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine “The impact of maintenance fees on students’ willingness to maintain bank accounts and establish credit profile” © The author(s) 2018. This publication is an open access article. businessperspectives.org Banks and Bank Systems, Volume 12, Issue 3, 2017 Ketsia Lorraine Motlhabane (South Africa) Abstract The purpose of this article is two-fold: to investigate how recurring maintenance fees levied on students’ accounts impact on university students’ willingness to maintain bank accounts and therefore begin creating the necessary credit profiles. Credit pro- files provide financial history that is useful for banks and other lenders to evaluate clients’ credit worthiness. Many students in South African universities are ignorant of this requirement and usually make choices detrimental to their accessing current and future financial products. The banks’ service quality and students’ expectations need to be harmonized at some point. The study was exploratory in nature, using expressive statements on banking costs to expose common causes of financial burden, the best and worst case scenarios of utilizing banking products including benefit accrual from their use. Pre-intervention data was collected using questionnaires N=60 conveniently sampled financial man- agement students. The post-intervention data was collected from the same students N=55 using similar questionnaires where five students did not show up. The study also assessed financial management tutorial influence on students’ decision making after being exposed to banking market demands and their costs. SPSS was used to analyze data collected. Cheaper once-off cardless services were found to be popular with students receiving money, citing its reasonableness and depositor charger rather than recipient shoul- dering transaction costs. Students confirmed their satisfaction with saving costly re- curring bank account maintenance fees, earning 0% interest on credit balances. Bank account holders increased in post-intervention compared to pre-tutorial with better understanding of client’s profile value for accessing credit. Other students remained reluctant to maintain bank accounts despite future economic benefits. Bank account maintenance fees discourage students from maintaining bank accounts. Maintenance fees may aid banks user cost recovery but losing clients to competing cheap cardless products may be more costly. © Ketsia Lorraine Motlhabane, 2017 The study contributes to relationship management in banking sector. Research debate focused on bank selection criteria based on established key quality factors and service. Little scholarly investigation exists on reasons for annulling bank accounts, replaced with once-off cardless services that is gaining popularity with university students over maintaining bank accounts. Highlighting what is critical to students, the paper may influence banks policies and developers to design innovative products suitable for university students market. Affordability and incentives are key attraction points for clients, mainly university students operating with limited resources. Loyalty prospects can surpass immediate recovery derived from maintenance fees. INTRODUCTION It is increasingly becoming necessary for university students to un- derstand and participate in financial markets, which may be initiated with maintaining bank accounts in order to access certain banking 283 Banks and Bank Systems, Volume 12, Issue 3, 2017 services, receive allowances and begin the culture of saving a portion of their income. In addition, stu- dents need to kick-start their relationships with banking institutions while still studying in order to understand their practice and continue its use, which will be required once they enter the job market. There is, however, limited evidence that addresses the reasons for students’ reluctance to commit to maintaining a bank account with one of the banking institutions. services, receive allowances and begin the culture of saving a portion of their income. In addition, stu- dents need to kick-start their relationships with banking institutions while still studying in order to understand their practice and continue its use, which will be required once they enter the job market. There is, however, limited evidence that addresses the reasons for students’ reluctance to commit to maintaining a bank account with one of the banking institutions. Banking institutions would ordinarily want to recover the costs of maintaining a bank account from the user, even though this may have unintended consequences for future client relationships adversely affecting their future prospects. It has become necessary for banking institutions to understand stu- dents’ behavior and attitudes in order to inform the development of attractive financial products and for retention strategies. Cardless services are financial products allowing once-off deposits and receipts of banking services, equivalent to using cash, mainly used by the poor as channels to deliver and exchange money from one person to the other (Lehman & Ledgerwood, 2013). These services are accessible at Computicket, E-wallet and Blue-wallet and others, enabling senders, by paying a minimum fee, to deposit money for the benefit of anyone with a registered mobile number in South Africa, whether or not the recipient possesses a bank account. A reference number would be send to the recipient’s mobile number, which they must punch in to withdraw the sent money. Dauda and Lee (2015), Mas and Rotman (2008) explain these electronic wallets as transactions carried out off-line, eliminating the direct involvement of finan- cial intermediaries together with their burden of high fixed costs of debit and credit cards. INTRODUCTION Recipients can avoid Automatic Teller Machine (ATM) and all card management costs (Bhosale & Sawant, 2012). Even though South Africa’s banks offer cardless services, they remain detrimental to both students (cre- ating no profile to access financial products) and the banks’ desire to attract and retain clients (contra- dicting clients’ attraction and retention strategy), as the once-off services require no recipient commit- ment once the money is collected. Banks’ participation in providing cardless services is self-defeating. While banks cannot guarantee the clients’ reuse of their service, the absence of a bank account offers no financial database, meaning that recipients are far away from building a profile that can attest to their financial behavior. It also means the students who prefer to use the cardless service would be exposed to unfavorable credit evaluation if needed. It is important for banking institutions to understand and cater for the diverse under-serviced university students market for their strategic planning to be realistic. The reality is that the service comes at a cost, as clients’ attraction costs are higher than those of retaining and selling services to existing clients (Driggs, Ramsey, & Nunes, 2006; Ogechukwu, 2012). Attracting university students would be even more expensive, as banks would depend on the prospects of these students becoming their future clients, requiring patience and futuristic strategies of sustaining busi- ness interest. Driggs, Ramsey, and Nunes (2006) reiterate that attracting customers and retaining their loyalty must be treated like team sport, with vital roles such as product development, marketing, pricing, sales and service pulling in the same direction. The question should not be whether or not to do it but how (Seelos & Mair, 2007). The objective and significance of the study The study’s objective is to explore whether or not the recurring maintenance fees charged to students’ bank accounts encourage or discourage their willingness to maintain bank accounts to begin building their credit profile. It intended to understand the reasons for university students’ option for cardless services or cash transactions over maintaining a bank account. The general assumption is that cardless services or cash transactions are once-off and cheap, however, they may cost the students’ current and future inclusion in the financial market. 284 Banks and Bank Systems, Volume 12, Issue 3, 2017 It is important to understand how recurring maintenance fees levied on university students’ accounts impact on their willingness to maintain bank accounts, serving as stepping-stones towards building the necessary credit profiles. Credit profiles are sources of financial records referred for use by banking institutions to judge clients’ creditworthiness at the time when accessing financial products is need- ed. It is important to identify ways to convince students to initiate minimal participation in finan- cial markets while still studying. Other financial products would be gradually introduced to orientate and build shock absorbers for greater services access once students become financially independent. At some point, the banks’ service quality and students’ expectations need to be harmonized, which was addressed through a series of personal financial management tutorials. The study also assessed tutorial impact after exposing students to financial market demands, their costs including the best and worst case scenarios influenced by clients’ knowledge level when utilizing banks’ products. The tutorial addressed students’ banking services perceptions, the significance and abilities to building credit profiles, budgeting and saving techniques. The trend is for students to start engaging with financial products access when they are financially independent, but this might be overwhelming, as many leave universities with education debt burdens, believed to be for a worthy course (Porter, 2002; Long & Riley, 2007). The burdens impact negatively on a person’s budget and credit ratings carried into adulthood. Unfavorable credit rating further excludes students from accessing financial services or if allowed to access the service, it will be offered at an inflated price. SA’s tertiary students should not be left trailing behind their peers who already enjoy extended benefits such as those in Kenya and other countries who have benefited from reduced fees or free maintenance students accounts. The objective and significance of the study Students in Australia and United Kingdom enjoy benefits like maintenance fees ex- emption, earn additional interest incentive on their credit balances/savings, can access minimal inter- est free loans and credit cards services unique to tertiary students accounts (Choe, 2009). The banks in these countries absorb the costs of recurring maintenance fees and other incentives to attract and retain students with the hope of establishing loyalty of customers with potential to earn high income (Njiri, 2016). The graduate clients have potential to bring high returns and increased business for the banks. These kinds of benefits are minimal in SA, and students’ access to most financial products normally re- quires collateral provision from parents or some financially independent person. Generally students perceive bank account maintenance to be more punitive than beneficial. Greater concern was raised with the expectation to pay monthly fees irrespective of use and the absence of in- centives. Credit balances earned 0% interest, which discourages students from savings. The banks’ lack of incentives and costlier maintenance fees mean students are better off without the service, making cardless services more sensible, given students’ financial situation. Such choices, however, have long- term detrimental effects on the students’ future access and participation in the banking markets. The paper is a part of a larger research project addressing students’ financial knowledge, financial markets participation and the motivation to save. The study intends to bridge the gap between students expectations and banking institutions processes by aligning both parties’ strategic positioning to identify value in each other’s support. The study intends to bridge the gap between students expectations and banking institutions processes by aligning both parties’ strategic positioning to identify value in each other’s support. healthcare and support system (Mkalala, 2016). The very youth is facing the plight of unemploy- ment and lack of opportunities averaged at 63% in SA and young graduates struggling to find suitable work with many competing for what is considered minimum wage (Mkalala, 2016). Without regular income, students will automatically fall short of 1. LITERATURE REVIEW healthcare and support system (Mkalala, 2016). The very youth is facing the plight of unemploy- ment and lack of opportunities averaged at 63% in SA and young graduates struggling to find suitable work with many competing for what is considered minimum wage (Mkalala, 2016). Without regular income, students will automatically fall short of SA’s youth population below the age of 25 years is estimated at 52%, placing the country at a good fu- ture position compared to other developed coun- tries dominated by ageing population adverse- ly impacting future plans and burdening state 285 Banks and Bank Systems, Volume 12, Issue 3, 2017 those which accrue from non-participation (Ennew, 1996). The extent to which both parties participate in a relationship impacts on over- all satisfaction and customer retention (Ennew, 1996). More often than not, bank switching or en- try and exit are caused by disillusionment in that such decisions will still have an adverse impact on the objectives of banking. Consequently, such decisions also adversely affect client satisfaction, specifically with fee pricing issues and lack of in- centives (Nordman, 2004; Gan, Cohen, Clemes, & Chong, 2006). meeting the bank’s demand for recurring main- tenance fees. What will become SA’s future when more than half the population is excluded from ac- tive participation in banking institutions or, as it is, have little understanding about how they func- tion? These constitute a generation that must take up the baton from the current management as the country’s economic custodians. The vast majority of students rely on their adult family members or guardians to access banking services, because the financial markets are focusing more on adults and less oriented towards the needs of the youth, the majority of whom are university students. Еngagement with potential banking student clients indicated general knowledge of the benefits of possessing a bank account, but it was less priori- tized given their other pressing issues (Atkinson & Messy, 2013). Anyway, developing countries’ banking systems and capital markets are often skewed towards affluent people and catering more for large enterprises (Claessens, 2006; Amaeshi, Ezeoha, Adi, & Nwafor, 2007). The market can be given costly, above average services to recover the cost of ordinary products offered to the broader community, particularly students who are not earning a regular income. 1.2. Building credit profile Strong credit histories are critical in accessing af- fordable credit needed to acquire expensive assets, which are also important tools for lenders to as- sess the risk factors involved (Birkenmaier, Curley, & Kelley, 2011). Inability to create a healthy credit profile with historical transactions, consequential to cash transactions, predisposes credit users to exploitative credit and financial services. The lack of credit profile inevitably leads to debt burdens (Birkenmaier, Curley, & Kelley, 2011). University students fit the profile of those exposed to exploitation risk, as the majority of them are fi- nancially illiterate, together with the families they come from. Financially excluded and illiterate families struggle to expose their growing children to financial matters, when they are not informed themselves (Atkinson & Messy, 2013). Personal fi- nance education and financial services are the least included in undergraduate university programs, more so in developing countries than developed countries (Sabitova, 2014). The lack thereof is det- 1. LITERATURE REVIEW A Standard Bank manager (Dr Molapo) advocates that bankers should think like fleet managers to go above and beyond client expectations (Molapo, 2016). He directs leaders in the field to listen care- fully to the needs of the client and be willing to innovate constantly. It has become necessary for banking institutions to understand students’ be- haviors and attitudes in order to inform the de- velopment of attractive financial products and for retention strategies. There is, however, little evi- dence that addresses the reasons for students’ re- luctance to foster lasting relationships with bank- ing institutions. Research debate has focused on bank selection criteria based on established key quality factors in banking such as service, prox- imity, attractiveness, recommendations, market- ing and pricing (Chigamba & Fatoki, 2011; Kheng, Mahamad, & Ramayah, 2010; Coetzee, van Zyl, & Tait, 2012; Mokhlis, Mat, & Salleh, 2008). In order to judge service quality, the person should have been a bank client or willing to foster continuous relationships even when they hop from one bank to the other, attracted or detracted by incentives offered. The debate neglects to investigate the rea- sons for annulling bank accounts, where once off cardless services are gaining popularity with stu- dents over mutual and enduring banking relation- ships maintaining bank accounts. 1.3. Barriers to banking services access Large price increases might motivate consumers to use fewer products or shift to another brand, discounted prices are mostly the reason for con- sumers’ choices (Litman, 2004). Poor services, high bank fees and failure to appreciate student needs are arguably three major factors frustrat- ing customers (Thwaites & Vere, 1995). Students are famous for being extremely sensitive to pric- es; their displeasure can cause huge consumption pattern changes (Litman, 2004). Djankov (2008) thus concludes that poorer house- holds are unbanked, having the least relationship with banks as the cost of maintaining a bank ac- count outweighs its benefits. In essence, this ex- plains SA’s wide-spread cheaper cardless services choice as the most suitable, although it compro- mises saving avenues and all the benefits associ- ated with banking services. The banks’ invest- ment in students market will be worthwhile look- ing at average future profit prospects which are greater from graduates’ higher income as com- pared to that of non-graduates (Carnoy, Loyalka, Androushchak, & Proudnikova, 2012). Developed financial system was found to reduce poverty and income inequality instead of the usual capitalist enterprise’s focus on handing over their largest possible profit to capital owners (Claessens, 2006). Lowering or eliminating maintenance fees and paying incentive could be a strong motivation to save, because households in developing coun- tries often cite high fees as a reason for not saving in bank accounts (Meyer, Zimmerman, & Boshara, 2008). If not saving in bank accounts where else would they save? It is thus necessary for banks to nurture relationships commencing from the time students are still studying, otherwise they risk los- ing them permanently. 1.5. How large is the cost of cash transaction? The uneducated and poor stay unbanked, as keep- ing a bank account is expensive for their infrequent financial transactions, even though informal sector is expensive in the long run (Djankov, 2008). Being unbanked means clients are excluded from paying online, but their transaction still means the hassle of standing in long queues to deposit money into the suppliers’ bank account, then send fax or scan and forward payment proof. This informal sector is costly with procedure and time consuming to perform a transaction. Furthermore, many sup- pliers, lately, charge additional fees to be included with cash deposits in order to recoup the cash han- dling fees usually charged by banks. All these costs are eliminated with electronic transfers, which are possible with maintaining a bank account. 1.1. The benefits of maintaining a bank account Every bank, like any other business, is dependent on its ability to attract and retain clients for its survival in the market. On the other hand, cli- ents must perceive reciprocal participation that is worthwhile and yielding benefits greater than 286 Banks and Bank Systems, Volume 12, Issue 3, 2017 These microloans are easily accessible and popu- lar with the poor, majority of who are unaware of the exorbitant finance costs attached to such risky transactions until they commit themselves. It may explain Barnhill, Papanagiotou, and Schumacher (2002) findings of the highest default rates to be among financial markets, charging borrowers high interest rates, a common problem also in South African society. These are the challenges facing poor communities in developing coun- tries who lack financial education, together with opportunities for viable and sustainable financial products. Pont and L. McQuilken (2005) attribute these social welfare issues to improper financial decisions and depravations, indicative of preju- dice that perpetuates the poor’s conditions and the benefits that the privileged take for granted. rimental to the individual’s social and economic well-being, including the personal relationships. It would thus be imperative for students to learn and develop healthy credit profile in order for them to access affordable credit, the benefits of which could filter through to their families and friends. These microloans are easily accessible and popu- lar with the poor, majority of who are unaware of the exorbitant finance costs attached to such risky transactions until they commit themselves. It may explain Barnhill, Papanagiotou, and Schumacher (2002) findings of the highest default rates to be among financial markets, charging borrowers high interest rates, a common problem also in South African society. These are the challenges facing poor communities in developing coun- tries who lack financial education, together with opportunities for viable and sustainable financial products. Pont and L. McQuilken (2005) attribute these social welfare issues to improper financial decisions and depravations, indicative of preju- dice that perpetuates the poor’s conditions and the benefits that the privileged take for granted. 1.4. The dilemma of the poor and unemployed A high percentage of low-income households in developing countries are without bank accounts with an even higher number of those below aver- age incomes such as those living in Mexico City and United States having no relationships with banks together with access to saving avenues (Djankov, 2008). Millions of potential clients in South Africa are said to be currently struggling to access the mainstream financial services, yet a high percentage of them have outstanding mi- croloan balances (Lafourcade, Isern, Mwangi, & Brown, 2005). 287 Banks and Bank Systems, Volume 12, Issue 3, 2017 Table 1. Participants gender Gender Number Percentage, % Female 28 46.67% Male 32 53.33% Total 60 100% Table 2. Age frequency of participants Age ranges Number Percentage, % 18-20 years 46 76.66% 21-23 years 11 18.33% 24-26 years 2 3.33% Above 26 years 1 1.67% Total 60 100% The researcher administered questionnaires col- lecting primary data pre-intervention from a population size of N=60 at the beginning of the semester. She then conducted a two day non-ex- aminable Personal Finance tutorial with these stu- dents. The tutorial covered income sources, bud- geting, spending and savings, debt and the credit profile role when accessing and negotiating finan- cial deals. It included expressive material such as statements displaying debt costs and penalties (omitting owner’s identity) and how to avoid them. These exposed the common causes of financial 2. THEORY OF RELATIONSHIP QUALITY AND PROFITABILITY burden, which can taint borrower’s image. After the tutorial, the students were mandated to experiment with banking market function and its demands. At the end of the semester, post-inter- vention data were collected from the same group N=55, because five students of the pre-interven- tion group were absent when post-intervention data were collected. Students responded to ques- tionnaires similar to those used in pre-interven- tion. SPSS was utilized to analyze the data. According to Nordman (2004), the theory provides the foundation for considering and understanding the world. Service quality and customer satisfac- tion (product offer and meeting customer needs) connect service management with its notion of perceived service quality to relationship market- ing, achieved with satisfied employees and cus- tomers (Yee, Yeung, & Cheng, 2008). Redefining quality in client-bank relationship is necessary though, as technology enables banking transac- tions in remote areas, making face to face least likely. With outdated strategies banks may lose student clients who have lost touch with historical classification of quality features. Unless services are redefined and clustered according to different market segments, students will be pushed further away from being financially included. Students’ fi- nancial literacy has been already classified as be- ing far below those of older generations, yet, many exhibit excessive credit utilization behavior that may push them further away from future inclu- sions (Atkinson & Messy, 2012; Devlin, 2005). 4. RESULTS The demographic information was intended for research population classification and their age ranges, because students’ bank accounts are cat- egorized according to age, separating high school pupils from tertiary students. The demographic information was, however, not used for distinct analysis in the study (See Appendix A). 3. RESEARCH METHOD A mini survey was conducted in a class of universi- ty students composed of those registered for busi- ness finance and some others registered for per- sonal finance for their degree qualifications. The group was conveniently sampled supposing these students would be conversant with the banking market practice. 5. DISCUSSION More than half of the student sample prefer to use cash transactions and cardless services to receive allowance money. The situation, however, improved 288 Banks and Bank Systems, Volume 12, Issue 3, 2017 2016). Banking services have been created with the adult in mind and students must just fit in and choose from what is offered. in post-intervention. In post-intervention, more students understood the value of building a credit profile, but some remained negative to maintaining a bank account, as they felt it was expensive. Little is known about building a credit profile to enable access to bank products. Students with the slightest idea of its significance assign the responsibility to the older generation, hoping to establish their own at the time of their financial independence. Standard Bank’s cheapest students’ account, charging no maintenance fees within prescribed limitations, is hardly known to students. The product value is eroded, as students tend to gener- alize their perceptions about the banks. Marketing variety of financial products and costs is not only needed to complement product developments (Dauda & Lee, 2015), but also requires going be- yond selecting the relevant medium for the target market. Secured and easily accessible technologies to students’ remote campus are the latest trend in- stead of ATMs and fancy branches suitable for the older generation and less appealing to students. The free Wi-Fi access that is common with univer- sity campuses could assist in convenient electronic banking rather than focusing on branch locations, which is potentially inaccessible and probably not attractive to students. The impact of the absence of a bank account and the credit profile would be felt more in post schooling period when the need to acquire expensive items such as properties and cars exist. Simple services such as renting a car from the rental companies would be next to impossible with cash transactions. It would mean students must postpone such acqui- sitions and needs until such time that a convinc- ing profile has been created. Alternatively, students will have to settle for unfavorable financial market terms, as they will be regarded as high risk clients or use the services of a third party meeting the re- quirements to acquire the service on their behalf. Perhaps tertiary students are judged through the lens of adults who have completely different abili- ties and needs. 5. DISCUSSION Even though students have limited or no resources, they still require that the banks cater for their unique needs to be worth having and that the relationship can be kept for longer periods. Drawing a budget was almost non-existent in pre- intervention; however, it improved in post-inter- vention. Another concern is from those who actu- ally draw a budget, but were failing to stick to what they have planned. Students seem to be less moti- vated to save with overall scores far below average than adult. The limited avenues for saving small sums of money may be the causes of discourag- ing the students from such practice. More must to be done to improve students’ willingness to main- tain bank accounts and develop the culture of sav- ing irrespective of the allowance amount received. Hopefully, the banking institutions would make it worth while for the students’ by rewarding their saving efforts. The least number of students are satisfied with the service quality they receive from banks. Students are neither satisfied with features, benefits and the period they need to wait before they can be served at the bank branches. Their peers’ preferential treatment paying a fraction or no maintenance fees in other countries does not go unnoticed. This means SA students are not only lagging behind, but also they are excluded from accessing services such as loans without the assistance of an adult or someone who is financially independent. They feel the maintenance fees on their accounts are unfair, as they are regularly charged whether or not the account has been utilized. In the absence of alter- natives to student accounts, cardless services will continue to grow and so will be the exclusion of university students from banking services. CONCLUSION Much as SA’s financial and banking markets place more emphasis on credit profile and security to ac- cess financial products, few students can provide it. Their level of ignorance regarding credit profile importance in the financial markets is usually exposed when access to financial products is denied, be- cause the students do not have a solid profile. The students may be cushioned by parents and guardians in accessing financial services and products on their behalf. Without the minimum required knowledge, the students would neglect managing their financial obligations or overcommit themselves, thus put- ting the very parents’ and guardians’ interest in jeopardy. Cash transactions are also becoming more and more expensive. Other than being an inconvenience to handle large sums of money, banking services would still be required to acquire expensive resources. The absence of a credit profile would be felt more in post schooling period when the need to acquire expensive items such as properties and cars exists. It would mean students must postpone such acquisi- tions up until such time that a convincing profile has been created. Alternatively, students will have to settle for unfavorable credit terms, as they will be regarded as high risk clients. Few students draw a budget which improved in post-intervention in the current study. Less of those who were budgeting stuck to the drawn budget out of those who said they do actually draw a budget. Other factors may be contributing to the students’ inability to stick to the budget, a subject that may be elabo- rated further in another study. The worrying factor is the low levels of saving, meaning there is a lack of motivation to save among the students community. More must be done to improve the willingness to save irrespective of the allowance amount. The banking institutions would hopefully avail incentives that will be worthwhile for the students’ effort to save. 6.1. Further studies account, however, the service can be self-defeat- ing to both parties and threatens the objective of banking institutions’ ability to attract and retain clients, including university students. The current focus of quality service and development may not necessarily be water under the bridge, as long as it can be presented in the manner that is appeal- ing to students distinct from adult products and services. The current banking institutions’ format of one size fits all quality dynamics, and upgrades would detract more students than attract them. Further studies could be undertaken with students in urban universities to assess how account main- tenance fees impact on the university students’ willingness to maintain bank accounts. Urban ar- eas and branches in big cities usually have above average services benefits resulting from the large market size, collective bargaining power and in- creased competition. Such collective power is lack- ing in semi-rural areas such as the one under study. 6. MANAGEMENT ІMPLICATIONS Majority of SA’s tertiary students do not under- stand financial management and neither are they financially savvy. It may be the reason for their sometimes unrealistic expectations and demands. Conceivably they have been judged through the lens of adults “failing to serve them fast, make the service fun and more favorable” (Motlhabane, Offering cardless services is a necessary service, seeing that less and less students maintain a bank 289 Banks and Bank Systems, Volume 12, Issue 3, 2017 RECOMMENDATION University students refers to the population sample in the study. RECOMMENDATION Service costs and desirable product variety is critical for students’ attraction and retention to replace obsolete products and their costs. To students, every cent counts thus the benefit of cardless services can be incorporated into formalized bank account holding in order to retain their continued usage. Understanding students’ needs will help banks in meeting their expectations for the benefit of both par- ties (Chigamba & Fatoki, 2011). The products design must enable students to earn points for meeting obligations timeously, and then qualify for additional products introduced gradually. Credit balances and continued savings need to be incentivized with interest earnings. Maintaining savings over time must also earn additional points to be utilized for favorable bank deals. The example may include higher interest earnings on savings. 290 Banks and Bank Systems, Volume 12, Issue 3, 2017 Credit utilization such as access to loans and credit-card facilities will have to be introduced gradually, but charged reduced or no interest in order to build students’ rapport for greater credit use when they attain financial independence. Credit utilization such as access to loans and credit-card facilities will have to be introduced gradually, but charged reduced or no interest in order to build students’ rapport for greater credit use when they attain financial independence. If there is any hope of harnessing the student market, banking institutions need to provide attractive, dedicated branch spaces exclusive for tertiary students use, serviced by informed peers who share simi- lar energies and passion. The services need to be delivered faster to cater for students’ time constraints, make branch visits fun and favorable for students to find value in fostering lasting relationships with the banks while still studying. The exercise may also serve multiple purposes. Students may find maintain- ing bank accounts a worthwhile exercise and continue the relationship when they are financially inde- pendent. The brand loyalty and bank preference may be passed on to other generations. The approach can be the banks’ powerful marketing tool, as well as a source of reliable updates. The like- lihood of keeping students in the service can foster lasting relations and loyalty with a particular bank. 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Youth and the 292 Banks and Bank Systems, Volume 12, Issue 3, 2017 customer satisfaction and loyalty across two divergent bank segments. Journal of Financial Services Marketing, 9(4), 344-359. 34. Porter, K. (2002). The Value of a College Degree. ERIC Digest. Retrieved from http://files.eric. ed.gov/fulltext/ED470038.pdf 35. Sabitova, N. M. (2014). Current issues of financial education in Russia. Procedia-Social and Behavioral Sciences, 152, 911-915. 36. Seelos, C., & Mair, J. (2007). Profitable business models and market creation in the context of deep poverty: A strategic view. The academy of management perspectives, 21(4), 49-63. 37. Thwaites, D., & Vere, L. (1995). Bank selection criteria—a student perspective. Journal of Marketing Management, 11(1-3), 133-149. 38. Yee, R. W., Yeung, A. C., & Cheng, T. E. (2008). PRE-TEST: ONE-SAMPLE STATISTICS error mean I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) 55 1.38 .490 .066 I use cash only to transact as I do not understand how credit works 55 1.45 .503 .068 I cannot keep-up a bank account because it is unaffordable to me 55 1.51 .505 .068 I understand bank account benefit for saving and building credit profile 55 1.58 .498 .067 I draw a budget to guide my spending (break down) monthly 55 1.56 .501 .067 I stick to (follow reliably) the budget according to plan regularly 55 1.56 .501 .067 I usually save a portion of my allowance monthly 55 1.55 .503 .068 I am satisfied with the service quality of banks to students 55 1.51 .505 .068 I am satisfied with the features, benefits and quick services of the banks 55 1.40 .494 .067 I trust that the banks have best financial products and safe electronic access services 55 1.62 .490 .066 I would prefer banks to have transparency and reliability in financial transactions and in other services 55 1.47 .504 .068 Bank charges for different services are fair 55 1.47 .504 .068 293 Banks and Bank Systems, Volume 12, Issue 3, 2017 ONE-SAMPLE TEST T df Sig. PRE-TEST: ONE-SAMPLE STATISTICS (2-tailed) Mean difference 95% confidence interval of the difference Lower Upper I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) 20.901 54 .000 1.382 1.25 1.51 I use cash only to transact as I do not understand how credit works 21.466 54 .000 1.455 1.32 1.59 I cannot keep-up a bank account because it is unaffordable to me 22.183 54 .000 1.509 1.37 1.65 I understand bank account benefit for saving and building credit profile 23.566 54 .000 1.582 1.45 1.72 I draw a budget to guide my spending (break down) monthly 23.169 54 .000 1.564 1.43 1.70 I stick to (follow reliably) the budget according to plan regularly 23.169 54 .000 1.564 1.43 1.70 I usually save a portion of my allowance monthly 22.808 54 .000 1.545 1.41 1.68 I am satisfied with the service quality of banks to students 22.183 54 .000 1.509 1.37 1.65 I am satisfied with the features, benefits and quick services of the banks 21.000 54 .000 1.400 1.27 1.53 I trust that the banks have best financial products and safe electronic access services 24.476 54 .000 1.618 1.49 1.75 I would prefer banks to have transparency and reliability in financial transactions and in other services 21.677 54 .000 1.473 1.34 1.61 Bank charges for different services are fair 21.677 54 .000 1.473 1.34 1.61 T df Sig. PRE-TEST: ONE-SAMPLE STATISTICS N Mean Std. deviation Std. error mean I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) 60 1.55 .502 .065 I use cash only to transact as I do not understand how credit works 60 1.48 .504 .065 I cannot keep-up a bank account because it is unaffordable to me 60 1.47 .503 .065 I understand bank account benefit for saving and building credit profile 60 1.48 .504 .065 I draw a budget to guide my spending (break down) monthly 60 1.35 .481 .062 I stick to (follow reliably) the budget according to plan regularly 60 1.43 .500 .065 I usually save a portion of my allowance monthly 60 1.43 .500 .065 I am satisfied with the service quality of banks to students 60 1.53 .503 .065 I am satisfied with the features, benefits and quick services of the banks 60 1.47 .503 .065 I trust that the banks have best financial products and safe electronic access services. 60 1.45 .502 .065 I would prefer banks to have transparency and reliability in financial transactions and in other services 60 1.52 .504 .065 Bank charges for different services are fair 60 1.48 .504 .065 POST-TEST: ONE-SAMPLE STATISTICS N Mean Std. deviation Std. PRE-TEST: ONE-SAMPLE STATISTICS (2-tailed) Mean difference 95% confidence interval of the difference Lower Upper I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) 20.901 54 .000 1.382 1.25 1.51 I use cash only to transact as I do not understand how credit works 21.466 54 .000 1.455 1.32 1.59 I cannot keep-up a bank account because it is unaffordable to me 22.183 54 .000 1.509 1.37 1.65 I understand bank account benefit for saving and building credit profile 23.566 54 .000 1.582 1.45 1.72 I draw a budget to guide my spending (break down) monthly 23.169 54 .000 1.564 1.43 1.70 I stick to (follow reliably) the budget according to plan regularly 23.169 54 .000 1.564 1.43 1.70 I usually save a portion of my allowance monthly 22.808 54 .000 1.545 1.41 1.68 I am satisfied with the service quality of banks to students 22.183 54 .000 1.509 1.37 1.65 I am satisfied with the features, benefits and quick services of the banks 21.000 54 .000 1.400 1.27 1.53 I trust that the banks have best financial products and safe electronic access services 24.476 54 .000 1.618 1.49 1.75 I would prefer banks to have transparency and reliability in financial transactions and in other services 21.677 54 .000 1.473 1.34 1.61 Bank charges for different services are fair 21.677 54 .000 1.473 1.34 1.61 CASE PROCESSING SUMMARY – POST INTERVENTION Cases Valid Missing Total N Percent N Percent N I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) I cannot keep-up a bank account because it is unaffordable to me 55 100.0% 0 0.0% 55 I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) * I cannot keep-up a bank account because it is unaffordable to me Cross-tabulation Count I cannot keep-up a bank account because it is unaffordable to me Total Yes No I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) Yes 8 15 23 No 18 14 32 Total 26 29 55 CASE PROCESSING SUMMARY – POST INTERVENTION Cases Valid Missing Total N Percent N Percent N I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) * I cannot keep-up a bank account because it is unaffordable to me 55 100.0% 0 0.0% 55 I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) * I cannot keep-up a bank account because it is unaffordable to me Cross-tabulation Count I cannot keep-up a bank account because it is unaffordable to me Total Yes No I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) Yes 8 15 23 No 18 14 32 Total 26 29 55 CASE PROCESSING SUMMARY – POST INTERVENTION 294 Banks and Bank Systems, Volume 12, Issue 3, 2017 CHI-SQUARE TESTS CHI-SQUARE TESTS Value df Asymptotic significance (2-sided) Exact sig. PRE-TEST: ONE-SAMPLE STATISTICS (2-sided) Exact sig. (1-sided) Pearson Chi-Square 2.474a 1 .116 Continuity Correctionb 1.688 1 .194 Likelihood Ratio 2.502 1 .114 Fisher's Exact Test .172 .097 Linear-by-Linear Association 2.429 1 .119 N of Valid Cases 55 Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 10.87; b. computed only for a 2x2 table. Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 10.87; b. computed only for a 2x2 table. CASE PROCESSING SUMMARY Cases Valid Missing Total N Percent N Percent N Percent Bank charges for different services are fair * Bank incentives are more and reasonable for students accounts 55 100.0% 0 0.0% 55 100.0% Bank charges for different services are fair * Bank incentives are more and reasonable for students accounts Cross tabulation Count Bank incentives are more and reasonable for students accounts Total Yes No Bank charges for different services are fair Yes 6 14 20 No 20 15 35 Total 26 29 55 CHI-SQUARE TESTS Value df Asymptotic significance (2-sided) Exact sig. (2-sided) Exact sig. (1-sided) Pearson Chi-Square 3.762a 1 .052 Continuity Correctionb 2.752 1 .097 Likelihood Ratio 3.844 1 .050 Fisher's Exact Test .091 .048 Linear-by-Linear Association 3.693 1 .055 N of Valid Cases 55 Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 9.45; b. computed only for a 2x2 table. CASE PROCESSING SUMMARY CASE PROCESSING SUMMARY Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 9.45; b. computed only for a 2x2 table. 295 Banks and Bank Systems, Volume 12, Issue 3, 2017 CASE PROCESSING SUMMARY Cases Valid Missing Total N Percent N Percent N Percent I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) * I cannot keep-up a bank account because it is unaffordable to me 60 100.0% 0 0.0% 60 100.0% I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) * I cannot keep up a bank account because it is unaffordable to me Cross-tabulation Count I cannot keep-up a bank account because it is unaffordable to me Total Yes No I receive my allowance through card-less transaction (E-wallet/ Shoprite /Blue wallet, etc.) Yes 13 15 28 No 18 14 32 Total 31 29 60 CHI-SQUARE TESTS Value df Asymptotic Significance (2-sided) Exact sig. (2-sided) Exact sig. PRE-TEST: ONE-SAMPLE STATISTICS (1-sided) Pearson Chi-Square 1. 744a 1 . 161 Continuity Correctionb 1. 886 1 . 419 Likelihood Ratio 2. 025 1 . 411 Fisher’s Exact Test .127 . 907 Linear-by-Linear Association 2. 294 1 .191 N of Valid Cases 60 Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 11.087; b. computed only for a 2x2 table. CHI-SQUARE TESTS Value df Asymptotic Significance (2-sided) Exact sig. (2-sided) Exact sig. (1-sided) Pearson Chi-Square 1. 744a 1 . 161 Continuity Correctionb 1. 886 1 . 419 Likelihood Ratio 2. 025 1 . 411 Fisher’s Exact Test .127 . 907 Linear-by-Linear Association 2. 294 1 .191 N of Valid Cases 60 Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 11.087; b. computed only for a 2x2 table. CASE PROCESSING SUMMARY Cases Valid Missing Total N Percent N Percent N Percent Bank charges for different services are fair * Bank incentives are more and reasonable for students accounts 60 100.0% 0 0.0% 60 100.0% Bank charges for different services are fair * Bank incentives are more and reasonable for students’ accounts Cross-tabulation Count Bank incentives are more and reasonable for students accounts Total Yes No Bank charges for different services are fair Yes 11 14 25 No 20 15 35 Total 31 29 60 CASE PROCESSING SUMMARY CASE PROCESSING SUMMARY 296 Banks and Bank Systems, Volume 12, Issue 3, 2017 CHI-SQUARE TESTS CHI-SQUARE TESTS Value df Asymptotic Significance (2-sided) Exact sig. (2-sided) Exact sig. (1-sided) Pearson Chi-Square 2.162a 1 .152 Continuity Correctionb 1. 527 1 .071 Likelihood Ratio 2. 484 1 .041 Fisher's Exact Test .019 .084 Linear-by-Linear Association 2. 936 1 .065 N of Valid Cases 60 Note: a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 9.45; b. computed only for a 2x2 table. cells (0.0%) have expected count less than 5. The minimum expected count is 9.45; b. computed only for a 2x2 table. 297
https://openalex.org/W4239800066	https://www.atmos-chem-phys.net/15/10149/2015/acp-15-10149-2015.pdf	English	null	Long-term real-time measurements of aerosol particle composition in Beijing, China: seasonal variations, meteorological effects, and source analysis	null	2,015	cc-by	16,696	Correspondence to: Y. L. Sun (sunyele@mail.iap.ac.cn) Received: 14 April 2015 – Published in Atmos. Chem. Phys. Discuss.: 22 May 2015 Revised: 31 August 2015 – Accepted: 1 September 2015 – Published: 11 September 2015 Abstract. High concentrations of ﬁne particles (PM2.5) are frequently observed during all seasons in Beijing, China, leading to severe air pollution and human health problems in this megacity. In this study, we conducted real-time mea- surements of non-refractory submicron aerosol (NR-PM1) species (sulfate, nitrate, ammonium, chloride, and organics) in Beijing using an Aerodyne Aerosol Chemical Speciation Monitor for 1 year, from July 2011 to June 2012. This is the ﬁrst long-term, highly time-resolved (∼15 min) measure- ment of ﬁne particle composition in China. The seasonal av- erage (±1σ) mass concentration of NR-PM1 ranged from 52 (±49) µg m−3 in the spring season to 62 (±49) µg m−3 in the summer season, with organics being the major frac- tion (40–51 %), followed by nitrate (17–25 %) and sulfate (12–17 %). Organics and chloride showed pronounced sea- sonal variations, with much higher concentrations in winter than in the other seasons, due to enhanced coal combustion emissions. Although the seasonal variations of secondary in- organic aerosol (SIA, i.e., sulfate + nitrate + ammonium) concentrations were not signiﬁcant, higher contributions of SIA were observed in summer (57–61 %) than in winter (43– 46 %), indicating that secondary aerosol production is a more important process than primary emissions in summer. Or- ganics presented pronounced diurnal cycles that were similar among all seasons, whereas the diurnal variations of nitrate were mainly due to the competition between photochemical production and gas–particle partitioning. Our data also indi- cate that high concentrations of NR-PM1 (> 60 µg m−3) are usually associated with high ambient relative humidity (RH) (> 50 %) and that severe particulate pollution is character- ized by different aerosol composition in different seasons. All NR-PM1 species showed evident concentration gradients as a function of wind direction, generally with higher val- ues associated with wind from the south, southeast or east. This was consistent with their higher potential as source ar- eas, as determined by potential source contribution function analysis. A common high potential source area, located to the southwest of Beijing along the Taihang Mountains, was ob- served during all seasons except winter, when smaller source areas were found. Correspondence to: Y. L. Sun (sunyele@mail.iap.ac.cn) These results demonstrate a high potential impact of regional transport from surrounding regions on the formation of severe haze pollution in Beijing. 1 Introduction Severe haze pollution episodes, characterized by high con- centrations of ﬁne particles (PM2.5), occur frequently dur- ing all seasons in China (Sun et al., 2013b; Guo et al., 2014; Zheng et al., 2015), not only reducing visibility sig- Long-term real-time measurements of aerosol particle composition in Beijing, China: seasonal variations, meteorological effects, and source analysis Y. L. Sun1, Z. F. Wang1, W. Du1,2, Q. Zhang3, Q. Q. Wang1, P. Q. Fu1, X. L. Pan4, J. Li1, J. Jayne5, and D. R. Worsnop5 1State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry, Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing 100029, China 2Department of Resources and Environment, Air Environmental Modeling and Pollution Controlling Key Laboratory of Sichuan Higher Education Institutes, Chengdu University of Information Technology, Chengdu 610225, China 3Department of Environmental Toxicology, University of California, 1 Shields Ave., Davis, CA 95616, USA 4Research Institute for Applied Mechanics, Kyushu University, Fukuoka, Japan 5Aerodyne Research, Inc., Billerica, MA 01821, USA Correspondence to: Y. L. Sun (sunyele@mail.iap.ac.cn) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10150 niﬁcantly, but also exerting harmful effects on public health (Cao et al., 2012). The mass concentrations of PM2.5 often far exceed the China National Ambient Air Quality Standard (NAAQS; 75 µg m−3 as a 24 h average), particularly in the economically developed regions of Beijing–Tianjin–Hebei and Yangtze River Delta (YRD). According to Beijing En- vironmental Statements, the annual average mass concentra- tion of PM2.5 was 89.5 and 85.9 µg m−3 in 2013 and 2014, respectively, 2.5 times the NAAQS (35 µg m−3 as an annual average), indicating that Beijing is still facing severe ﬁne par- ticle pollution. While extensive studies have been conducted in recent years to characterize severe haze pollution (e.g., Guo et al., 2014; Huang et al., 2014; Sun et al., 2014; Zheng et al., 2015), most were carried out in a particular season. In reality, the very different compositions, sources, and evolu- tion processes of severe haze pollution among the different seasons mean that a longer-term approach is needed to meet the challenge of mitigating ﬁne particle pollution in Beijing. month or even less were conducted for each season, due to the high cost and maintenance of the AMS. The recently de- veloped Aerosol Chemical Speciation Monitor (ACSM) is specially designed for long-term routine measurements of submicron aerosol composition (Ng et al., 2011). The ACSM has been proven reliable by several recent long-term ﬁeld measurements, e.g., in Paris (Petit et al., 2015), north-central Oklahoma (Parworth et al., 2015), and Santiago de Chile (Carbone et al., 2013). Although the ACSM has been de- ployed at various sites in China (Sun et al., 2012, 2013b; Zhang et al., 2015), long-term measurements have yet to be reported. In this study, the ﬁrst of its kind, we conducted long-term, real-time measurements of non-refractory submicron aerosol (NR-PM1) composition with an ACSM in Beijing, China, from July 2011 to June 2012. The seasonal variations of mass concentration and composition of submicron aerosol were characterized, and the diurnal cycles of aerosol species during the four seasons elucidated. The effects of meteoro- logical parameters, particularly relative humidity and tem- perature, on aerosol composition and formation mechanisms were investigated. Published by Copernicus Publications on behalf of the European Geosciences Union. Finally, the potential source areas leading to high concentrations of aerosol species during the four sea- sons were investigated via potential source contribution func- tion (PSCF) analysis. A number of long-term measurements and source analy- ses have been conducted in Beijing during the last decade. Zhao et al. (2009) reported pronounced seasonal variations of PM2.5, with higher concentrations in winter than sum- mer. Similarly, Yang et al. (2011) conducted a long-term study of carbonaceous aerosol from 2005 to 2008 in urban Beijing. Both organic carbon (OC) and elemental carbon (EC) showed pronounced seasonal variations, with the high- est concentrations occurring in winter and the lowest values in summer. A more detailed investigation of the chemical composition and sources of PM2.5 in urban Beijing can be found in Zhang et al. (2013a). Sources of ﬁne particles also vary greatly among the different seasons; for instance, coal combustion during periods requiring more domestic heating, biomass burning in harvest seasons, and dust storms in spring (Zheng et al., 2005; R. Zhang et al., 2013). Despite this, most previous long-term studies either focused on limited aerosol species, relied upon weekly ﬁlter samples, or used 1 month’s worth of data to represent an entire season (R. Zhang et al., 2013; Y. Zhang et al., 2013). Therefore, our understanding of the full spectrum of seasonal variations of aerosol species and sources remains quite poor. 2.2 Aerosol and gas measurements The submicron aerosol particle composition including or- ganics, sulfate, nitrate, ammonium, and chloride was mea- sured in situ by the ACSM at a time resolution of ∼15 min. The ACSM, built upon previous AMSs (Jayne et al., 2000; Drewnick et al., 2005; DeCarlo et al., 2006), is specially de- signed for long-term routine measurements of ﬁne particle composition (Ng et al., 2011). The ACSM has been success- fully deployed at various sites for chemical characterization of submicron aerosol (Ng et al., 2011; Budisulistiorini et al., 2013; Carbone et al., 2013; Sun et al., 2013b; Parworth et al., 2015). In this study, ambient aerosol particles were delivered to the sampling room through a stainless steel tube (outer diameter: 1.27 cm) with a ﬂow rate of ∼3 L min−1, out of which ∼84 cc min−1 was sampled into the ACSM. A PM2.5 URG cyclone (URG-2000-30ED) was installed in front of the sampling line to remove coarse particles (> 2.5 µm). To reduce the uncertainties of collection efﬁciency (CE), a sil- ica gel diffusion dryer was set up in the front of the ACSM to ensure that the aerosol particles sampled were dry (< 40 %). The ACSM was calibrated routinely with pure ammonium nitrate particles for the response factor following the proce- dures detailed in Ng et al. (2011). A more detailed descrip- tion of the ACSM calibration is given in Sun et al. (2012). It should be noted that we did not calibrate the ACSM with (NH4)2SO4 to determine the relative ionization efﬁciency Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10151 0.1 0.2 0.1 0.2 0 45 90 135 180 225 270 315 0 - 2 2 - 4 4 - 6 6 - 8 8 - 10 10+ 0 45 90 135 180 225 270 315 0 45 90 135 180 225 270 315 0 45 90 135 180 225 270 315 Summer Winter Fall Spring IAP (a) (b) Figure 1. (a) Map of the sampling site (IAP). (b) Wind rose plots, color-coded by wind speed (m s−1) for each season. The frequencies are set to the same scales for all seasons. 0.1 0.2 0.1 0.2 0 45 90 135 180 225 270 315 0 - 2 2 - 4 4 - 6 6 - 8 8 - 10 10+ 0 45 90 135 180 225 270 315 Summer Winter (b) IAP (a) 0 45 90 135 180 225 270 315 Fall Summer (b) 45 180 180 Spring Winter 0 45 90 135 180 225 270 315 Spring 0 45 45 135 180 180 Figure 1. (a) Map of the sampling site (IAP). (b) Wind rose plots, color-coded by wind speed (m s−1) for each season. The frequencies are set to the same scales for all seasons. (RIE) of sulfate since such an approach was only proposed recently. Using the method suggested by Budisulistiorini et al. (2014), the RIE of sulfate was estimated to be 1.1–1.6 dur- ing four seasons, leading to a highest uncertainty of 35 % in sulfate quantiﬁcation. Considering that aerosol particle acid- ity may vary largely between different seasons, the method of Budisulistiorini et al. (2014) may introduce additional un- certainties in sulfate quantiﬁcation. Therefore, we kept the default RIE of sulfate for the data analysis in this study. showed similar seasonal cycles, with high values in summer and low values in winter. The monthly variations of WS were relatively ﬂat, yet slightly higher values in spring were ob- served. In addition, a considerable amount of precipitation was observed from June to August, yet it was negligible dur- ing wintertime. 2.3 ACSM data analysis The ACSM data were analyzed for the mass concentrations of NR-PM1 species including organics, sulfate, nitrate, am- monium, and chloride using ACSM standard data analy- sis software. The RH in the sampling line, aerosol particle acidity and the fraction of ammonium nitrate (fAN) in NR- PM1 are three major factors affecting the uncertainties of CE (Huffman et al., 2005; Matthew et al., 2008; Middle- brook et al., 2012). Because aerosol particles were dry and overall neutralized for most of the time, except some pe- riods when the ratio of measured NH+ 4 to predicted NH+ 4 (i.e., 2 × SO2− 4 /96 × 18 + NO− 3 / 62 × 18 + Cl−/35.5 × 18) (Zhang et al., 2007) was less than 0.8, the composition- dependent CE recommended by Middlebrook et al. (2012), which is CE = max(0.45, 0.0833 + 0.9167 × fAN), was used in this study. The validity of the ACSM data using variable CE in summer and winter was reported previously in Sun et al. (2012, 2013b) by comparing the NR-PM1 with PM2.5 mass concentration measured by a TEOM system. The cor- relation between NR-PM1 and PM2.5 for the entire year is shown in Fig. S1 in the Supplement. The variation of mea- sured NR-PM1 tracked well with that of PM2.5 overall, and yet showed different slopes in different seasons. The aver- 2.1 Sampling site The ACSM was deployed on the roof of a two-story build- ing (∼8 m) at the Institute of Atmospheric Physics (IAP), Chinese Academy of Sciences (39◦58′28′′ N, 116◦22′16′′ E, Fig. 1a) from July 2011 to June 2012. The sampling site is lo- cated between the north third and fourth ring road in Beijing, which is a typical urban site with inﬂuences from local trafﬁc and cooking sources (Sun et al., 2012). The wind rose plots (Fig. 1b) show that southwesterly winds dominate all sea- sons except winter, when northwesterly and northerly winds prevail. The spring and fall seasons are also characterized by high frequencies of northwesterly and northerly winds. Also note that the prevailing winds with high wind speeds are more frequent during winter and spring than summer. The Aerodyne aerosol mass spectrometer (AMS) is unique in its ability to provide real-time, online measurements of size-resolved submicron aerosol composition (Jayne et al., 2000; Canagaratna et al., 2007). While the AMS has been widely used in China in recent years (J. Xu et al., 2014, and references therein), real-time, long-term measurements of aerosol particle composition are still rare. Y. Zhang et al. (2013) conducted a 4-month measurement campaign of submicron aerosol composition and size distributions using a quadrupole AMS in urban Beijing. Their results showed higher concentration of organics during wintertime and sec- ondary inorganic species in summer. Furthermore, positive matrix factorization (PMF) analysis of organic aerosol (OA) showed higher primary OA (POA) in winter and secondary OA (SOA) in summer. However, measurements over only 1 The meteorological parameters, including wind speed (WS), wind direction, relative humidity (RH), and tempera- ture (T ) were obtained from a 325 m meteorological tower at the same location. The parameters of pressure (P), so- lar radiation (SR), and precipitation were obtained from a ground meteorological station located nearby. The monthly variations of these meteorological parameters are presented in Fig. 2. Pronounced seasonal variations were observed for all meteorological parameters except WS. RH aver- aged at > ∼60 % in summer and presented its minimum value (< 30 %) in February. Temperature and solar radiation Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10152 10152 Y. L. Sun et al.: Long-term real-time me 80 60 40 20 0 RH (%) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun 30 20 10 0 -10 T (°C) 300 200 100 0 Precip. (mm) 40 30 20 10 0 O3 (ppb) 200 150 100 50 0 SR (W m -2) 100 80 60 40 20 0 NOy (ppb) 5 4 3 2 1 0 WS (m s -1) 103 102 101 100 99 P (kPa) (a) (c) (d) (b) Figure 2. Monthly variation of (a) gaseous O3 and NOy, (b) pre- cipitation (Precip.) and solar radiation (SR), (c) wind speed (WS) and pressure (P ), and (d) relative humidity (RH) and temperature (T ). The WS at the heights of 8 m (solid gray circles) and 240 m (solid black circles) are shown in (c). 80 60 40 20 0 RH (%) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun 30 20 10 0 -10 T (°C) 300 200 100 0 Precip. (mm) 40 30 20 10 0 O3 (ppb) 200 150 100 50 0 SR (W m -2) 100 80 60 40 20 0 NOy (ppb) 5 4 3 2 1 0 WS (m s -1) 103 102 101 100 99 P (kPa) (a) (c) (d) (b) Each trajectory contained a series of latitude–longitude co- ordinates every 1 h backward in time for 72 h. If a trajectory end point falls into a grid cell (i, j), the trajectory is assumed to collect material emitted in the cell (Polissar et al., 1999). The number of end points falling into a single grid cell is nij. Some of these trajectory end points are associated with the data with the concentration of aerosol species higher than a threshold value. The number of these points is mij. The potential source contribution function (PSCF) is then calcu- lated as the ratio of the number of points with concentration higher than a threshold value (mij) to the total number of points (nij) in the ijth grid cell. Higher PSCF values indicate higher potential source contributions to the receptor site. In this study, the domain for the PSCF was set in the range of (34–44◦N, 110–124◦E). Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China The 75th percentile for each aerosol species during the four seasons (Table S1) was used as the threshold value to calculate mij. To reduce the uncertain- ties of mij/nij for those grid cells with a limited number of points, a weighting function (wij) recommended by Polissar et al. (1999) was applied to the PSCF in each season. Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Figure 2. Monthly variation of (a) gaseous O3 and NOy, (b) pre- cipitation (Precip.) and solar radiation (SR), (c) wind speed (WS) and pressure (P ), and (d) relative humidity (RH) and temperature (T ). The WS at the heights of 8 m (solid gray circles) and 240 m (solid black circles) are shown in (c). wij =    1.00 80 < nij 0.70 20 < nij ≤80 0.42 10 < nij ≤20 0.05 nij ≤10    (1) wij =    1.00 80 < nij 0.70 20 < nij ≤80 0.42 10 < nij ≤20 0.05 nij ≤10    (1) (1) age ratio of NR-PM1 / PM2.5 for the entire year was 0.77 (r2 = 0.66). It should be noted that the PM2.5 was mea- sured by a heated TEOM (50 ◦C), which might have caused signiﬁcant losses of semi-volatile species, e.g., ammonium nitrate and semi-volatile organics. For example, Docherty et al. (2011) found an average loss of ∼44 % PM2.5 mass through use of the heated TEOM compared to that measured with a ﬁlter dynamics measurement system. Assuming that the average loss of PM2.5 mass by the heated TEOM is 30– 50 %, the NR-PM1 / PM2.5 ratio for the entire study would be ∼0.5–0.6, which is close to that reported in Y. Zhang et al. (2013). Figure S1 also shows large variations of NR- PM1 / PM2.5 ratios in the different seasons. The reasons for the variations include: (1) the ACSM cannot detect refrac- tory black carbon, mineral dust, and metals. 3.1 Mass concentration and chemical composition The average mass concentration of NR-PM1 was 62 µg m−3 in summer (Fig. 3), which is higher than the 50 µg m−3 for July–August 2011 reported in Sun et al. (2012) due to the biomass burning impacts in June 2012 (Fig. S2). The summer NR-PM1 level is close to that measured by a high-resolution aerosol mass spectrometer during the Beijing 2008 Olympic Games (Huang et al., 2010), but ∼20 % lower than that de- termined in summer 2006 (Sun et al., 2010). The average NR-PM1 mass concentrations were relatively similar during the other three seasons, varying from 52 to 59 µg m−3 and with slightly higher concentration during wintertime (Fig. 3). The NR-PM1 measured in urban Beijing is overall higher than those previously reported in the Yangtze River Delta (YRD) region (27–43 µg m−3) (Huang et al., 2012, 2013; Zhang et al., 2015) and Pearl River Delta (PRD) region (31– 48 µg m−3) (He et al., 2011; Huang et al., 2011; Gong et al., 2012), indicating more severe submicron aerosol pollu- tion in Beijing compared to other places in China. Indeed, the annual average NR-PM1 concentration (57 µg m−3) was much higher than the China NAAQS of PM2.5 (35 µg m−3 as an annual average). Assuming a similar PM2.5 level as that (89.5 µg m−3) in Beijing in 2013, NR-PM1 on average ac- counted for 64 % of PM2.5, which is overall consistent with Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China For example, low ratios of NR-PM1 / PM2.5 (< 0.3) were observed during dust storm periods, when mineral dust is the dominant com- ponent of ﬁne particles; (2) the contribution of semi-volatile species to PM2.5 varied greatly among the different seasons; and (3) the contribution of particles in the range of 1–2.5 µm to the total PM2.5 might also be different among different pollution episodes. age ratio of NR-PM1 / PM2.5 for the entire year was 0.77 (r2 = 0.66). It should be noted that the PM2.5 was mea- sured by a heated TEOM (50 ◦C), which might have caused signiﬁcant losses of semi-volatile species, e.g., ammonium nitrate and semi-volatile organics. For example, Docherty et al. (2011) found an average loss of ∼44 % PM2.5 mass through use of the heated TEOM compared to that measured with a ﬁlter dynamics measurement system. Assuming that the average loss of PM2.5 mass by the heated TEOM is 30– 50 %, the NR-PM1 / PM2.5 ratio for the entire study would be ∼0.5–0.6, which is close to that reported in Y. Zhang et al. (2013). Figure S1 also shows large variations of NR- PM1 / PM2.5 ratios in the different seasons. The reasons for the variations include: (1) the ACSM cannot detect refrac- tory black carbon, mineral dust, and metals. For example, low ratios of NR-PM1 / PM2.5 (< 0.3) were observed during dust storm periods, when mineral dust is the dominant com- ponent of ﬁne particles; (2) the contribution of semi-volatile species to PM2.5 varied greatly among the different seasons; and (3) the contribution of particles in the range of 1–2.5 µm to the total PM2.5 might also be different among different pollution episodes. www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 2.4 PSCF analysis he results reported in previous studies (Sun et al., 2012, 013b; Y. Zhang et al., 2013). As indicated in Fig. 4, the summer season showed the ighest frequency with NR-PM1 loading in the range of 0–60 µg m−3 (36 % of the time), while the winter sea- on presented the highest frequency of low mass loadings westerly winds (Fig. 1b). However, high NR-PM1 l (> 90 µg m−3) occurred 31 % of the time during th ter season, substantially more than during any of the seasons (25, 25, and 21 % during summer, fall, and respectively), indicating that heavy pollution occurre frequently during winter than the other seasons T Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 1 Table 1. Summary of mass concentrations of NR-PM1 species, gaseous pollutants and meteorological parameters during the four se and entire study period. 2.4 PSCF analysis The 72 h back trajectories arriving at the IAP study site at a height of 300 m were calculated every 3 h for the entire study period using the National Oceanic and Atmospheric Administration Hybrid Single-Particle Lagrangian Integrated Trajectory model, version 4.8; Draxler and Rolph, 2003). Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ . L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China able 1. Summary of mass concentrations of NR-PM1 species, gaseous pollutants and meteorological parameters during the four nd entire study period. Entire study Summer Fall Winter Spring mean SD mean SD mean SD mean SD mean SD Org (µg m−3) 25.7 22.1 24.5 20.7 26.8 24.7 29.6 24.8 21.7 16.0 SO2− 4 (µg m−3) 8.1 8.3 10.6 8.2 6.5 7.5 7.7 9.2 7.3 7.6 NO− 3 (µg m−3) 12.6 12.8 15.6 14.4 11.4 12.7 10.3 9.5 13.1 13.4 NH+ 4 (µg m−3) 8.5 7.9 10.2 8.2 6.9 7.3 8.1 7.4 8.8 8.1 Cl−(µg m−3) 1.8 2.5 0.8 1.5 1.7 2.7 3.0 3.0 1.5 1.9 NR-PM1 (µg m−3) 56.6 48.2 61.6 48.8 53.3 49.7 58.7 50.5 52.3 42.7 SO2 (ppb) 16.2 14.0 5.4 0.8 25.3 16.0 11.5 8.3 CO (ppm) 1.5 1.3 1.8 1.3 1.7 1.6 1.2 1.0 NO (ppb) 30.0 43.0 7.8 10.8 41.9 51.2 50.9 50.9 19.8 30.0 NOy (ppb) 64.0 55.5 35.6 17.9 77.8 63.1 89.1 66.6 54.0 43.3 O3 (ppb) 21.2 23.8 33.3 29.1 20.3 24.4 7.9 8.5 20.8 19.3 RH (%) 47.0 23.4 62.7 18.9 52.7 20.0 35.6 20.3 36.5 22.5 T (◦C) 13.3 11.6 26.3 3.6 14.1 7.0 −1.3 3.4 14.6 8.4 WS, 8 m (m s−1) 1.2 0.8 1.0 0.5 0.9 0.7 1.4 1.0 1.4 0.9 WS, 240 m (m s−1) 4.4 3.0 3.5 2.3 4.1 2.7 4.6 3.4 5.3 3.3 200 150 100 50 0 Mass Concentration (µg m -3) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Summer Fall Winter Spring Summer Heating Season 40% 17% 25% 17% 1% 62 μg m-3 53 μg m-3 50% 12% 21% 13% 3% 59 μg m-3 51% 13% 17% 14% 5% 52 μg m-3 41% 14% 25% 17% 3% Org SO4 2− NO3 − NH4 + Cl− igure 3. Time series of NR-PM1 species for the entire year. The pie charts show the average chemical composition of NR-PM1 du ur seasons (summer, fall, winter, and spring). Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10153 Table 1. Summary of mass concentrations of NR-PM1 species, gaseous pollutants and meteorological parameters during the four seasons and entire study period. concentrations of NR-PM1 species, gaseous pollutants and meteorological parameters during the four seaso 59 μg m-3 51% 13% 17% 14% 5% 52 μg m-3 41% 14% 25% 17% 3% 40% 17% 25% 17% 1% 62 μg m-3 53 μg m-3 50% 12% 21% 13% 3% 59 μg m-3 51% 13% 17% 14% 5% 52 μg m-3 41% 14% 25% 17% 3% 200 150 100 50 0 Mass Concentration (µg m -3) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Summer Fall Winter Spring Summer Heating Season Org SO4 2− NO3 − NH4 + Cl− Figure 3. Time series of NR-PM1 species for the entire year. The pie charts show the average chemical composition of NR-PM1 during the four seasons (summer, fall, winter, and spring). Figure 3. Time series of NR-PM1 species for the entire year. The pie charts show the average chemical composition of NR-PM1 during the four seasons (summer, fall, winter, and spring). the results reported in previous studies (Sun et al., 2012, 2013b; Y. Zhang et al., 2013). westerly winds (Fig. 1b). However, high NR-PM1 loading (> 90 µg m−3) occurred 31 % of the time during the win- ter season, substantially more than during any of the other seasons (25, 25, and 21 % during summer, fall, and spring, respectively), indicating that heavy pollution occurred more frequently during winter than the other seasons. The fall and spring seasons showed similar variations of frequencies, As indicated in Fig. 4, the summer season showed the highest frequency with NR-PM1 loading in the range of 30–60 µg m−3 (36 % of the time), while the winter sea- son presented the highest frequency of low mass loadings (< 20 µg m−3, 34 % of the time) due to the prevailing north- 2.4 PSCF analysis Entire study Summer Fall Winter Spring mean SD mean SD mean SD mean SD mean SD Org (µg m−3) 25.7 22.1 24.5 20.7 26.8 24.7 29.6 24.8 21.7 16.0 SO2− 4 (µg m−3) 8.1 8.3 10.6 8.2 6.5 7.5 7.7 9.2 7.3 7.6 NO− 3 (µg m−3) 12.6 12.8 15.6 14.4 11.4 12.7 10.3 9.5 13.1 13.4 NH+ 4 (µg m−3) 8.5 7.9 10.2 8.2 6.9 7.3 8.1 7.4 8.8 8.1 Cl−(µg m−3) 1.8 2.5 0.8 1.5 1.7 2.7 3.0 3.0 1.5 1.9 NR-PM1 (µg m−3) 56.6 48.2 61.6 48.8 53.3 49.7 58.7 50.5 52.3 42.7 SO2 (ppb) 16.2 14.0 5.4 0.8 25.3 16.0 11.5 8.3 CO (ppm) 1.5 1.3 1.8 1.3 1.7 1.6 1.2 1.0 NO (ppb) 30.0 43.0 7.8 10.8 41.9 51.2 50.9 50.9 19.8 30.0 NOy (ppb) 64.0 55.5 35.6 17.9 77.8 63.1 89.1 66.6 54.0 43.3 O3 (ppb) 21.2 23.8 33.3 29.1 20.3 24.4 7.9 8.5 20.8 19.3 RH (%) 47.0 23.4 62.7 18.9 52.7 20.0 35.6 20.3 36.5 22.5 T (◦C) 13.3 11.6 26.3 3.6 14.1 7.0 −1.3 3.4 14.6 8.4 WS, 8 m (m s−1) 1.2 0.8 1.0 0.5 0.9 0.7 1.4 1.0 1.4 0.9 WS, 240 m (m s−1) 4.4 3.0 3.5 2.3 4.1 2.7 4.6 3.4 5.3 3.3 200 150 100 50 0 Mass Concentration (µg m -3) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Summer Fall Winter Spring Summer Heating Season 40% 17% 25% 17% 1% 62 μg m-3 53 μg m-3 50% 12% 21% 13% 3% 59 μg m-3 51% 13% 17% 14% 5% 52 μg m-3 41% 14% 25% 17% 3% Org SO4 2− NO3 − NH4 + Cl− Figure 3. Time series of NR-PM1 species for the entire year. The pie charts show the average chemical composition of NR-PM1 durin our seasons (summer, fall, winter, and spring). Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China A higher concentration of nitrate than sulfate has also been frequently observed at urban and rural sites in China in recent years, e.g., Nanjing, in the YRD region (Zhang et al., 2015), and Changdao Island (Hu et al., 2013). g The NR-PM1 species varied dramatically and differently during the four seasons (Fig. 3). Overall, organics dominated NR-PM1 during all seasons, accounting for 40–51 % on aver- age. The dominance of organics in NR-PM1 has been widely observed at various sites in China, e.g., 31–52 % in the YRD region (Huang et al., 2012, 2013; Zhang et al., 2015), 36– 46 % in the PRD region (He et al., 2011; Huang et al., 2011; Gong et al., 2012), and 47 % in northwest China (J. Xu et al., 2014). Organics showed the largest contribution to NR-PM1 in winter due to a large amount of carbonaceous aerosol emit- ted from coal combustion (Chen et al., 2005; Zhang et al., 2008). This is also consistent with the highest contribution of chloride, with coal combustion being a major source in win- ter (Zhang et al., 2012). High concentrations of organics were also observed during late June and early October, due to the impacts of agricultural burning in these 2 months. Secondary inorganic aerosol (SIA, i.e., sulfate + nitrate + ammonium) contributed the largest fraction of NR-PM1 during the sum- mer season (59 %) and the lowest fraction during the win- ter season (44 %). Such seasonal differences in PM compo- sition reﬂect the different roles played by primary emissions and secondary formation. While photochemical production of secondary aerosol associated with higher O3 and stronger solar radiation (Fig. 2) plays a dominant role in affecting aerosol composition in summer, primary emissions play an Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10154 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 150 120 90 60 30 0 25 20 15 10 5 0 150 120 90 60 30 0 NR-PM1 (µg m -3) Frequency (%) (a) Summer (d) Spring (c) Winter (b) Fall Figure 4. Frequency of NR-PM1 mass loadings during the four sea- sons: (a) summer; (b) fall; (c) winter; (d) spring. Note that the fre- quency was calculated with 15 min average data. 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 150 120 90 60 30 0 25 20 15 10 5 0 150 120 90 60 30 0 NR-PM1 (µg m -3) Frequency (%) (a) Summer (d) Spring (c) Winter (b) Fall Figure 4. Frequency of NR-PM1 mass loadings during the four sea- sons: (a) summer; (b) fall; (c) winter; (d) spring. Note that the fre- quency was calculated with 15 min average data. hi h ll d d t i ll f ti f NR 120 80 40 0 50 40 30 20 10 0 20 15 10 5 0 Mass Conc. (µg m -3) 40 30 20 10 0 25 20 15 10 5 0 5 4 3 2 1 0 7 8 9 10 1112 1 2 3 4 5 6 Month of Year 7 8 9 10 1112 1 2 3 4 5 6 NR-PM1 Org SO4 2– NO3 – NH4 + Cl – 2011 2012 2012 2011 Figure 5. Seasonal variation of non-refractory submicron aerosol species. The bars represent the 25th and 75th percentiles, and the lid i l h l 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 150 120 90 60 30 0 25 20 15 10 5 0 150 120 90 60 30 0 NR-PM1 (µg m -3) Frequency (%) (a) Summer (d) Spring (c) Winter (b) Fall Figure 4 Frequency of NR PM mass loadings during the four sea 120 80 40 0 50 40 30 20 10 0 20 15 10 5 0 Mass Conc. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China (µg m -3) 40 30 20 10 0 25 20 15 10 5 0 5 4 3 2 1 0 7 8 9 10 1112 1 2 3 4 5 6 Month of Year 7 8 9 10 1112 1 2 3 4 5 6 NR-PM1 Org SO4 2– NO3 – NH4 + Cl – 2011 2012 2012 2011 Figure 4. Frequency of NR-PM1 mass loadings during the four sea- sons: (a) summer; (b) fall; (c) winter; (d) spring. Note that the fre- quency was calculated with 15 min average data. Figure 5. Seasonal variation of non-refractory submicron aerosol species. The bars represent the 25th and 75th percentiles, and the solid circles show mean values. which overall decreased monotonically as a function of NR- PM1 loadings. Note that heavily polluted events, with NR- PM1 mass concentrations larger than 150 µg m−3, occurred during all seasons, on average accounting for 3–7 % of the total time. Such heavily polluted events were mainly caused by agricultural burning in summer and fall, and coal com- bustion in winter, particularly under stagnant meteorological conditions (Sun et al., 2013b; Cheng et al., 2014). enhanced role in winter when photochemical processing is weaker (Sun et al., 2013b). It is interesting to note that ni- trate, on average, showed a higher contribution than sulfate during the four seasons. Compared to previous AMS mea- surements in Beijing (Huang et al., 2010; Sun et al., 2010), the nitrate contribution to NR-PM1 appears to show an in- creasing trend. The ratio of NO− 3 / SO2− 4 varied from 1.3 to 1.8 in this study, which is overall higher than those (0.8–1.5) observed during the four seasons in 2008 (Y. Zhang et al., 2013). This result likely indicates a response of secondary inorganic aerosol composition to the variations of precursors of NOx and SO2 in recent years. For instance, a continuous effort to reduce SO2 emissions is accompanied with a gradual increase in NOx emissions (S. X. Wang et al., 2014), which results in an increasingly more important role played by ni- trate in PM pollution in Beijing. Indeed, a recent model anal- ysis of the response of SIA to their precursors from 2000 to 2015 showed that the increase of nitrate would exceed the re- duction of sulfate in northern China, assuming no change to NH3 emissions (Wang et al., 2013). www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 urements of aerosol particle composition in Beijing, China 120 80 40 0 50 40 30 20 10 0 20 15 10 5 0 Mass Conc. (µg m -3) 40 30 20 10 0 25 20 15 10 5 0 5 4 3 2 1 0 7 8 9 10 1112 1 2 3 4 5 6 Month of Year 7 8 9 10 1112 1 2 3 4 5 6 NR-PM1 Org SO4 2– NO3 – NH4 + Cl – 2011 2012 2012 2011 Figure 5. Seasonal variation of non-refractory submicron aerosol species. The bars represent the 25th and 75th percentiles, and the solid circles show mean values. 3.2 Seasonal variation The monthly average NR-PM1 mass concentration stayed relatively constant throughout the year, with the average value ranging from 46 to 60 µg m−3, except in June 2012 Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Y. L. Sun et al.: Long-term real-time measurements of aer 80 60 40 20 0 Mass Conc. (µg m -3) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Org SO4 2– NO3 – NH4 + Cl – 1.0 0.8 0.6 0.4 0.2 0.0 Mass Fraction Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun (a) (b) Figure 6. Monthly variation of (a) mass concentrations and (b) mass fractions of NR-PM1 species. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10155 80 60 40 20 0 Mass Conc. (µg m -3) Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Org SO4 2– NO3 – NH4 + Cl – (a) (b) pared to the other three seasons, was also observed in Bei- jing (R. Zhang et al., 2013; Zhao et al., 2013). The seasonal variation of organics is primarily driven by emissions from various sources and secondary production. While the POA, particularly from coal combustion emissions, is signiﬁcantly elevated during wintertime, the photochemically processed SOA dominates OA in summer (Sun et al., 2012, 2013b). In the present study, chloride showed a similar seasonal vari- ation to that of organics. The chloride concentration during wintertime (2.8–3.3 µg m−3) was approximately 6 times that (0.5 µg m−3) in summer. The contribution of chloride to NR- PM1 showed a similar seasonal trend, with the lowest con- tribution in summer (∼1 %) and the highest in winter (∼5– 6 %) (Fig. 6). High concentrations of chloride in winter are associated with enhanced coal combustion emissions (Sun et al., 2013b), but also with low ambient temperature, which fa- cilitates the formation of particle-phase ammonium chloride. Also note that chloride showed a twice as high concentration and contribution in June than the other 2 months in summer because agricultural burning is also a large source of chloride (Viana et al., 2008; Cheng et al., 2014). 3.2 Seasonal variation (b) 1.0 0.8 0.6 0.4 0.2 0.0 Mass Fraction Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun ( ) ( g ) The seasonal variation of sulfate is different from organics and chloride. The sulfate concentration gradually decreased from 10.1 µg m−3 in August to 4.9 µg m−3 in November, which was associated with a synchronous decrease in so- lar radiation and O3 (Fig. 2). The contribution of sulfate to NR-PM1 showed a corresponding decrease from 19 to 10 %. The sulfate concentration then increased to 8.3–8.8 µg m−3 in December and January, likely due to a signiﬁcant increase of precursor SO2 associated with an increased demand for domestic heating during the winter season, which can be oxidized to form sulfate via either gas-phase oxidation or aqueous-phase processing (W. Y. Xu et al., 2014). Sulfate showed the highest concentration in June (13.5 µg m−3) due to secondary production, but possibly the impact of biomass burning as well. Indeed, a recent study in the YRD re- gion also found a large enhancement of sulfate in biomass burning plumes in summer (Zhang et al., 2015). Nitrate showed minor seasonal variation, with the monthly average concentration ranging from 8 to 15 µg m−3, except in June (23 µg m−3). It is interesting that a higher concentration of nitrate was observed in summer and spring than in winter. On average, nitrate accounted for ∼25 % of NR-PM1 dur- ing summertime, but decreased to ∼15 % during wintertime (Fig. 6). Although high temperatures in summer favor the dissociation of ammonium nitrate particles to gas-phase am- monia and nitric acid, the correspondingly high RH and ex- cess gaseous ammonia facilitate the transformation of nitric acid to aqueous NH4NO3 particles (Meng et al., 2011; Sun et al., 2012). The lowest concentration of nitrate during winter- time might be primarily caused by the weak photochemical production associated with low solar radiation and oxidants (e.g., O3). In addition, the higher particle acidity in winter (Liu, 2012) and lower mixing ratio of gaseous ammonia may also suppress the formation of ammonium nitrate particles Figure 6. Monthly variation of (a) mass concentrations and (b) mass fractions of NR-PM1 species. (Fig. 5). The month of June presented the highest NR-PM1 (89 µg m−3) due to the impact of agricultural burning. 3.2 Seasonal variation Con- sistently, a higher concentration of NR-PM1 was observed in the summer of 2008 (5 June–3 July) than the other seasons in Beijing (Y. Zhang et al., 2013). Zhao et al. (2009) also observed the highest concentration of PM2.5 in June 2007, due to the inﬂuences of agricultural burning. These results indicate that agricultural burning is a large source of PM pollution in Beijing in summer. The lowest concentration of NR-PM1 in summer occurred in July, mainly due to abun- dant precipitation and high temperatures, which facilitated wet scavenging and convection of PM, respectively (Fig. 2). Similarly lower concentrations of PM2.5 in summer than in the other seasons were also observed previously at an urban site in Beijing (Zhao et al., 2009). Among the NR aerosol species, organics and chloride pre- sented pronounced seasonal variations, showing higher con- centrations in winter than in the other seasons (Fig. 5). The concentration of organics increased from 17 µg m−3 in July to ∼30 µg m−3 in October, and then remained relatively sta- ble across the whole of wintertime. The concentration of or- ganics reached a minimum in April (17 µg m−3), and then rapidly increased to 37 µg m−3 in June. Correspondingly, the contribution of organics to NR-PM1 increased from ∼40 % in summer to above 50 % during wintertime (Fig. 6). A higher concentration of carbonaceous aerosol in winter, com- Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10156 24 20 16 12 8 4 0 24 20 16 12 8 4 0 24 20 16 12 8 4 0 24 20 16 12 8 4 0 60 40 20 0 60 40 20 0 60 40 20 0 50 40 30 20 10 0 20 10 0 10 8 6 4 2 0 12 8 4 0 12 8 4 0 30 20 10 0 20 10 0 16 12 8 4 0 20 10 0 2.5 2.0 1.5 1.0 0.5 0.0 5 4 3 2 1 0 5 4 3 2 1 0 4.0 3.0 2.0 1.0 0.0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Mass Concentration (µg m -3) (a) Organics (b) SO4 2– (c) NO3 – (d) Cl – Hour of Day Summer Fall Winter Spring Figure 7. Monthly average diurnal cycle of (a) organics, (b) sulfate, (c) nitrate, and (d) chloride during the four seasons. Figure 7. Monthly average diurnal cycle of (a) organics, (b) sulfate, (c) nitrate, and (d) chloride during the fo verage diurnal cycle of (a) organics, (b) sulfate, (c) nitrate, and (d) chloride during the four seasons. (Zhang et al., 2007). The seasonal variation of ammonium is similar to that of sulfate and nitrate because ammonium primarily exists in the form of NH4NO3 and (NH4)2SO4. (Zhang et al., 2007). The seasonal variation of ammonium is similar to that of sulfate and nitrate because ammonium primarily exists in the form of NH4NO3 and (NH4)2SO4. that the daytime photochemical production of sulfate from gas-phase oxidation of SO2 might be masked by an elevated planetary boundary layer (PBL). Considering the dilution ef- fect of the PBL, Sun et al. (2012) found that sulfate increased gradually from morning to late afternoon, demonstrating the daytime photochemical production of sulfate. In this study, sulfate in May, June and October showed an evident day- time increase until late afternoon, indicating an important role played by gas-phase photochemical processing in driv- ing the sulfate diurnal cycle. www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10157 40 30 20 10 0 50 40 30 20 10 0 60 40 20 0 30 20 10 0 24 20 16 12 8 4 0 12 8 4 0 10 8 6 4 2 0 12 8 4 0 12 8 4 0 24 20 16 12 8 4 0 25 20 15 10 5 0 16 12 8 4 0 16 12 8 4 0 16 12 8 4 0 24 20 16 12 8 4 0 1.6 1.2 0.8 0.4 0.0 3.0 2.0 1.0 0.0 6 4 2 0 2.5 2.0 1.5 1.0 0.5 0.0 24 20 16 12 8 4 0 Mass Concentration (µg m -3) (a) Organics (b) SO4 2– (c) NO3 – (d) Cl – Hour of Day Summer Fall Winter Spring Weekdays Weekends Figure 8. Comparison of the average diurnal cycles of (a) organics, (b) SO2− 4 , (c) NO− 3 , and (d) Cl−between weekdays and weekends during the four seasons. Note that the periods with NR-PM1 < 20 µg m−3 are excluded. Figure 8. Comparison of the average diurnal cycles of (a) organics, (b) SO2− 4 , (c) NO− 3 , and (d) Cl−between weekdays and weekends during the four seasons. Note that the periods with NR-PM1 < 20 µg m−3 are excluded. mand, which usually starts on 15 November and ends on 15 March. Coal combustion has been found to be a large source of chloride (Zhang et al., 2012; Sun et al., 2013b). Therefore, the diurnal cycle of chloride is likely dominantly driven by coal combustion emissions that are intensiﬁed at night for domestic heating. orative loss of NH4NO3 associated with high temperatures, which overcomes the amount of photochemical production, plays the major role in driving such diurnal cycles. The ris- ing PBL plays an additional role in the low concentrations of nitrate during daytime (Sun et al., 2012). The diurnal cycle of nitrate in May and September was also signiﬁcant, charac- terized by a pronounced morning peak occurring at ∼10:00, when photochemical production dominated over the gas– particle partitioning of NH4NO3. Nitrate showed a relatively ﬂat diurnal cycle in April, indicating a combined effect of various nitrate formation mechanisms. 3.4 Weekend effects Because the switch between clean periods and pollution episodes arising from different source areas happens fre- quently in Beijing (Sun et al., 2013b; Guo et al., 2014), the diurnal cycles of aerosol species can vary greatly due to the inﬂuences of different occurrences of clean periods between weekdays and weekends (Sun et al., 2013b). Therefore, peri- ods with low aerosol loadings (NR-PM1 < 20 µg m−3) were excluded from the results (Fig. 8) for a better investigation of the weekend effects (for the average diurnal cycles with clean periods included, see Fig. S3). As shown in Fig. 8, there were no clear weekend effects in the summer, except for slightly lower concentrations of organics, sulfate, and ni- trate in the late afternoon on weekends. This suggests that there are no signiﬁcant differences in anthropogenic activ- ity between weekdays and weekends in summer. Although some enhanced trafﬁc emissions between 00:00 and 06:00 on weekends might have occurred, as indicated by the higher Chloride in this study was primarily detected as ammo- nium chloride because ACSM is insensitive to refractory NaCl and/or KCl at its vaporizer temperature of 600 ◦C. As shown in Fig. 7, two different diurnal cycles were observed throughout different months. For the months of July, Au- gust, September, April, and May, chloride presented a morn- ing peak when both temperatures and the PBL were at their lowest, and then rapidly decreased to a low ambient level at ∼18:00. Such a diurnal cycle was likely primarily driven by temperature-dependent gas–particle partitioning (Hu et al., 2008). The diurnal cycles of chloride during the remaining months were also signiﬁcant, all of which were character- ized by high concentrations at night. Coincidentally, these months fell during the season of high domestic-heating de- 3.3 Diurnal variations As demonstrated in Fig. 7, the diurnal cycles of organ- ics during the four seasons were overall similar, character- ized by two pronounced peaks occurring at noon and during the evening. PMF analysis of OA suggested that the noon peak was primarily caused by cooking emissions, while the evening peak was driven by different primary emissions (e.g., cooking, trafﬁc, and coal combustion emissions) among the different seasons (Sun et al., 2012, 2013b). It should be noted that the noon peak in summer was more signiﬁcant than in fall and winter. Indeed, the cooking emissions, de- termined by subtracting the background (10:00–11:00) from the noon peak (12:00–13:00), were ∼1.5–2 µg m−3 from September to the following March, which were lower than the ∼3.5 µg m−3 calculated for June and July. This seasonal trend agreed with that of temperature, indicating that cook- ing emissions are temperature-dependent, probably because of increased cooking activity in hot summers than cold win- ters. g y Nitrate showed substantially different diurnal cycles among different months. A clear daytime increase starting from about 08:00 to 19:00 was found in the 5 months of January, February, March, November and December, indi- cating that such a diurnal pattern is more signiﬁcant during wintertime compared to the fall and spring seasons. Figure 2 shows that the temperature during these 5 months was gener- ally low (< 10 ◦C), under which the partitioning of NH4NO3 into gaseous NH3 and HNO3 would not be signiﬁcant. As a result, photochemical production would be the primary fac- tor driving the diurnal variations. The photochemical pro- duction rate calculated from the daytime increase was 0.6– 0.8 µg m−3 h−1 during winter and ∼0.2–0.3 µg m−3 h−1 in November and March. Nitrate presented pronounced diurnal cycles in summer (June, July, and August), with the concen- trations gradually decreasing during daytime and reaching a minimum at ∼16:00. Similar diurnal cycles have been ob- served on many occasions in summer in Beijing (Huang et al., 2010; Sun et al., 2012; Zhang et al., 2015). The evap- Relatively ﬂat diurnal cycles were observed for sulfate during most months, indicating the regional characteristics of sulfate. In fact, multi-day build-up of sulfate was frequently observed during all seasons (Fig. 3), supporting the notion of regional inﬂuences on sulfate in Beijing. It should be noted Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ While the diurnal variations of organics and chloride were similar be- tween weekdays and weekends during the fall season, sul- fate and nitrate showed pronounced weekend effects, with persistently higher concentrations at weekends throughout the day. An explanation for this is the stronger photochem- ical production of secondary species associated with higher O3 and solar radiation on weekends (Fig. S4). Consistently, SOA showed similar weekend effects as those of secondary inorganic species, while POA did not (Sun et al., 2015). Be- cause of the regional characteristics of secondary aerosols, further analysis is needed to address the impacts of regional transport on the weekend effects of secondary species. Win- ter showed the most pronounced weekend effects for all aerosol species. All aerosol species showed much lower con- centrations on weekends than on weekdays across the en- tire day, which was consistent with those of NO, SO2, and CO (Fig. S4). These results clearly indicate much reduced anthropogenic activity on weekends during wintertime be- cause of low ambient temperature (−4 to −3 ◦C). Further evidence is provided by the diurnal cycles of organics, which presented pronounced noon peaks on weekends during all seasons except winter. This observation was consistent with much reduced cooking activity on weekends during winter- time. Similar to summer, no evident weekend effects were observed in spring. The weekend effects of aerosol species in this study are overall consistent with those observed by Han et al. (2009), in which similar diurnal cycles of primary ele- mental carbon, CO, and CO2 between weekdays and week- ends under weak wind conditions were observed during the three seasons other than winter. y The RH- and T -dependent distributions of major aerosol species (Fig. 10) allow us to further investigate the RH/T im- pacts on the formation of aerosol species. While all aerosol species showed similar concentration gradients as a func- tion of RH to that of NR-PM1, the T -dependent patterns varied greatly. Organics generally showed the highest con- centrations under low T (< 6 ◦C) and high humidity con- ditions – very similar to the behavior of chloride, which is mainly derived from combustion sources, e.g., coal combus- tion or biomass burning (Zhang et al., 2012; Cheng et al., 2014). www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10158 36 30 24 18 12 6 0 -6 -12 T (°C) 90 80 70 60 50 40 30 20 10 RH (%) 6 5 4 3 2 1 WS (m/s) 36 30 24 18 12 6 0 -6 -12 T (°C) 90 80 70 60 50 40 30 20 10 RH (%) 60 40 20 0 NR-PM1 (μg m -3) (a) (b) Figure 9. RH/T dependence of (a) NR-PM1 mass concentration and (b) WS for a whole year. The data are grouped into grids with increments of RH and T being 5 % and 3 ◦C, respectively. Grid cells with a number of data points fewer than 10 are excluded. T (°C) 36 30 24 18 12 6 0 -6 -12 T (°C) 90 80 70 60 50 40 30 20 10 RH (%) 60 40 20 0 NR-PM1 (μg m -3) (a) 36 30 24 18 12 6 0 -6 -12 T (°C) 90 80 70 60 50 40 30 20 10 RH (%) 6 5 4 3 2 1 WS (m/s) (b) (a) Figure 9. RH/T dependence of (a) NR-PM1 mass concentration and (b) WS for a whole year. The data are grouped into grids with increments of RH and T being 5 % and 3 ◦C, respectively. Grid cells with a number of data points fewer than 10 are excluded. erally less than 20 µg m−3 at RH < 20 %, and had no clear dependence on T . This can be explained by the high WS (often larger than 5 m s−1; Fig. 9b) at low RH levels asso- ciated with clean air masses from the north and/or north- west. Previous studies have also found a strong association between low aerosol loading and high WS in Beijing (Han et al., 2009; Sun et al., 2013b). NR-PM1 showed moder- ately high concentrations (∼20–40 µg m−3) at low RH (20– 40 %), which rapidly increased to a high concentration level (> 60 µg m−3) at RH > 50 %. These results indicate that se- vere haze episodes in Beijing mostly occur under high hu- midity conditions, when WS is low as well. Two different regions with high concentrations of NR-PM1 are apparent in Fig. www.atmos-chem-phys.net/15/10149/2015/ The results suggest that high concentrations of organ- ics during wintertime are primarily caused by coal combus- tion emissions during the domestic-heating season, particu- larly from residential coal combustion (Zhang et al., 2008). In fact, a previous study by our group found that nearly one- third of OA during wintertime is primary coal combustion OA (CCOA) (Sun et al., 2013b). In contrast, organics showed much lower concentrations under the conditions of higher RH and higher T , for which one of the reasons was prob- www.atmos-chem-phys.net/15/10149/2015/ 9a: one in the top-right region with high temperature (> ∼15 ◦C), and another in the bottom-right region with low ambient temperature (< ∼6 ◦C). Such a difference in dis- tribution illustrates the severity of PM pollution in different seasons. Note that low concentrations of NR-PM1 sometimes occurred at RH > 90 %, likely due to the scavenging of par- ticles by rain or winter snow. concentration of NO (Fig. S4), they appeared to have neg- ligible impacts on secondary sulfate and nitrate. While the diurnal variations of organics and chloride were similar be- tween weekdays and weekends during the fall season, sul- fate and nitrate showed pronounced weekend effects, with persistently higher concentrations at weekends throughout the day. An explanation for this is the stronger photochem- ical production of secondary species associated with higher O3 and solar radiation on weekends (Fig. S4). Consistently, SOA showed similar weekend effects as those of secondary inorganic species, while POA did not (Sun et al., 2015). Be- cause of the regional characteristics of secondary aerosols, further analysis is needed to address the impacts of regional transport on the weekend effects of secondary species. Win- ter showed the most pronounced weekend effects for all aerosol species. All aerosol species showed much lower con- centrations on weekends than on weekdays across the en- tire day, which was consistent with those of NO, SO2, and CO (Fig. S4). These results clearly indicate much reduced anthropogenic activity on weekends during wintertime be- cause of low ambient temperature (−4 to −3 ◦C). Further evidence is provided by the diurnal cycles of organics, which presented pronounced noon peaks on weekends during all seasons except winter. This observation was consistent with much reduced cooking activity on weekends during winter- time. Similar to summer, no evident weekend effects were observed in spring. The weekend effects of aerosol species in this study are overall consistent with those observed by Han et al. (2009), in which similar diurnal cycles of primary ele- mental carbon, CO, and CO2 between weekdays and week- ends under weak wind conditions were observed during the three seasons other than winter. concentration of NO (Fig. S4), they appeared to have neg- ligible impacts on secondary sulfate and nitrate. 3.5 Meteorological effects Figure 9 shows the RH- and T -dependent distributions of NR-PM1 and WS for the entire year. The distribution of NR- PM1 showed an obvious concentration gradient as a func- tion of RH. NR-PM1 showed the lowest mass loading, gen- Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 RH (%) 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 RH (%) 50 40 30 20 10 0 Org (μg m -3) 30 20 10 0 SO4 2- (μg m -3) 30 20 10 0 NO3 - (μg m -3) 6 4 2 0 Cl - (μg m -3) 0.6 0.5 0.4 0.3 0.2 0.1 Org/NR-PM1 0.25 0.20 0.15 0.10 0.05 SO4 2-/NR-PM1 0.25 0.20 0.15 0.10 0.05 0.00 NO3 -/NR-PM1 0.06 0.04 0.02 0.00 Cl -/NR-PM1 Temperature ( oC) (a) (b) (c) (d) Figure 10. RH/T dependence of mass concentrations and mass fractions of aerosol species for a whole year: (a) organics; (b) sulfate; (c) nitrate; (d) chloride. The data are grouped into grids with increments of RH and T being 5 % and 3 ◦C, respectively. Grid cells with a number of data points fewer than 10 are excluded. Figure 10. RH/T dependence of mass concentrations and mass fractions of aerosol species for a whole year: (a) organics; (b) sulfate; (c) nitrate; (d) chloride. The data are grouped into grids with increments of RH and T being 5 % and 3 ◦C, respectively. Grid cells with a number of data points fewer than 10 are excluded. The RH/T dependence of secondary inorganic species showed somewhat different behaviors from that of organics. Sulfate presented two high concentration regions, with the highest values occurring during wintertime when T was be- low 0 ◦C and RH was above 70 %. Aqueous-phase oxidation, mostly fog processing, has been found to play a dominant role in sulfate formation under such meteorological condi- tions (Sun et al., 2013a). Surprisingly, the semi-volatile ni- trate showed a relatively homogeneous distribution across different temperatures at RH > 40 %. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China n et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10159 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 RH (%) 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 36 24 12 0 -12 90 80 70 60 50 40 30 20 10 RH (%) 50 40 30 20 10 0 Org (μg m -3) 30 20 10 0 SO4 2- (μg m -3) 30 20 10 0 NO3 - (μg m -3) 6 4 2 0 Cl - (μg m -3) 0.6 0.5 0.4 0.3 0.2 0.1 Org/NR-PM1 0.25 0.20 0.15 0.10 0.05 SO4 2-/NR-PM1 0.25 0.20 0.15 0.10 0.05 0.00 NO3 -/NR-PM1 0.06 0.04 0.02 0.00 Cl -/NR-PM1 Temperature ( oC) (a) (b) (c) (d) Figure 10. RH/T dependence of mass concentrations and mass fractions of aerosol species for a whole year: (a) organics; (b) sulfat nitrate; (d) chloride. The data are grouped into grids with increments of RH and T being 5 % and 3 ◦C, respectively. Grid cells with a nu of data points fewer than 10 are excluded. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Despite high temper- ature in summer, high humidity facilitates the transforma- tion of gaseous species into aqueous-phase nitrate particles (Sun et al., 2012), particularly in the presence of high abun- dance of gaseous ammonia (Ianniello et al., 2010). In fact, ni- trate showed the highest contribution (> ∼25 %) to NR-PM1 mass under high T and high RH conditions, which were also the conditions under which high concentrations of NR-PM1 were observed. The fact that nitrate contributed more than sulfate (∼15–20 %) to NR-PM1 mass during these condi- ably far fewer coal combustion emissions during summer- time (Zheng et al., 2005; R. Zhang et al., 2013). Consis- tently, CCOA has not yet been resolved from PMF analyses of AMS OA in summer in Beijing (Huang et al., 2010; Sun et al., 2010). Note that the region with a high concentration of organics corresponded to a high concentration of NR-PM1. In this region, organics accounted for the largest fraction of NR-PM1 (approximately 40–50 %), indicating that severe PM pollution under low temperature and high humidity con- ditions is dominantly contributed to by organics. The mass fraction of organics, however, showed an opposite distribu- tion to that of mass loading. As shown in Fig. 10, organics presents the highest contribution to NR-PM1 (∼> 50 %) in the left-hand region with low RH, indicating the dominance of organics during periods with low NR-PM1 mass loadings. Such a distribution is independent of temperature, suggesting a ubiquitously organics-dominant composition during clean days in all seasons. www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China The fall season showed a similar aerosol composition dependence as that in summer, with higher concentrations from the E, SE, and S. However, the gradients of wind sec- tors appeared to be smaller. For example, the average NR- PM1 concentration ranged from 46.3 to 72.7 µg m−3 in all eight sectors except NW. Organics showed a similar domi- nance in NR-PM1, accounting for 47–55 %, and the contri- bution was ubiquitously higher than in summer for all wind sectors. It should be noted that the NW sector showed the largest difference between mean and median values for all species. The much lower median values suggest a dominance of clean days for most of the time in this sector. In con- trast, the summer season showed higher median concentra- tions from the NW, indicating a higher regional background during this season. The winter season showed consistently high concentrations of PM across the different wind sec- tors, except for NW, where the mass concentrations were ap- proximately half of those in the other sectors. The average NR-PM1 ranged from 55.0 to 84.4 µg m−3, with organics be- ing the major fraction, accounting for 46–54 %. The spring tions suggests an important role played by nitrate in summer haze formation. While the concentration of nitrate at various temperatures was similar, its contribution to NR-PM1 was generally lower at low temperatures due to the greater en- hancement of organics during wintertime. Also note that the two semi-volatile species, i.e., nitrate and chloride, show the lowest contributions to NR-PM1 in the top-left region with the highest T and lowest RH. This illustrates the evapora- tive loss process of ammonium nitrate and ammonium chlo- ride under high temperatures in summertime. However, sul- fate shows a relatively higher contribution in this region since ammonium sulfate is less volatile than ammonium nitrate and chloride (Huffman et al., 2009). Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10160 60 40 20 0 80 60 40 20 0 60 40 20 0 50 40 30 20 10 0 N NE E SE S SW W NW Wind Dir ( o) 25 20 15 10 5 0 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 N NE E SE S SW W NW Wind Dir ( o) 40 30 20 10 0 30 20 10 0 30 20 10 0 30 20 10 0 N NE E SE S SW W NW Wind Dir ( o) 3.0 2.0 1.0 0.0 5 4 3 2 1 0 8 6 4 2 0 5 4 3 2 1 0 N NE E SE S SW W NW Wind Dir ( o) Mass Concentration (µg m -3) (a) Organics (b) SO4 2– (c) NO3 – (d) Cl – Summer Fall Winter Spring Figure 11. Box plots of mass concentrations of (a) organics, (b) SO2− 4 , (c) NO− 3 , and (d) Cl−as a function of wind direction (Dir) sectors. All the data were segregated into eight wind sectors representing north (N), northeast (NE), east (E), southeast (SE), south (S), southwest (SW), west (W), and northwest (NW). The mean (cross), median (horizontal line), 25th and 75th percentiles (lower and upper box), and 10th and 90th percentiles (lower and upper whiskers) are shown. Figure 11. Box plots of mass concentrations of (a) organics, (b) SO2− 4 , (c) NO− 3 , and (d) Cl−as a function of wind direction (Dir) sectors. All the data were segregated into eight wind sectors representing north (N), northeast (NE), east (E), southeast (SE), south (S), southwest (SW), west (W), and northwest (NW). The mean (cross), median (horizontal line), 25th and 75th percentiles (lower and upper box), and 10th and 90th percentiles (lower and upper whiskers) are shown. mental Statement 2013). These results suggest an inhomoge- neous distribution of air pollution around the IAP sampling site in summer. Organics dominated NR-PM1 across differ- ent sectors (37–43 %), followed by nitrate (21–28 %), sulfate (15–20 %), and ammonium (15–17 %). While chloride con- tributed a small fraction of NR-PM1 (0.7–1.8 %), the mass concentration showed the largest difference between SE and NW. www.atmos-chem-phys.net/15/10149/2015/ Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 (c) 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 (d) Figure 12. PSCF of NR-PM1 species during four seasons: (a) organics; (b) sulfate; (c) nitrate; (d) chloride. The cities marked in each panel are Beijing (BJ), Tianjing (TJ), Langfang (LF), Baoding (BD), Shijiazhuang (SJZ), and Hengshui (HS). The color scales indicate the values of PSCF. The high potential source area to the southeast of Beijing was mainly caused by open agricultural burning in June in north- ern China. Sulfate showed a distinct source region character- ized by a narrow high PSCF band along Hengshui–Baoding– Langfang–Beijing. Such a pollution band agrees well with the topography of the North China Plain, with the Taihang Mountains to the west and Yan Mountains to the north. The wide area of high PSCF for sulfate also indicates a regional characteristic of sulfate that is formed from gas-phase oxida- tion or cloud processing of precursor SO2, which is particu- larly high in Hebei Province (Ji et al., 2014). Secondary ni- trate showed a similar, yet much smaller, PSCF region com- pared to sulfate. One reason for this might be the evaporative loss of ammonium nitrate during the long-range transport in summer. The high potential source area to the southeast of Beijing was mainly caused by open agricultural burning in June in north- ern China. Sulfate showed a distinct source region character- ized by a narrow high PSCF band along Hengshui–Baoding– Langfang–Beijing. Such a pollution band agrees well with the topography of the North China Plain, with the Taihang Mountains to the west and Yan Mountains to the north. The wide area of high PSCF for sulfate also indicates a regional characteristic of sulfate that is formed from gas-phase oxida- tion or cloud processing of precursor SO2, which is particu- larly high in Hebei Province (Ji et al., 2014). Secondary ni- trate showed a similar, yet much smaller, PSCF region com- pared to sulfate. One reason for this might be the evaporative loss of ammonium nitrate during the long-range transport in summer. season showed a similar wind sector dependence on aerosol composition as the fall season. Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China The average NR-PM1 ranged from 49.0 to 74.4 µg m−3 for all of the wind sectors, except the N (38.5 µg m−3) and NW (24.7 µg m−3) sectors which had much lower mass concentrations. Similar to other sea- sons, organics dominated NR-PM1 throughout the different sectors (36–53 %), followed by nitrate (19–27 %), and sulfate (11–16 %). As Fig. 12 shows, the potential source areas for aerosol species varied among the four seasons. In summer, high potential source areas were mainly located to the south, southwest, and southeast of Beijing. Organics had a rela- tively small potential source region in the south of Beijing (< 100 km) and a small source region located around Baod- ing – one of the most polluted cities in Hebei Province. A narrow and visible source area to the southeast of Beijing, including Tianjin and the Bohai Sea, was also observed. Ni- trate and chloride showed similar source areas to organics. All aerosol species showed similar PSCF spatial distribu- tions during the fall season, with high potential source re- gions located in a narrow area from Hengshui, Baoding to Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10161 g p p j g, 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 SJZ BD LF TJ HS BJ 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.5 0.4 0.3 0.2 0.1 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.35 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.5 0.4 0.3 0.2 0.1 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.30 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.25 0.20 0.15 0.10 0.05 44 42 40 38 36 34 124 122 120 118 116 114 112 110 0.20 0.16 0.12 0.08 (a) (b) (c) (d) Summer Fall Winter Spring Figure 12. PSCF of NR-PM1 species during four seasons: (a) organics; (b) sulfate; (c) nitrate; (d) chloride. The cities marked in each panel are Beijing (BJ), Tianjing (TJ), Langfang (LF), Baoding (BD), Shijiazhuang (SJZ), and Hengshui (HS). The color scales indicate the values of PSCF. 3.6 Source analysis In summer, all NR-PM1 species showed evident wind sec- tor gradients, with higher concentrations in association with winds from the east (E) and southeast (SE), and lower con- centrations with northwest (NW) wind (Fig. 11). The aver- age NR-PM1 concentration from the SE was 89.5 µg m−3, which was more than twice that (39.4 µg m−3) from the NW. All aerosol species increased as wind sectors changed along the N–NE–E–SE gradient, and then decreased along the SE– S–SW–W gradient. Such wind sector dependence of aerosol composition is remarkably consistent with the spatial distri- bution of ﬁne particles in Beijing in 2013 (Beijing Environ- Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Atmos. Chem. Phys., 15, 10149–10165, 2015 Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Although Hebei Province often has worse air pollution than Beijing during wintertime (Ji et al., 2014), the cities far away from Beijing appear not to be a very important source of winter- time air pollution in Beijing. One explanation for this is that stagnant meteorological conditions occur more frequently in winter due to low WS and T inversions. Thus, local emis- sions and transport from nearby regions would play a more signiﬁcant role in affecting the pollution level in Beijing. While the spring season showed similarly small potential source regions to those during wintertime, an obvious high potential source area in Hebei Province was also observed. The transport of air pollution from the SW to the NE along the Taihang Mountains in northern China has been observed many times in previous studies (L. T. Wang et al., 2014; Z. Wang et al., 2014). Given that many cities located on this pathway are often highly polluted, such as Shijiangzhuang, Baoding, and Hengshui, regional transport from these areas would have a potentially high impact on the formation of se- vere haze pollution in Beijing. g Meteorological conditions play an important role in the formation of severe PM pollution in Beijing. In this study, we have illustrated the inﬂuences of RH and T on aerosol load- ing and chemistry in different seasons. All aerosol species increased signiﬁcantly under stagnant meteorological condi- tions associated with high RH and low WS. NR-PM1 showed two high concentration regions (> 60 µg m−3) at RH > 60 %. While organics comprised a major fraction of NR-PM1 in these two regions, the abundances of sulfate and nitrate and air temperature were largely different, suggesting they play different roles in causing PM pollution during different sea- sons. Under drier conditions (RH < 30 %), the NR-PM1 con- centration was generally low and organics contributed more than 50 % of its mass, indicating the importance of organics during clean periods. The semi-volatile nitrate presented the largest contribution under high RH and high T , highlighting the importance of nitrate formation via aqueous-phase pro- cessing in summer. All NR-PM1 species showed obvious de- pendence on wind direction, with higher concentrations com- monly associated with winds from the S, E, and SE. This was consistent with the results from PSCF analysis, which showed that the high potential source areas were mainly lo- cated to the S and SW of Beijing. 4 Conclusions This paper presents the results from a year-long, real-time measurement study of submicron aerosol particle composi- tion using an ACSM, conducted at an urban site in Beijing from July 2011 to June 2012. The mass concentration of NR- PM1 varied dramatically, with the seasonal average concen- tration ranging from 52 to 62 µg m−3. Organics comprised a major fraction of NR-PM1 during all seasons, accounting for 40–51 % on average. The average contribution of nitrate to NR-PM1 (17–25 %) exceeded that of sulfate (12–17 %) during all seasons, suggesting an enhanced role of nitrate in PM pollution in recent years. Organics and chloride were two species showing pronounced seasonal variations in both mass concentrations and mass fractions. The higher con- centrations of organics and chloride in winter than summer were largely due to enhanced coal combustion emissions. We also observed high concentrations of organics and chloride in June and October – 2 months with strong agricultural burn- ing impacts. The seasonal variations of secondary sulfate and nitrate were not signiﬁcant because of the large variations of precursor concentrations, photochemical production, and also meteorological effects in different seasons. However, higher contributions of SIA in summer (57–61 %) than in winter (43–46 %) were still observed, indicating a more sig- niﬁcant role of secondary production in summer. The diurnal cycles of organics were similar during all seasons, all char- acterized by two pronounced peaks. While the diurnal cycles Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China The high potential source areas varied differently during the four seasons. A common high potential source area to the SW of Beijing, along the Taihang Mountains, was observed during all seasons except winter, demonstrating the potentially high impact of regional transport on severe PM pollution in Beijing. The winter sea- son showed a much smaller source region compared to the other seasons, indicating that local and regional transport over a smaller regional scale are more important. High po- tential source areas to the SE of Beijing were also observed for organics, nitrate, and chloride in summer, likely due to agricultural burning. The Supplement related to this article is available online at doi:10.5194/acp-15-10149-2015-supplement. Acknowledgements. This work was supported by the National Key Project of Basic Research (2014CB447900; 2013CB955801), the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB05020501), and the National Natural Science Foundation of China (41175108). Atmos. Chem. Phys., 15, 10149–10165, 2015 www.atmos-chem-phys.net/15/10149/2015/ Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China Y. L. Sun et al.: Long-term real-time measurements of aerosol particle composition in Beijing, China 10162 Beijing. These results suggest that regional transport from the southwest plays a dominant role in formation of se- vere haze pollution in fall. The wintertime results showed largely different PSCF distributions from the other seasons. High PSCF values were mainly located in a small region (< 50 km) in the south and southeast of Beijing. Although Hebei Province often has worse air pollution than Beijing during wintertime (Ji et al., 2014), the cities far away from Beijing appear not to be a very important source of winter- time air pollution in Beijing. One explanation for this is that stagnant meteorological conditions occur more frequently in winter due to low WS and T inversions. Thus, local emis- sions and transport from nearby regions would play a more signiﬁcant role in affecting the pollution level in Beijing. While the spring season showed similarly small potential source regions to those during wintertime, an obvious high potential source area in Hebei Province was also observed. The transport of air pollution from the SW to the NE along the Taihang Mountains in northern China has been observed many times in previous studies (L. T. Wang et al., 2014; Z. Wang et al., 2014). Given that many cities located on this pathway are often highly polluted, such as Shijiangzhuang, Baoding, and Hengshui, regional transport from these areas would have a potentially high impact on the formation of se- vere haze pollution in Beijing. of secondary sulfate were overall relatively ﬂat during most months of the year, those of nitrate varied greatly in different seasons. It was evident that the diurnal cycles of nitrate are driven by gas-particle partitioning and daytime photochemi- cal production in summer and winter, respectively. The win- ter season showed substantially different concentrations of aerosol species between weekdays and weekends, with much lower concentrations on weekends. However, no signiﬁcant weekend effects were observed during the other seasons. Beijing. These results suggest that regional transport from the southwest plays a dominant role in formation of se- vere haze pollution in fall. The wintertime results showed largely different PSCF distributions from the other seasons. High PSCF values were mainly located in a small region (< 50 km) in the south and southeast of Beijing. www.atmos-chem-phys.net/15/10149/2015/ Y. L. 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https://openalex.org/W4296584472	https://zenodo.org/record/1244547/files/8216ijmit02.pdf	English	null	DIGITAL SOCIETY : A REVIEW OF E-SERVICE AND MOBILE TECHNOLOGY IN EARTHQUAKES RELIEF OPERATIONS	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	5,971	Kazeem Oluwakemi OSENI1, Dr. Kate DINGLEY2, Dr. Penny HART3, Ayoade Iyabode ADEWOLE4 and Oluwakayode Abayomi DAWODU5 1,2,3School of Computing, University of Portsmouth, United Kingdom 4Department of Mathematics, Tai Solarin University of Education, Ijagun, Nigeria 5Department of Computer Science, Michael Otedola College of Primary Education, Noforija, Epe, Nigeria Kazeem Oluwakemi OSENI1, Dr. Kate DINGLEY2, Dr. Penny HART3, Ayoade Iyabode ADEWOLE4 and Oluwakayode Abayomi DAWODU5 1,2,3School of Computing, University of Portsmouth, United Kingdom 4Department of Mathematics, Tai Solarin University of Education, Ijagun, Nigeria 5Department of Computer Science, Michael Otedola College of Primary Education, Noforija, Epe, Nigeria KEYWORDS Digital Society, E-Service, Mobile Technology, Earthquakes, Disaster Operations ABSTRACT Developing countries lacking technology infrastructures experience most disasters such as tsunami, hurricane Katrina, earthquakes. Even though, many lives are saved in developed countries through the use of high-level or sophisticated technology, only the technology that is easy to use, inexpensive, durable and field-tested should be introduced and used during the relief operation. The effect of climate change and rapid population growth are probably exposed people inhabiting areas to substantial environmental risks. The authors highlight and identify current practices, particularities, and challenges in earthquakes relief operations with the aims of reviewing the roles E-Service and Mobile Technology tools in real-life situations and practices could do to help in disaster operations. This study focuses on victims of earthquakes worldwide. The study will review and provide insights into the roles of E-Service and Mobile Technologies in earthquakes relief operations and how the internally displaced person could benefit from these services. The limitations of these services will be discussed and how the technology could be used to further predict natural disaster such as earthquakes is also considered. International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 1. INTRODUCTION Natural disasters such as earthquake though not new strike nations, countries and this is causing massive damages, generating sufferings for habitats and creating negative impacts on economies worldwide. Disaster is a tragic interruption to the development process. Many lives are not spare, capital investments are demolished and social networks are interrupted. Disaster permeates every corner of human settlement. It does not spare the rich neither does it spares the poor. It affects individuals, businesses and organizations. The economic losses due to disaster can be both direct and indirect [2]. These disasters are so prominent in the areas vulnerable to heavy volcanic and seismic activity and in the coastal regions where life-threatening weather actions and flood are likely to occur 15 DOI : 10.5121/ijmit.2016.8202 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 with growing frequency. Humanitarian crises will likely increase in such tensed condition, assistance are usually got worldwide from individual and private bodies including the United Kingdom military [2]. Disasters nowadays such as earthquakes are both natural and artificial which are caused by human as they are normally measured to be events on the spatial and sequential parameters such as wildfires, public unrests, terrorist attacks, epidemics, oil spillages, famine [2] and this turned most inhabitants to internally displaced person (IDP). Effect of the earthquakes could result in the collapse of communications infrastructure, antennas, buildings, power are always affected etc. But, immediately after the disaster, due to the emergency, communications are vital. There are two aspects in this scenario, the first is to instantly trace the affected persons in order to offer emergency assistance and the second scenario is to provide earthquakes relief operations during and after the incident. It should be noted that most of the local communication infrastructures might not be unavailable in both described scenarios. A typical example is the destructions of some base stations by the earthquakes [2]. The most rewarding aspect of the technology is that government and the public have started to appreciate and accept e-Service as the values are being demonstrated considering the effectiveness, cost cutting, accountability and transparency [17]. E-Service and mobile technology will provide the avenue for developing new services that will enhance the disaster relief operations and aid in quick and effective re-settle of the displaced citizens. 2.1 INTRODUCTION There are related research papers on e-service and mobile technology, disaster relief operations such as relief operations for the earthquake’s victims [7]; [14]; [16]; [18]. The roles of non- governmental organisations (NGOs), public and private organisation’s in giving assistance to the earthquake victims and the equipment needed to facilitate the operations will be discussed. At present, there is no research paper that specifically and majorly focus on E-Service and Mobile technology in earthquakes relief operations, this shows the important of this study within the research domain. 1. INTRODUCTION This paper reviews the roles of E-Service and Mobile Technologies in disaster relief operations and how the internally displaced person could benefit from these services. The limitations of these services will be discussed and how the technology could be used to further predict natural disaster is also considered. The paper has a well-laid structure; they are Introduction, Literature Review, Research Method, Finding and Analysis, Limitations, Technology in Future Prediction, Conclusion and Future Work, Acknowledgement and Reference. 2.2 E-SERVICE AND MOBILE TECHNOLOGY The interactive information service nature of E-Services have made it broadly accepted as the services are being provided electronically or via internet based equipment [17]. The accessibility of these services through mobile for organisations and consumers to use information accessed in creating a better experience [17]. 16 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 The effective delivery of E-Services electronically and through the use of mobile has smoothed the interaction among individuals, organisations, general public and earthquake’s relief operations staff [17]. While the aims of disaster relief are to alleviate the results of calamity, safe life, limit damages and the restoration of important services to some level, the need e-Service and mobile tools to succeed in the operations. The services also help both the private relief organisations and the local authority to quickly resettle the displaced or injured citizens in any natural disaster. Instead of the internally displaced person (IDP) who is forced to flee his or her home but who remains within his or her country's borders, the can be resettled within the shortest period with the availability of e-Service and mobile equipment [15]. 2.3 DISASTER RELIEF OPERATIONS TECHNOLOGY - EARTHQUAKES The disaster which could be termed as an event causes injuries, destructions, hardships even death to people, it could be natural or man-made disasters and regardless of the causes, it bring sadness to people [18]. Among the most devastating natural disaster in the world today, earthquakes are known as a leading natural disaster. According to [18], earthquakes in the society happen when the enormous plates that make up the earth’s surface slip over or crash into each other. Moreover, [14] substantiated the claims in their paper analysis about the two earthquakes that occurred in Indonesia in 2009. The earthquakes of 7.6 and 6.2 magnitude on the Richter scale smashed off the coast of West Sumatra occurred within 22 minutes apart and it affected estimated inhabitant of 1.2 million people. In Africa, despite the continents are being the most rapidly urbanizing nations, the disaster is now increasingly becoming a phenomenon worldwide [16]. The bottom line here is that the strongest earthquakes could open a deep crack in the earth’s surface causing massive and destructive landslides. It can tear down buildings and when this happen, it could be difficult to contain as only a few people who are lucky enough may survive it [18]. The use of e-service and mobile technology will help in giving quick relief and it will reduce the loss of many lives. The next section of this paper will review various E-Service and Mobile Technology tools, their use, approach and effectiveness in disaster relief operations, they are: 2.31 GPS Global Positioning Systems are the inexpensive hand-held tools that are based on satellite technology. It consists of a network of twenty-four orbiting satellites that are eleven thousand nautical miles in space and in six different orbit paths [12]. They use signals from three or more satellites rotating around the earth to track down the geographical location (in terms of latitude and longitude coordinate) of a person, vehicle, house, road etc. anywhere on the earth during earthquakes relief operations [8]. The satellites are constantly moving, making two complete orbits around the earth in less than twenty-four hours. Most GPS receivers can store up to hundred data recordings. In order to display accurate maps at the end of the day, GPS data needs to be applied to Geography Information System (GIS). It is on the record that the first GPS satellite was launched in February 1978 with the transmitter power of about fifty watts [1]. 17 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 LUETOOTH 2.32 BLUETOOTH Bluetooth is a short-range (up to 10m) radio technology for Wireless Personal Area Networking (WPAN-802.15) operating in the 2.4 GHz ISM (Industrial-Scientific-Medicine) frequency band. It was developed by Ericsson and originally intended to replace cables. A total of 79 RF channel is present where the raw data rate is 1Mbits/s. “A time division multiplexing (TDD) technique divides the channel into 625µs slots and with a 1 Mbits/s symbol rate a slot will transmit up to 625 bits [13]”. A Bluetooth Personal Area Network consists of piconets. Every piconet is a cluster of up to eight Bluetooth devices. The devices are either a master or a slave according to their designation. Master device will intermittently poll the slave device and slave is allowed to transmit only after receiving the poll from the master. The master for a specific set of connections is defined as the device that initiated the connections [13]. During the earthquakes relief operations, two piconets can be connected through a common Bluetooth device, gateway or bridge, to form a scatter net. The interconnected piconets will form a backbone for the Mobile Ad Hoc Networks (MANET), and can enable devices which are not communicating with each other directly, or out of range of another device, to exchange data through several hops in the scatter net [13]. 2.33 VSAT VSAT stands for Very Small Aperture Terminal. VSATs have the capability of simultaneously handling multiple telephone calls and providing a connection to the Internet, they are therefore ideally suited to larger scale operations such as when relief gets underway. TDM/TDMA star network is the most common VSAT configuration, with a high bit rate outbound carrier (TDM) from the hub to the remote earth stations. Also, it has one or more low/medium bit rate Time Division Multiple Access (TDMA) inbound carriers. Interactive VSAT technology is appropriate for any organization with centralized management and data processing considering its star configuration network architecture, and this is very useful during the earthquakes relief operations [2]. 2.35 GIS Geographic Information Systems (GIS) and Remote Sensing is considered to be an improvement in Information Technology in the form of Internet, GIS, Remote Sensing, satellite communication, etc. It plays important roles in the planning and implementation of hazards reduction measures in earthquakes relief operations. GIS can improve both the power of analysis, quality of natural hazards assessments, guide development activities, assist planners in the selection of mitigation measures during the implementation of emergency preparedness and response action. Furthermore, remote sensing is a tool that effectively contribute towards the identification of hazardous areas, it helps in monitoring the planet for change on a real time basis and early warning is giving to many impending earthquake disasters [1]. 3. RESEARCH METHOD Research Methodology approach in information system has been given a lot of discussions, as making a good approach choice by the researchers will have a major impact on the study. Most researchers in the literature review made use of either the qualitative or quantitative approach while some researchers prefer combining both approaches in their studies known as mix-method to obtain better results which could also serve as triangulation in evaluating results of their studies [3]. In the light of the above, the researchers, therefore, choose to use systematic literature review. The method of investigating relevant conference papers and journals from many research engines including science direct and google scholar was adopted as there is a need to use a robust search method which led to the use of systematic literature review. Researchers adopted the literature review analysis and techniques used by [22] because of the uniqueness in the process, synthesis and participation in presenting evidence. 2.34 RFID The Radio Frequency Identification (RFID) technology is a technique to remotely store, retrieve data using a small microchip with an antenna called the RFID tag [5]. Each tag holds a unique identification and other information. This makes the identification, authentication of objects and people possible without any physical contact between a tag and its reader during earthquakes relief operations. This also uses radio waves to read an object’s marking in the form of a unique identifying number stored on the embedded or attached silicon chip [5]. An RFID system includes three components: 1. A tag is also known as transponder located on the object to be identified [11]. 2. An interrogator (reader) which may be read or write/read device, and 3. An antenna that emits radio signals to activate the tag and read/write data to it. [11]. 18 18 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 Operating frequency ranges could be used to distinguished RFID systems. There are four frequency ranges: • Low (LF) frequency band -125KHz and 134.2KHz • High Frequency (HF)band-13.56MHz • Ultra High Frequency (UHF)band- 869MHz, 915MHz, 950MHz • Microwave band -2.45GHz and 5.8GHz [21]. • Microwave band -2.45GHz and 5.8GHz [21]. RFID system can be further classified into two categories, depending upon their source of electrical power-Active and passive. Active RFID tags contain their power source, usually an onboard battery. Power is obtained from passive tags from the signal of an external reader [23]. 4. FINDING AND ANALYSIS The finding and analysis section provide the authors the opportunity to elaborate further how these technologies emphasised in the literature review are used in disaster relief operations. They are: 19 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 4.1 GPS can be used in many ways during disaster relief operations which inclu 4.1 GPS can be used in many ways during disaster relief operations which includes: 4.1 GPS can be used in many ways during disaster relief operations which includes: 4.11 Tracking: GPS is used to track people in disaster operations so that an accurate estimation of people affected by the disaster could be collated. This device was used by the “U.S army where they developed a GPS Truth Data Acquisition, Recording, and Displaying System (TDARDS) for its operation and it helped in tracking down personnel during earthquakes relief operations” [4]. This mobile technology GPS-based tracking system uses up-to-date GPS data, radio data link, and computer technology to provide highly accurate, real-time, time-space position information (TSPI) on up to ten test objects such as ground vehicles, helicopters, and fixed-wing aircraft [4]. The system is highly flexible and is built with commercial off-the-shelf hardware, it is easily adjustable to meet any special needs of individual testing and tracking applications during earthquakes relief operations [4]. 4.12 Rescue and Emergency Response: GPS is used to determine the location of casualties during earthquakes relief operations which make the emergency response teams to reduce response time. The US air force for an example developed a GPS-based technology called a Combat Survivor Evader Locator (CSEL) system, this new technology integrates the GPS receiver with a communication radio so that search and rescue teams can trace downed aircrew members faster and more accurately to give immediate assistance [1]. The GPS receivers will locate the survivors during earthquakes relief operations and at the same time allows the rescue and emergency teams to obtain physical location information such as latitude, longitude, altitude, velocity, and precise time traceable to Coordinated Universal Time (UTC) [6]. 4.2 Bluetooth: The continued call for the use of wireless technology in relief operations has been on the increase especially with recent disasters such as terrorist attacks on September 11, Tsunami [24]. Wireless ad-hoc networking such as Mobile Ad Hoc Network (MANET) could be effectively deployed in the lack of fixed communication systems [24]. 4. FINDING AND ANALYSIS Ad-hoc networking is defined as communication concept in which users willing to communicate with each other form a temporary network without help or existence of any infrastructure or centralized administration [24]. The variety of nodes that can be used in an ad hoc network is great for PDAs, laptops, as well as medical equipments, sensors, actuators, etc. The node has a wireless access interface- Bluetooth, WLAN, HiperLAN2, for communication, Bluetooth features are very useful in setting up ad hoc networks [10]. The possible ways in which the blue technology can be employed in disaster situation are as follows: Telemedicine - Information regarding a victim like his present state can be communicated to an expert via the satellite communication device on the ambulance to seek advice. Telemedicine - Information regarding a victim like his present state can be communicated to an expert via the satellite communication device on the ambulance to seek advice. It can help in locating survivors trapped in the rubble of structural collapse during the earthquakes relief operations through sensitive listening devices or through signals from their mobile phones. It can help in locating survivors trapped in the rubble of structural collapse during the earthquakes relief operations through sensitive listening devices or through signals from their mobile phones. The Bluetooth technology can also be used by the rescue team to transmit audio and voice data to communicate with each other [9]. Other possible application of Bluetooth technology can be ambulatory wireless medical monitoring. It can help in connecting medical monitoring devices to a patient wirelessly. This Other possible application of Bluetooth technology can be ambulatory wireless medical monitoring. It can help in connecting medical monitoring devices to a patient wirelessly. This 20 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 would make the patient more at ease. This also reduces the risk of cable’s infection in earthquakes relief operations. would make the patient more at ease. This also reduces the risk of cable’s infection in earthquakes relief operations. 4.3 Very Small Aperture Terminal (VSAT): Remote user sites have a number of personal computers, dumb terminals and printers connected to the VSAT terminal, which connects them to a centralized host computer either at the organization’s head office or data processing centre in a typical VSAT network settings. 4. FINDING AND ANALYSIS VSAT terminal receives data from the DTEs and later buffered, transmitted to the hub in packets [2]. It has the capability of simultaneously handling multiple telephone calls during the earthquakes relief operations and it provide a connection to the internet in order to retrieve and collect data from other partner relief agency during this operation. They are therefore ideally suitable for large scale operations such as when relief assistance gets underway [2]. In practice, they can as well form the basis of a communication centre or ‘Cyber Café’ which many different agencies and other workers can make telephone calls and connect to the internet during relief operations [2]. Internet bandwidth can be provided to a site at any data rate required. Telephone traffic can be routed in and out of the International Public Switched Telephone Network (PSTN). Alternatively, a privately directed link can be established between the site and the coordination centre or NGO’s headquarters during relief operations [2]. As a result of the above, there are two types of satellite internet connections namely: One-Way satellite internet connection Two-Way satellite internet connection The One-way connection is also known as offline connection because in one way we can only download the files like, movies, pictures, sounds etc. Meanwhile, in a one-way system, internet content like webpage could be distributed by "pushing" them out to local storage at end user sites [2]. The Two-way satellite internet connection service is absolutely autonomous setup and it does not require any IP connectivity from the subscriber. It can provide satellite internet access anywhere within the satellite coverage map. This is best explained that almost in any point on the planet, there is a chance to get a high-quality, affordable broadband Internet access [2]. The two-way satellite internet access subscribers are required to purchase and install the earth station equipment which is generally known as VSAT (Very Small Aperture Terminal) equipment [2]. The VSAT earth station, consequently, creates two-way radio circuit with the satellite spacecraft and is used both for downloading and uploading data from and to the internet [2] as shown in Fig. 1 below. 21 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 ternational Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 Figure 1. Two-way Satellite Internet [2] Figure 1. 4. FINDING AND ANALYSIS Two-way Satellite Internet [2] 4.4 RFID: The following are the potential applications of RFID technology during earthquakes relief operations: 4.4 RFID: The following are the potential applications of RFID technology during earthquakes relief operations: 4.41 Victim and resource tracking: Rescue personnel can be supplied with PDA’s equipped with radio-frequency (RF) cards and interrogator attachments. They can attach the RF tags to victims during earthquakes relief operations. This enables instantaneous tracking of victims. In addition RF tags can be attached to resources like equipments, supplies, responding personnel etc., to track them as well. These tags can also be used in the hospitals and emergency centres to keep track of medical equipments and medicines [21]. 4.42 Medical care in hospitals: Paramedics on the field can use identification tags to store primary data about his medical condition during earthquakes relief operations and upload this data to a hospital or an emergency centre. Whenever the victim arrives at the emergency centre, he can get appropriate medical care as data about him can be accessed through his identification tag [21]. 4.43 Location of disasters survivors: RFID tags can be used to track missing persons and disasters survivors. Recently, the Japanese government has decided to sprinkle RFID-tagged sensors over disaster-prone areas. The sensors scattered in disaster areas will make up a mesh network that shall detect heat and vibration in a particular place [21]. 4.5 Geographic Information Systems (GIS) and Remote Sensing: This device can be used effectively in any earthquakes relief operation by providing the first-hand information about the 4.5 Geographic Information Systems (GIS) and Remote Sensing: This device can be used effectively in any earthquakes relief operation by providing the first-hand information about the 22 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 damage magnitude, the areas affected and to give direction to the rescue and earthquake relief operations staff [8]. In a hypothetical earthquake event, the first information that would be needed is the location of the epicentre and the extent of the worst affected areas. According to [8], the Internet GIS and its applications would assist the emergency managers to have the knowledge and map of the affected area with other statistics such as a number of houses, the population, estimation of the causalities and damage were done. 4. FINDING AND ANALYSIS The online information stored becomes widely available to the concerned agencies and people, and with interconnections through a wide area network, control rooms can be established [8]. Hazard mapping as GIS applications in the field of risk assessment will assist in showing earthquakes, landslides, floods or fire hazards [8]. Cities, districts or even countries and the tropical cyclone could use this map. Remote sensing makes an observation of any object from a distance and without coming into actual contact. Remote sensing can gather data much faster than ground-based observation, can cover a large area at one time to give a synoptic view [8]. Remote sensing comprises aerial remote sensing which records information such as photographs and images from the sensor on aircraft, satellite remote sensing which consists of the remote sensing system can be used to integrate natural hazard assessments into development planning studies [8]. These are SPOT Satellite, Landsat, Satellite Radar System, and advanced very high-resolution radio [1]. 5.2 BLUETOOTH: The following are some of the numerous limitations of Bluetooth technology, they are: 5. LIMITATIONS E-Service and Mobile Technology tools in earthquakes relief operations have setbacks just like many other types of equipment and research studies. However, despite the important roles these tools offer in disaster relief operations, they have the following limitations. 5.1 GPS: There are many technical limitations facing GPS as a technology used in disaster relief operations which includes: 5.11 Multipath: This is as a result of the radio signal being reflected off an object. Multipath also causes ghost images on a television set, though not common on television technology of today as most new televisions make use of cable and not old days antennas [12]. Multipath is caused when the signal bounces off a building before the GPS receiver’s antenna and could add errors to the overall positioning of GPS receiver [12]. 5.12 Satellite Geometry: The location of satellites relatively to each other from the perspective of the GPS receiver is called satellite geometry. Satellite geometry may be poor if GPS is locked into four satellites and all facing north r west of the receiver [12]. This is due to the fact that all the distance measurements coming from the same general direction, which makes triangulation be poor and the common area where these distance measurements intersect are fairly large [12]. 5.13 Positioning: GPS positioning information occasionally contain errors [1]. GPS errors primarily depend on both physical and artificial errors such as selective availability [1]. 5.2 BLUETOOTH: The following are some of the numerous limitations of Bluetooth technology, they are: 23 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 5.21 Capacity Limitations: The interference in Bluetooth wireless communication should increase the number of piconets. This is so because Bluetooth communication uses a frequency hopping system that hops randomly from amongst a total of 79 frequencies. As the number of piconets increase, the probability that two frequencies in adjacent piconets are the same increases. This leads to packet collisions requiring retransmission [19]. 5.22 Device Discovery in Bluetooth: In a Bluetooth environment the procedure of device discovery must be performed before communication can be done. The device discovery includes inquiry and paging procedures. Sometimes it can take a long time before devices are discovered and data transmission begins [19]. 5.23 Low Battery life: Bluetooth devices normally have a very short a battery life. 5.24 Short-range: Bluetooth is a short-range technology, it can work up to a range of 10m. 5. LIMITATIONS 5.3 Very Small Aperture Terminal (VSAT): The principle perceived drawback is that there is an ongoing cost element for using the service. The limited battery power of laptops, satellite mobile phones and other handheld devices issues about power for VSAT devices [2]. One of the few drawbacks with satellite internet is that there is high latency or a transmission delay. The distance between the satellite and end user is usually around 22,000 miles compared to the distance between antenna and end user [2]. The satellite's transmissions must traverse this distance coming from the user and go back down to the hosting facility. This usually causes a delay of around 1 second [2]. 5.4 RFID: This technology has the following limitations, they are: 5.4 RFID: This technology has the following limitations, they are: The carrier frequency bands for RFID systems are limited. The read range of RFID tags is too small. RFID technology has a low tolerance to fluid and metal environments. RFID technology has a low tolerance to fluid and metal environments. Reader collision occurs when two readers simultaneously try to read the tags [11]. 5.5 Geographic Information Systems (GIS) and Remote Sensing: A major problem with Geographic Information System getting an accurate date and it is being reported [8]. Vital and accurate information about victims of earthquakes disaster could be difficult to obtain and this is a big setback for the relief operations [8]. 6. TECHNOLOGY IN PREDICTING FUTURE NATURAL DISASTER To achieve the aim of fast and reliable natural disaster prediction such as earthquakes, a software utility is needed that must be able to do many things. The effects of disaster must be displayed pictorially on geo-referenced maps, photographs, satellite images etc. to determine their spatial and temporal extents [20]. Therefore, researchers will review the Consequences Assessment Toolset technology that can be used in prediction of further natural disaster. 24 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 ternational Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 6.1 CATS: The Consequences Assessment Toolset was created in response to the need to predict damage and analyse consequences from natural and technological disasters. According to [20], CATs was created by the science applications international corporation (SAIC) in conjunction with the Federal Emergency Management Agency (FEMA) and the Defence Threat Reduction Agency (DTRA) in America. CATs, a user-friendly and PC-based software package offers a wide range of capabilities includes the latest communications, satellite, and weather analysis technologies, computer modelling of natural and technological hazards, and a Geographic Information System (GIS) [20]. The CATs system combines damage prediction models and databases in a customised GIS as displayed in Fig. 2 below to allow for a broad range of consequence assessments. It takes advantage of existing ground-based and satellite communications networks for receiving hazard/disaster warning messages, meteorological data, and site reports; and for distributing the hazard/disaster damage predictions and estimated resources reports to the local area commanders and emergency operation centres [20]. Figure 2. Conceptual View of powerful Analysis Tool (CATs) [20] Figure 2. Conceptual View of powerful Analysis Tool (CATs) [20] 7. CONCLUSION AND FUTURE WORK Earthquake disasters cannot be prevented, if the accurate procedures in giving proper warnings and disaster after recovery are used, the effects could be reduced. More research work is required to establish more advanced ways in which the use of E-Service and Mobile technology is encouraged in disaster relief operations. Future research is needed to identify and implement cost- effective and timely earthquakes disaster relief operations worldwide. Decent leadership management, awareness and availability of e-services and mobile technology devices are recommended in order to have a more robust and effective earthquakes disaster relief operations [16]. More indicators of good governance could be added to these ideas for a new realistic finding and further acceptable outcomes. A secured and effective Geographical information system (GIS) database is needed for environmental and disaster such as an earthquake, this will 25 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 aid effective monitoring and management [16]. Above all, a strong political will and technological knowledge are required to cope with current disaster relief operations. aid effective monitoring and management [16]. Above all, a strong political will and technological knowledge are required to cope with current disaster relief operations. aid effective monitoring and management [16]. Above all, a strong political will and technological knowledge are required to cope with current disaster relief operations. ACKNOWLEDGEMENT Special thanks to the co-authors for their usual assistance, School of Computing, the University of Portsmouth, United Kingdom, Tai Solarin University of Education, Ijagun, Nigeria and Michael Otedola College of Primary Education, Noforija, Epe, Nigeria for providing the enabling environment for this research study. REFERENCE [1] Bloom G. (2002) The Right Equipment in Working Order, World Health forum, Vol. 10, pp. 3-27, 2002 [2] Chris Bell (2003) The Role of Satellite Communications in Disaster Management, Journal Presented at African Aid, Disaster Management and Relief, Johannesburg. [3] Creswell J.W (1999) Research Design: Qualitative, Quantitative and Mixed Methods Approaches. (Third Edition) SAGE Publication. [4] Gilkey J, Galijan R, Palomino A, (1994) The Army GPS Truth Data Acquisition, Recording, and Display System (TDARDS) at the White Sand Missile Range (WSMR). [5] Günther, O ( 2005) RFID: Tagging the world Source Communications of the ACM Archive, Communications of the ACM, Special issue, Volume 48 Issue 9. [6] Hofmann-Wellenhof .B, Lichtenegger. H and Collins. J, (1997) Global Positioning System: Theory and Practice, 4th edition, Springer-verlag, New York. [7] JDP (2008) Disaster Relief OPERATIONS, Joint Doctrine Publication 3-52 (JDP 3-52), 2nd Edition. [8] Jensen, J. R., Hodgson, M. E., Garcia-Quijano, M., Im, J., & Tullis, J. A. (2009). A remote sensing and GIS-assisted spatial decision support system for hazardous waste site monitoring. Photogrammetric Engineering & Remote Sensing, 75(2), 169-177 [9] Kargl, F; Ribhegge, S; Schlott, S; and Weber, M (2003) Bluetooth-based ad-hoc networks for voice transmission. System Sciences, Proceedings of the 36th Annual Hawaii, International Conference on 6-9 Jan 2003 Page(s):9 pp. [10] Krco, S; Dupcinov, M and Cooney, K (2001) Initial Experiences in Building an Ad-hoc Test Network - Production Of the TELFOR. [11] Loc Ho, Melody Moh, Zachary Walker, Takeo Hamada, Ching-Fong Su (2005) A Prototype on RFID and sensor networks for elder healthcare, Proceeding of the 2005 ACM SIGCOMM. [12] Marrow, R.H and Smith P.G, (1990) Field Trials of Health Interventions in Developing Countries, A Toolbox, Macmillan. [13] McDermott-Wells, P., (2005) “What is Bluetooth?” Potentials, IEEE Volume 23, Issue 5, Page(s):33 – 35 26 International Journal of Managing Information Technology (IJMIT) Vol.8, No.2, May 2016 [14] Moroney, Jennifer D. P; Stephanie Pezard; Laurel E. Miller; Jeffrey Engstrom and Abby Doll (2013) Lessons from Department of Defence Disaster Relief Efforts in the Asia-Pacific Region, The RAND Corporation. [15] Oduwole Tajudeen. A and Fadeyi Adebayo. O (2013) Issues of Refugees and Displaced Persons in Nigeria, the Journal of Sociological Research, ISSN: 1948-5468, Vol. 4, Number 1. [16] Pelling, M., & Wisner, B. (2012) Disaster risk reduction: Cases from urban Africa. Routledge. [17] Rowley, J. (2006) An analysis of the E-Service Literature: Towards a Research Agenda Internet Research, 16(3): 339-359. REFERENCE [18] Roza, Greg (2007) Earthquake: True Stories of Survival, Published by Rosen Group, New York, first edition. [19] Shorey, R and Miller, B.A (2000) The Bluetooth technology: merits and limitations. Personal Wireless Communications, IEEE International Conference, Page(s):80 – 84 [20] Swiatek, Joseph A. (1999) Hazards Assessment and Crisis Prediction Disaster Management, The Consequences Assessment Tool Set (CATS). [21] Tedjini, S; Tan-Phu, Vuong; Vincent Beroulle (2005) Antennas for RFID tags, Proceedings of the 2005 Joint Conference on Smart Objects and Ambient intelligence, Innovative Context-aware Services, Usages and Technologies. [22] Watson, R. and Webster, J. (2002) ‘Analysing the Past to Prepare for the Future; Writing A Literature Review’. MIS Quarterly, 26(2). [23] Weinstein, Ron (2005) RFID: A Technical Overview and Its Application to the Enterprise, Published by the IEEE Computer Society, Volume 7, Issue 3, Pages 27 – 33, [24] Zussman, G.; Segall, A (2003) Energy Efficient Routing in Ad-hoc Disaster Recovery Networks. INFOCOM 22nd Annual Joint Conference of the IEEE Computer and Communications Societies. Volume 1, Pages 682 - 691 AUTHORS Mr. Kazeem Oluwakemi OSENI is a PhD Candidate in the School of Computing, University of Portsmouth, United Kingdom (E-mail: Kazeem.oseni@port.ac.uk) Dr. Kate DINGLEY is a Principal Lecturer in the School of Computing, University of Portsmouth, United Kingdom (E-mail: Kate.dingley@port.ac.uk) Dr. Penny HART is a Senior Lecturer in the School of Computing, University of Portsmouth, United Kingdom (E-mail: Penny.hart@port.ac.uk) Mrs Ayoade Iyabode ADEWOLE is a lecturer in the Department of Mathematics, Tai Solarin University of Education, Ijagun, Nigeria (Email: hayorhade2005@gmail.com) Mr. Oluwakayode Abayomi DAWODU is a lecturer in the Department of Computer Science, Michael Otedola College of Primary Education, Noforija, Epe, Nigeria (Email: dawoolami33@gmail.com) 27 27
https://openalex.org/W2802563695	https://escholarship.org/content/qt5fj8r18r/qt5fj8r18r.pdf?t=pfm3b6	English	null	Predictors for patients understanding reason for hospitalization	PloS one	2,018	public-domain	5,190	UCLA UCLA Previously Published Works Title Predictors for patients understanding reason for hospitalization Permalink https://escholarship.org/uc/item/5fj8r18r Journal PLOS ONE, 13(4) ISSN 1932-6203 Authors Weerahandi, Himali Ziaeian, Boback Fogerty, Robert L et al. Publication Date 2018 DOI 10.1371/journal.pone.0196479 Peer reviewed UCLA UCLA Previously Published Works Title Predictors for patients understanding reason for hospitalization Permalink https://escholarship.org/uc/item/5fj8r18r Journal PLOS ONE, 13(4) ISSN 1932-6203 Authors Weerahandi, Himali Ziaeian, Boback Fogerty, Robert L et al. Publication Date 2018 DOI 10.1371/journal.pone.0196479 Peer reviewed Title Predictors for patients understanding reason for hospitalization Himali Weerahandi1,2, Boback Ziaeian3,4, Robert L. Fogerty5, Grace Y. Jenq6, Leora I. Horwitz1,2,7 1 Department of Medicine, NYU School of Medicine, New York, NY, United States of America, 2 Department of Population Health, NYU School of Medicine, New York, New York, United States of America, 3 Division of Cardiology, UCLA Medical Center, Los Angeles, CA, United States of America, 4 Division of Cardiology, Veteran Affairs Greater Los Angeles Healthcare System, Los Angeles, CA, United States of America, 5 Division of General Internal Medicine, Yale School of Medicine, New Haven, CT, United States of America, 6 Division of Geriatrics and Palliative Medicine, University of Michigan, Ann Arbor, MI, United States of America, 7 Center for Healthcare Innovation and Delivery Science, NYU Langone Medical Center, New York, New York, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Himali.Weerahandi@nyumc.org Powered by the California Digital Library University of California eScholarship.org RESEARCH ARTICLE Results Among 377 patients, 59.8% of patients completely understood their diagnosis. Bivariate analyses demonstrated that outpatient provider being aware of admission and having more than a day notice prior to discharge were not associated with patient understanding diagno- sis. Presence of jargon was not associated with increased likelihood of understanding in a multivariable analysis. Patients on housestaff and cardiology teams were more likely to understand diagnosis compared to non-teaching teams (OR 2.45, 95% CI 1.30–4.61, p<0.01 and OR 3.83, 95% CI 1.92–7.63, p<0.01, respectively). Data Availability Statement: Data are from the DISCHARGE study whose authors may be contacted at leora.horwitz@nyumc.org. Funding: At the time this study was conducted, Dr. Horwitz was supported by the CTSA grants UL1 RR024139 and KL2 RR024138 from the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH), and NIH roadmap for Medical Research, and was a Centers of Excellence Scholar in Geriatric Medicine by The John A. Hartford Foundation and the American Federation for Aging Editor: Iratxe Puebla, Public Library of Science, UNITED KINGDOM Primary outcome was complete understanding of diagnosis, based on post-discharge patient interview. Predictors assessed were the following: jargon on discharge instructions, type of medical team, whether outpatient provider knew if the patient was admitted, and whether the patient reported more than one day notice before discharge. Received: April 17, 2017 Accepted: April 13, 2018 Published: April 27, 2018 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. OPEN ACCESS To examine predictors for understanding reason for hospitalization. Citation: Weerahandi H, Ziaeian B, Fogerty RL, Jenq GY, Horwitz LI (2018) Predictors for patients understanding reason for hospitalization. PLoS ONE 13(4): e0196479. https://doi.org/10.1371/ journal.pone.0196479 Citation: Weerahandi H, Ziaeian B, Fogerty RL, Jenq GY, Horwitz LI (2018) Predictors for patients understanding reason for hospitalization. PLoS ONE 13(4): e0196479. https://doi.org/10.1371/ journal.pone.0196479 * Himali.Weerahandi@nyumc.org Methods This was a retrospective analysis of a prospective, observational cohort study of patients 65 years or older admitted for acute coronary syndrome, heart failure, or pneumonia and dis- charged home. Predictors for patients understanding reason for hospitalization Himali Weerahandi1,2*, Boback Ziaeian3,4, Robert L. Fogerty5, Grace Y. Jenq6, Leora I Horwitz1,2,7 Study cohort This study is a retrospective chart review of data collected from DISCHARGE. DISCHARGE was a prospective, observational cohort study of patients 65 years or older admitted to a single academic medical center for acute coronary syndrome, heart failure, or pneumonia between May 2009 and April 2010 who were subsequently discharged to home.[4,5,7] Additional eligi- bility criteria included speaking English or Spanish, not being in hospice care, and participat- ing in a telephone interview; caregivers could also take part on behalf of patients. Patients were excluded if they appeared delirious or failed the Mini-Cog mental status screen while admitted (defined as a score <3).[8] DISCHARGE was approved by the Yale’s Human Investigation Committee. Verbal informed consent was obtained from all study participants to participate in a telephone interview and included separate permission to the investigators to review their medical records. Verbal informed consent was approved by the ethics committee at Yale. Data was de-identified prior to data analysis by non-Yale collaborators. The DISCHARGE study included a telephone interview with patients or their caregivers within one week of discharge. The interview included questions about the reason for admis- sion and the amount of notice patients had prior to discharge. This interview was conducted by trained, non-clinical personnel. A medical record review was also conducted by experi- enced nurse abstractors. Predictors for understanding reason for hospitalization Introduction Research. Dr. Horwitz was also supported by the National Institute on Aging (K08 AG038336) and by the American Federation for Aging Research through the Paul B. Beeson Career Development Award Program. This work was also supported by a grant from the Claude D. Pepper Older Americans Independence Center at Yale University School of Medicine (P30AG021342 NIH/NIA). Dr. Weerahandi is supported by the NYU CTSA Grant UL 1TR001445 and KL2 TR001446. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Almost one in five Medicare beneficiaries are readmitted within 30 days of hospital discharge, making the transition from hospital back to community a particularly vulnerable time for patients.[1] Inpatient discharge typically occurs when patients have improved sufficiently to no longer require inpatient care. However, at this point the acute illness typically is not completely resolved and patients may still need additional care in the immediate post-dis- charge period. Patient education, including the reason for admission, helps prepare the patient for such post-discharge care requirements. Understanding diagnosis is an important part of framing a treatment and self-monitoring plan to a patient, and previous studies have demon- strated that patients’ understanding of their treatment plan impacts self-management behav- iors.[2] Poor understanding of the reason for admission may negatively affect patients’ ability to understand discharge instructions and their importance, and may impede patients from successfully carrying out discharge instructions. Thus, ensuring patients understand their diagnosis is an important area to target to reduce readmissions.[3] Competing interests: The authors have declared that no competing interests exist. We recently demonstrated that only about 60% of patients were able to accurately describe their diagnosis in post-discharge interviews,[4] and the majority of patients do not understand medication changes on discharge [5], which is consistent with prior studies.[6] The reasons for this failure, however, are uncertain. In order to effectively intervene in improving patient understanding of diagnosis and other post-discharge care requirements, it is essential to iden- tify systems-level, modifiable factors that affect patient comprehension. To examine systems-level contributors to patient understanding, we examined data from the DIagnosing Systemic failures, Complexities and HARm in GEriatric discharges study (DISCHARGE). Conclusions Non-teaching team patients were less likely to understand their diagnosis. Further investiga- tion of how provider-patient interaction differs among teams may aid in development of tools to improve hospital to community transitions. 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Measures Complete understanding of diagnosis was the primary outcome. Verified patient understand- ing of reason for hospitalization was performed by comparing patients’ responses to the PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 2 / 10 Predictors for understanding reason for hospitalization question “please tell me the reason you were in the hospital” with administrative billing data of principal diagnosis, the wording in the diagnosis section of the discharge instructions, and investigators’ assignment of the patient to the heart failure, pneumonia, or acute coronary syn- drome cohort. If the patient had several main diagnoses, description of any of them was defined as complete understanding. Patient understanding of discharge diagnosis was consid- ered to be complete if the patient’s language would make it clear to a medical professional what the diagnosis for the hospitalization was, even without technical language. For example, if a patient had been admitted for heart failure, we gave full credit for understanding both for responses such as “heart failure” and for “fluid in the legs” or “weak heart.” We gave partial credit for provision only of symptoms consistent with the diagnosis (such as “trouble breath- ing” for heart failure), and no credit for vague symptoms (“weakness,” “not feeling well”) or lack of knowledge (“don’t know,” “my doctor told me to come in”). For this study we dichoto- mized understanding as complete or not complete. Modifiable systems predictors assessed for complete understanding of diagnosis were the presence of jargon on patient discharge instructions, type of medical team, whether outpatient provider knew if the patient was admitted, and whether the patient had more than more than one day notice prior to discharge. Non-modifiable predictors were age, race, sex, diagnosis, education, and income. To define jargon on discharge instructions, every reason for hospitalization was recorded and categorized by study investigators. Disagreements were resolved through iterative discussion. Intelligible language was defined by any term that was commonly used in spoken English (e.g., pneumonia or heart attack), any medical jargon for chronic diseases that the investigators believed that patients would likely be familiar with (e.g., congestive heart failure or atrial fibrillation), and any medical jargon for acute events that was commonly used by patients in the study to describe their hospitalization (e.g., catheterization). Remaining terms were defined as medical jargon (S1 Appendix). Type of medical team (non-teaching, housestaff, or cardiology) was abstracted from the chart. Measures At this institution, non-teaching teams are composed of a hospitalist working with either a physician assistant or a nurse practitioner. Housestaff teams are composed of medical stu- dents, interns, residents, and attending physicians. The attending on the housestaff team may be either a hospitalist or primarily outpatient faculty physician. Cardiology teams are composed of a cardiologist and either housestaff, a nurse practitioner, or a physician assistant. Data on whether the outpatient provider knew if the patient was admitted and whether the patient had more than one day notice prior to discharge was obtained from the post-discharge interview. Data on age, race, and length of stay were obtained from hospital administrative databases. Statistical analysis The characteristics of the study population were summarized with descriptive analyses. We summarized dichotomous variables as proportions and continuous variables with a mean and standard deviation if they were normally distributed, if not, with a median and interquartile range. We examined the relationship of systems predictors to fully understanding/not fully understanding hospital diagnosis using a multivariable logistic regression model controlling for age, race, sex, diagnosis, education, and income. Data were analyzed using SAS, version 9.3 (SAS Institute, Cary, NC). Sample characteristics A total of 3743 patients over 64 years old were discharged home from the medical service at our medical center during the study period, and 3028 patients were screened for eligibility 3 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Predictors for understanding reason for hospitalization Fig 1. Flow diagram of enrolled patients. https://doi.org/10.1371/journal.pone.0196479.g001 Fig 1. Flow diagram of enrolled patients. https://doi.org/10.1371/journal.pone.0196479.g001 within 24 hours of admission. Screening identified 635 eligible admissions and we enrolled 395 patients (62.2%) in the study. Of these, 377 provided permission for chart review and there was data on understanding diagnosis on 374 patients (Fig 1). within 24 hours of admission. Screening identified 635 eligible admissions and we enrolled 395 patients (62.2%) in the study. Of these, 377 provided permission for chart review and there was data on understanding diagnosis on 374 patients (Fig 1). In this group, 222 (59.8%) patients completely understood the diagnosis. The mean age in both groups was about 77, and sex distribution was similar between the two groups (Table 1). Patients who did not completely understand their diagnoses were more likely to have been admitted for HF (50.7% vs 30.2%, p<0.01), have less formal education (p<0.01), and have a lower income (p<0.01). Predictors for understanding reason for hospitalization Table 1. Baseline characteristics of the study cohort (n = 374). Characteristic Completely understood diagnosis (n = 222) Did not completely understand diagnosis (n = 152) p-value Condition (n = 374) Acute Coronary Syndrome 125 (56.3%) 70 (46.1%) 0.05 Community-Acquired pneumonia 54 (24.3%) 36 (23.7%) 0.89 Heart Failure 67 (30.2%) 77 (50.7%) <0.01 Age, mean (SD) (n = 366) 76.9 (7.6) 77.0 (7.5) 0.98 Male sex (n = 366) 123 (56.2%) 78 (53.1%) 0.56 English-speaking (n = 366) 212 (98.2%) 143 (97.3%) 0.47 Race/ethnicity (n = 369) 0.16 Non-Hispanic white 188 (85.8%) 116 (78.9%) Non-Hispanic black 18 (8.2%) 23 (15.7%) Hispanic 8 (3.7%) 7 (4.8%) Other 5 (2.3%) 1 (0.7%) Education (n = 365) <0.01 <9th grade 19 (8.8%) 21 (14.5%) 9th-12th grade 34 (15.7%) 22 (15.2%) High school diploma or GED 51 (23.5%) 53 (36.6%) College degree 78 (35.9%) 28 (19.3%) Graduate degree 35 (16.1%) 21 (14.5%) Yearly Income (n = 340) <0.01 0-$18,000 48 (23.5%) 51 (37.5%) $18,000-$30,000 25 (12.3%) 26 (19.1%) $30,000-$45,000 23 (11.3%) 8 (5.9%) $45,000-$65,000 19 (9.3%) 5 (3.7%) >$65,000 50 (24.5%) 20 (14.7%) No response 39 (19.1%) 26 (19.1%) https://doi.org/10.1371/journal.pone.0196479.t001 Table 1. Baseline characteristics of the study cohort (n = 374). compared with patients without jargon on their discharge instructions (70.5% vs 56.3% respec- tively, p = 0.01, see Table 2). As this was an unexpected finding, we performed a stratified anal- ysis by diagnosis to examine whether this relationship was disease specific. Stratified analysis by diagnosis demonstrated that HF patients with jargon were more likely to understand their diagnosis (66.7% vs 43.5%, p = 0.05). Most of these patients (57%) had “coronary artery dis- ease/unstable angina” listed on their discharge instructions. Jargon was not associated with understanding diagnosis for patients who had ACS (p = 0.34) or patients who had pneumonia (p = 0.76). Patients with jargon on their discharge instructions were more likely to be white, but otherwise had similar baseline characteristics compared to patients without jargon on their discharge instructions (S2 Appendix). In a multivariate model controlling for age, race, sex, diagnosis, education, income, and medical team, jargon was no longer a significant predictor for completely understanding diag- nosis (OR 1.53, 95% CI 0.83–2.82, p = 0.17, see Table 3). However, patients admitted with HF were still less likely to understand their diagnosis (OR 0.37, 95% CI 0.19–0.72, p<0.01). Predictors for understanding diagnosis In this cohort, 367 patients had a diagnosis written on their discharge instructions, which was coded for the presence of jargon. A bivariate analysis demonstrated that patients with jargon on their discharge instructions were more likely to completely understand their diagnosis PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 4 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Type of medical team was known for 363 patients. A bivariate analysis demonstrated that housestaff and cardiology team patients were more likely to completely understand their diag- nosis (62.5% and 70.8% respectively, see Table 2) compared to non-teaching teams (43.4%, p<0.01). This relationship remained significant in a multivariable analysis (housestaff OR 2.45, 95% CI 1.30–4.62, p<0.01; cardiology 3.83, 95% CI 1.92–7.63, p<0.01). While cardiology teams were more likely to have patients with ACS and less likely to have patients with 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Predictors for understanding reason for hospitalization Table 2. Bivariate analysis examining systems predictors to completely understanding hospital diagnosis. Predictor Completely understands diagnosis p value Jargon (n = 367) 0.01 Yes 67 (70.5%) No 153 (56.3%) Medical team (n = 363) <0.01 Non-teaching 46 (43.4%) Housestaff 75 (62.5%) Cardiology 97 (70.8%) Outpatient provider aware (n = 328) 0.63 Yes 168 (59.0%) No 22 (55.0%) More than one day notice prior to discharge (n = 374) 0.71 Yes 111 (60.3%) No 111 (58.4%) https://doi.org/10.1371/journal.pone.0196479.t002 Table 2. Bivariate analysis examining systems predictors to completely understanding hospital diagnosis. community-acquired pneumonia, the patients across these different teams had similar baseline characteristics (S3 Appendix). community-acquired pneumonia, the patients across these different teams had similar baseline characteristics (S3 Appendix). Data on whether outpatient provider knew whether or not the patient was admitted was available for 328 patients. Outpatient provider being aware of admission was not associated with patient completely understanding diagnosis (59.0% vs 55.0%, p = 0.64). Data on whether the patient had more than day notice prior to discharge was available on 374 patients. Having more than one day notice prior to discharge was not associated with completely understanding diagnosis (60.3% vs 58.4%, p = 0.71). PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Discussion In this single site study of patients admitted with ACS, pneumonia, and HF, our analysis found that jargon on discharge instructions, outpatient provider being aware of admission, and having more than one day notice prior to discharge were not associated with completely understanding diagnosis. However, non-teaching team patients were less likely to understand their diagnoses. While previous studies have revealed that a significant portion of patients do not understand their reason for hospitalization [4,6,9] this is the first study, to our knowledge, to explore systems-level, modifiable predictors for understanding reason for hospitalization. The use of medical jargon has been postulated to worsen physician-patient communica- tion and result in poor patient understanding of their medical condition.[10] This associa- tion was demonstrated in a study of diabetes patients by Schillinger et al.[11] However, in our multivariate analysis, we found no association between jargon on discharge instructions and lack of understanding of diagnosis. Schillinger defined jargon based on patient self- report (“How often did your regular doctor use medical words that you did not under- stand?”) while we defined jargon a priori based on specific terminology. We may have made Table 3. Association of jargon and medical team with completely understanding diagnosis (n = 336). Predictor Adjusted OR (95% CI) p-value Medical Team (compared to Non-teaching team) Housestaff 2.45 (1.30–4.61) <0.01 Cardiology 3.83 (1.92–7.63) <0.01 Presence of Jargon 1.53 (0.83–2.82) 0.17 https://doi.org/10.1371/journal.pone.0196479.t003 Table 3. Association of jargon and medical team with completely understanding diagnosis (n = 336). Predictor Adjusted OR (95% CI) p-value Medical Team (compared to Non-teaching team) Housestaff 2.45 (1.30–4.61) <0.01 Cardiology 3.83 (1.92–7.63) <0.01 Presence of Jargon 1.53 (0.83–2.82) 0.17 https://doi.org/10.1371/journal.pone.0196479.t003 Table 3. Association of jargon and medical team with completely understanding diagnosis (n = 336). 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Predictors for understanding reason for hospitalization incorrect assumptions about what type of medical language would be difficult for patients to understand. Specifically, “coronary artery disease” on the discharge instructions was associated with increased understanding, which we did not expect. Moreover, we based our definition of jargon on language in the patient discharge instructions. Patients are educated in many forms, including verbal discussions, and non-discharge instruction education may have contributed to patient understanding despite confusing language in the discharge instructions. Discussion We found non-teaching team patients were less likely to completely understand their rea- son for hospitalization compared to housestaff and cardiology teams. We hypothesize that patients on housestaff teams may benefit from being rounded on by multiple providers from the same team. This may result in more face-to-face time with the primary team and further reinforcement of the reason for hospitalization. Bedside teaching may also result in collateral education for the patient. There may be other unadjusted factors that may also be contributing, such as the patient’s clinical complexity, cognition, and health literacy. Further work should investigate how these different teams approach patient education. Our findings appear to be consistent with prior literature on hospitalists that has demon- strated that patients cared for by hospitalists were more likely to have emergency department visits and readmissions after discharge.[12–14] Our study adds to this body of literature, as these prior studies did not investigate factors that may be driving utilization after discharge. We have found that there is decreased understanding of diagnosis on non-teaching teams, and this may be driving this disparate utilization. Further studies should examine whether under- standing of diagnosis is linked with utilization. We investigated outpatient provider knowledge of patient admission, as patients whose out- patient providers knew about their hospitalization may represent those patients whose care was better coordinated, specifically with regards to diagnosis. If both inpatient and outpatient providers communicate to stay “on message” regarding the patient’s reason for hospitalization, this may enhance patients’ ability to understand why they were hospitalized. Outpatient pro- viders who know their patients are hospitalized may also visit or communicate with patients during hospitalization. Patients may feel more comfortable asking their regular outpatient pro- vider questions about their hospital course than their inpatient team. We did not, however, find this to be a significant association with understanding. Similarly, patients that had more than one day notice prior to discharge may have longer discharge preparation, which could present an opportunity for the patient to have more ques- tions answered about their illness before leaving. However, advance notice was also not associ- ated with better understanding. Our findings highlight the fact that even in cases in which the outpatient provider was aware of hospitalization and there was adequate notice for discharge, many patients still do not understand their reason for admission. PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Predictors for understanding reason for hospitalization in Ireland where the patient population may be different from that in the United States. In addition, that study did not differentiate between ACS and HF. Lower income and less formal education were other patient level factors that were associ- ated with not completely understanding diagnosis in a bivariate analysis. These socioeconomic factors were not significant in the multivariable analysis, which may reflect that systems-level factors may mitigate these factors with respect to understanding diagnosis. Further studies should investigate which educational interventions are effective in the inpatient setting, and if they can be generalized to different diagnoses. Limitations Our study has several limitations. This is a single site study at an academic center, which limits its generalizability. In addition, our study population was limited to only older patients who were admitted with either ACS, pneumonia, or HF, and thus our findings may not apply to younger patients or those who are admitted with other illnesses. Given that this is an older cohort, in-hospital delirium may affect patients’ ability to understand why they were admitted; however, this study excluded patients who failed a mental status screen. Data on whether the outpatient provider knew if the patient was admitted and if the patient had more than one day notice prior to discharge was obtained from the patient themselves, and may be inaccurate. Our assessment of complete understanding of diagnosis may be considered lenient, and over- estimate understanding. In addition, we did not control for the quality of communication patients received. There were an insufficient number of readmitted patients in this study to examine whether these predictors of interest were associated with readmission and we were also underpowered for jargon. Conclusions In summary, this study focused on systems-level, modifiable factors that may impact patients’ ability to understand why they were admitted. We found that non-teaching team patients were less likely to completely understand their diagnosis compared to housestaff and cardiology teams. Heart failure patients were also less likely to understand their reason for admission. These findings have quality improvement implications as potential targets to reduce read- missions. Further investigation of how provider-patient interaction differs among these groups may aid in the development of effective patient education tools to improve the transition from hospital to community. Discussion Several patient level factors were noted to be associated with not completely understanding diagnosis. In particular, we found HF diagnosis was associated with not completely under- standing diagnosis. We were surprised by this finding because we had hypothesized that patients might better understand chronic diseases, in which they have had more opportunity over time for education. In fact, the patients with HF who did understand their diagnosis appeared to have largely had concomitant ACS, even though the diagnoses written on their discharge instructions were largely classified as medical jargon. It may be that specific diagno- ses such as ACS may initiate an institutional-level cascade of patient education unique to that diagnosis, improving patient understanding. ACS patients also may be more likely to be admitted to an intensive care unit. Our analysis did not control for increased level of care dur- ing hospitalization. Our findings contrast with a previous study[9] that found cardiac diagno- sis was associated with poor understanding of diagnosis. However, this study was conducted PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 7 / 10 Supporting information S1 Appendix. Definition of intelligible terms for diagnosis section of patient discharge instructions. (DOCX) S2 Appendix. Baseline characteristics comparing patients with jargon on their discharge instructions to patients without jargon on their discharge instructions. (DOCX) S3 Appendix. Baseline characteristics comparing patients on hospitalist, housestaff and cardiology teams. (DOCX) 8 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 Predictors for understanding reason for hospitalization Acknowledgments At the time this study was conducted, Dr. Horwitz was supported by the CTSA Grant UL1 RR024139 and KL2 RR024138 from the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH), and NIH roadmap for Medical Research, and was a Centers of Excellence Scholar in Geriatric Medicine by The John A. Hartford Foundation and the American Federation for Aging Research. Dr. Horwitz was also supported by the National Institute on Aging (K08 AG038336) and by the American Fed- eration for Aging Research through the Paul B. Beeson Career Development Award Program. This work was also supported by a grant from the Claude D. Pepper Older Americans Inde- pendence Center at Yale University School of Medicine (P30AG021342 NIH/NIA). Dr. Weer- ahandi is supported by the NYU CTSA Grant UL 1TR001445 and KL2 TR001446. Author Contributions Conceptualization: Himali Weerahandi, Leora I. Horwitz. Conceptualization: Himali Weerahandi, Leora I. Horwitz. Data curation: Grace Y. Jenq, Leora I. Horwitz. Data curation: Grace Y. Jenq, Leora I. Horwitz. Formal analysis: Himali Weerahandi, Leora I. Horwitz. Formal analysis: Himali Weerahandi, Leora I. Horwitz. Funding acquisition: Leora I. Horwitz. Funding acquisition: Leora I. Horwitz. Funding acquisition: Leora I. Horwitz. Investigation: Himali Weerahandi, Boback Ziaeian, Robert L. Fogerty, Grace Y. Jenq, Leora I. Horwitz. Investigation: Himali Weerahandi, Boback Ziaeian, Robert L. Fogerty, Grace Y. Jenq, Leora I. Horwitz. Methodology: Himali Weerahandi, Leora I. Horwitz. Project administration: Robert L. Fogerty, Leora I. Horwitz. Resources: Robert L. Fogerty, Leora I. Horwitz. Resources: Robert L. Fogerty, Leora I. Horwitz. Software: Himali Weerahandi. Software: Himali Weerahandi. Supervision: Leora I. Horwitz. Supervision: Leora I. Horwitz. Writing – original draft: Himali Weerahandi, Leora I. Horwitz. Writing – original draft: Himali Weerahandi, Leora I. Horwitz. Writing – review & editing: Himali Weerahandi, Boback Ziaeian, Robert L. Fogerty, Grace Y. Jenq, Leora I. Horwitz. 5. Makaryus AN, Friedman EA. Patients’ understanding of their treatment plans and diagnosis at dis- charge. Mayo Clin Proc. 2005; 80: 991–994. S0025-6196(11)61579-6 [pii]. https://doi.org/10.4065/80.8. 991 PMID: 16092576 PLOS ONE \| https://doi.org/10.1371/journal.pone.0196479 April 27, 2018 References References 1. Jencks SF, Williams MV, Coleman EA. Rehospitalizations among patients in the medicare fee-for-ser- vice program. N Engl J Med. 2009; 360: 1418–1428. https://doi.org/10.1056/NEJMsa0803563 PMID: 19339721 2. Pearson M.L., Mattke S., Shaw R., Ridgely M.S., Wiseman S.H. Patient Self-Management Support Pro- grams: An Evaluation. 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Rehospitalizations among patients in the medicare fee-for-ser- vice program. N Engl J Med. 2009; 360: 1418–1428. https://doi.org/10.1056/NEJMsa0803563 PMID: 19339721 2. Pearson M.L., Mattke S., Shaw R., Ridgely M.S., Wiseman S.H. Patient Self-Management Support Pro- grams: An Evaluation. Final Contract Report (Prepared by RAND Health under Contract No. 282-00- 0005). Rockville, MD: Agency for Healthcare Research and Quality; 2007. 3. Kripalani S, Jackson AT, Schnipper JL, Coleman EA. Promoting effective transitions of care at hospital discharge: a review of key issues for hospitalists. J Hosp Med. 2007; 2: 314–323. https://doi.org/10. 1002/jhm.228 PMID: 17935242 4. Horwitz LI, Moriarty JP, Chen C, Fogerty RL, Brewster UC, Kanade S, et al. Quality of discharge prac- tices and patient understanding at an academic medical center. JAMA Intern Med. 2013; 173: 1715– 1722. 1729535 [pii]. https://doi.org/10.1001/jamainternmed.2013.9318 PMID: 23958851 5. Makaryus AN, Friedman EA. 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https://openalex.org/W3187329908	https://khidmah.ikestmp.ac.id/index.php/khidmah/article/download/346/289	Indonesian	null	PENYULUHAN MASYARAKAT MENGENAI EFEKTIVITAS PEMERIKSAAN RAPID TEST ANTIBODI DAN ANTIGEN DALAM MENDETEKSI COVID-19	Khidmah	2,021	cc-by-sa	2,250	Abstrak Jumlah kematian yang terkait dengan COVID-19 saat ini sangat melampaui dua corona virus sebelumnya (SARS-CoV dan MERS-CoV), dan wabah ini masih berlangsung, yang merupakan ancaman besar bagi kesehatan dan ekonomi masyarakat global. Berbagai teknik dapat digunakan untuk mengetahui risiko COVID-19 pada pasien dan memberikan bukti diagnosis infeksi secara akurat. Saat ini, pemeriksaan yang digunakan di Indonesia guna mendeteksi dan menangani infeksi COVID-19 meliputi Rapid Test Diagnostic (RDT) Antibodi dan/atau Antigen pada kasus kontak dari pasien positif. Tujuan pengabdian ini adalah untuk memberikan pengetahuan dan menambah wawasan masyarakat daerah PALI mengenai efektivitas dari pemeriksaan rapid test antibodi dan rapid test antigen dalam mendeteksi COVID-19. Metode yang digunakan adalah dengan memberikan materi kepada masyarakat Desa Pangabuan Kecamatan Tanah Abang Kabupaten PALI. Berdasarkan hasil penyuluhan kesehatan yang dilakukan didapatkan bahwa Masyarakat Desa Pangabuan Kecamatan Tanah Abang Kabupaten PALI Provinsi Sumatera Selatan lebih memahami dan bertambah pengetahuan mengenai pentingnya Pemeriksaan Rapid Test Antibodi dan Rapid Test Antigen dalam Mendeteksi Covid-19 dilihat dari hasil penilaian pre test dan post test yang mengalami peningkatan dari 20 responden yang mengikuti penyuluhan kesehatan tersebut. Kata Kunci: Antibodi, Antigen, COVID-19 Kata Kunci: Antibodi, Antigen, COVID-19 Abstract The current number of deaths associated with COVID-19 greatly exceeds the two previous coronaviruses (SARS-CoV and MERS-CoV), and the outbreak is still ongoing, posing a major threat to global public health and economy. Various techniques can be used to determine the risk of COVID-19 in patients and provide evidence of an accurate diagnosis of infection. Currently, the tests used in Indonesia to detect and treat COVID-19 infection include Rapid Test Diagnostic (RDT) Antibodies and/or Antigens in contact cases from positive patients. The purpose of this service is to provide knowledge and add insight to the people of the PALI area regarding the effectiveness of the rapid antibody test and antigen rapid test in detecting COVID-19. The method used is to provide material to the people of Pangabuan Village, Tanah Abang District, PALI Regency. Based on the results of the health education conducted, it was found that the Pangabuan Village Community, Tanah Abang District, PALI Regency, South Sumatra Province better understood and increased knowledge about the importance of Antibody Rapid Test and Antigen Rapid Test Examination in Detecting Covid-19 seen from the results of pre test and post test assessments that experienced an increase of 20 respondents who participated in the health education. 294 Keywords: Antibody, Antigen, COVID-19 Keywords: Antibody, Antigen, COVID-19 PENDAHULUAN Pada akhir tahun 2019, tepatnya pada bulan Desember, terjadi peningkatan kasus pneumonia di Wuhan, Hubei, China. Berdasarkan studi analisis yang dilakukan oleh para epidemiologis menunjukkan bahwa penyebaran penyakit ini diperkirakan terkait dengan Pasar Makanan Laut Cina Selatan di Wuhan. Pada bulan Januari 2020, World Health Organization (WHO) memberi virus ini nama baru yaitu SARS-COV-2 dan penyakitnya yaitu COVID-19 (Alsuliman, 2020). Jumlah kematian yang terkait dengan COVID-19 saat ini sangat melampaui dua corona virus sebelumnya (SARS-CoV dan MERS-CoV), dan wabah ini masih berlangsung, yang merupakan ancaman besar bagi kesehatan dan ekonomi masyarakat global. Berdasarkan bukti ilmiah bahwa COVID-19 dapat menular dari manusia ke manusia melalui percikan batuk/bersin (droplet), tidak melalui udara. Orang yang paling berisiko tertular penyakit ini adalah orang yang kontak erat dengan pasien COVID-19 termasuk yang merawat pasien COVID-19. Orang dengan gejala dan orang tanpa gejala memiliki peluang yang sama untuk menulari virus ini ke lingkungan sekitarnya. Sehingga salah satu cara terbaik mengendalikan penularan adalah dengan melakukan karantina mandiri atau isolasi di rumah (Rosyanti, 2020). Tingkat infeksi yang tinggi, metode diagnostik yang cepat dan akurat sangat diperlukan untuk mengidentifikasi, mengisolasi dan merawat pasien sesegera mungkin guna mengurangi angka kematian dan risiko penyebaran infeksi di populasi. Banyak uji diagnostik yang dapat digunakan untuk mendeteksi corona virus 2019 (COVID-19) yang telah tersedia sejauh ini, dengan lebih banyak memperoleh persetujuan darurat setiap harinya. Teknik uji molekuler lebih direkomendasikan daripada pemeriksaan klinis dan CT untuk diagnosis yang akurat dikarenakan dapat mengidentifikasi patogen target. Pengembangan teknik molekuler berkaitan pada pemahaman mengenai komposisi proteomik dan genom patogen serta induksi perubahan ekspresi protein / gen dalam inang selama dan setelah infeksi (Scohy et al., 2020). Dalam melakukan diagnosis diperlukannya uji diagnostik yang memiliki tingkat sensitifitas dan spesifisitas yang tinggi. Hal tersebut sangatlah penting dan krusial untuk mendeteksi dan mengendalikan infeksi COVID-19 dan juga berguna sebagai implementasi dalam penilaian, kontrol dan membatasi terjadinya penyebaran wabah 295 (Scohy et al., 2020). Pemeriksaan laboratorium diagnostik yang dapat dilakukan untuk mengidentifikasi Novel Corona virus SARS-COV-2 ini dapat dilakukan dengan tiga teknik pendekatan, yaitu pemeriksaan pengurutan genom (whole genome sequencing), Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) dan pemeriksaan serologi (Ahn et al., 2020). Saat ini, pemeriksaan yang digunakan di Indonesia guna mendeteksi dan menangani infeksi COVID-19 meliputi Rapid Test Diagnostic (RDT) Antibodi dan/atau Antigen pada kasus kontak dari pasien positif. Rapid Test (RT) Antibodi juga digunakan untuk deteksi kasus infeksi pada kasus suspek di wilayah yang tidak mempunyai fasilitas untuk pemeriksaan RT-PCR. MASALAH Berdasarkan analisis situasi di atas, disimpulkan permasalahan yang dihadapi oleh masyarakat saat ini adalah kurangnya pemahaman masyarakat tentang pemeriksaan rapid test antibodi dan antigen dalam mendeteksi Covid-19. Kondisi ini menyebabkan masyarakat masih bingung dalam mendeteksi Covid-19, dikarenakan adanya banyak metode dalam mendeteksi virus ini, selain itu biaya dalam pemeriksaan rapid test Covid-19 ini pun beragam. Dari beberapa metode yang digunakan, yang paling sering dilakukan adalah dengan cara rapid test antibodi dan antigen. Tetapi dalam masalah efektivitasnya kebanyakan masyarakat belum paham mana yang lebih baik dalam mendeteksi Covid-19. Sehingga dengan adanya penyuluhan ini diharapkan akan meningkatkan pengetahuan dan juga kesadaran tentang pentingnya pemeriksaan rapid test dalam mendeteksi Covid-19. METODE PELAKSANAAN Pelaksanaan kegiatan pengabdian kepada masyarakat ini dilakukan dengan metode pemberian materi kepada masyarakat Desa Pangabuan Kabupaten PALI sebanyak 20 orang. Tahapan kegiatan pengabdian kepada masyarakat dilakukan sebagai berikut: 1. Persiapan Kegiatan Persiapan dimulai dari penyusunan proposal kegiatan yaitu rancangan kegiatan yang akan dilakukan, pembagian tugas pada saat pelaksanaan kegiatan, dan surat perijinan kepada Kepala Desa di PALI. Selain itu persiapan pencetakan leafleat pemeriksaan rapid test antibodi dan antigen untuk mendeteksi Covid-19. Persiapan dimulai dari penyusunan proposal kegiatan yaitu rancangan kegiatan yang akan dilakukan, pembagian tugas pada saat pelaksanaan kegiatan, dan surat perijinan kepada Kepala Desa di PALI. Selain itu persiapan pencetakan leafleat pemeriksaan rapid test antibodi dan antigen untuk mendeteksi Covid-19. PENDAHULUAN Namun, hasil pemeriksaan RT Antibodi tetap harus dikonfirmasi dengan melakukan pemeriksaan RT-PCR (ECDC, 2020). Salah satu uji antibodi yang dapat dilakukan yaitu RTD antibodi dari virus COVID-19. Pemeriksaan ini sudah sangat umum digunakan dan diperjual belikan. RTD antibodi ini dilakukan dengan mendeteksi keberadaan antibodi di dalam darah individu. Deteksi antibodi ini juga dapat terjadi reaksi silang dengan patogen lainnya seperti jenis corona virus yang menyerang manusia lainnya sehingga memberikan hasil positif palsu. Tes ini berperan penting dalam membantu upaya penemuan vaksin namun tidak untuk diagnosis klinis dikarenakan tidak dapat diketahui apakah sedang berlangsung infeksi akut pada pasien tersebut dan berdampak pada pengambilan keputusan klinis. WHO tidak merekomendasikan penggunaan tes diagnostik cepat berbasis deteksi antibodi untuk perawatan pasien, namun tes ini dapat membantu dalam surveilans penyakit dan penelitian epidemiologis. Dikarenakan pemeriksaan ini sangat cepat dilakukan, maka dapat dilakukan di laboratorium rumah sakit dan mempersingkat waktu diagnosis (Alsuliman, 2020). Salah satu uji antigen yang sering digunakan saat ini yaitu RDT antigen. Salah satu alat yang digunakan yaitu COVID-19 Ag Respi-Strip (Coris Bio Concept, Gembloux, Belgium). Pemeriksaan ini dilakukan dengan mendeteksi presensi dari protein virus (antigen) COVID-19 pada sampel yang berasal dari saluran pernapasan seseorang. Jika konsentrasi antigen sasaran pada sampel cukup, antigen tersebut akan mengikat antibodi yang terdapat pada strip uji dan akan menghasilkan tanda visual, hasil biasanya didapatkan dalam waktu 30 menit. Antigen yang terdeteksi hanya bisa diekspresikan saat virus aktif bereplikasi. Oleh karena itu, tes ini paling baik digunakan untuk mengidentifikasi infeksi pada fase akut atau tahap awal infeksi (Scohy et al., 2020). 296 a. Hasil Jumlah peserta yang mengikuti kegiatan pengabdian masyarakat ini berjumlah 20 orang masyarakat Desa Pengabuan Kecamatan Abab Kabupaten Penukal Abab Lematang Ilir. Evaluasi kegiatan penyuluhan kesehatan ini dilihat dari nilai pre test dan post test yang diberikan kepada 20 responden mendapatkan nilai pre test dengan nilai rerata 50 dan nilai post test dengan nilai rerata 82. Dengan hasil evaluasi nilai pre test dan post test kegiatan penyuluhan kesehatan ini mengalami peningkatan yang artinya masyarakat di Desa Pangabuan Kecamatan Tanah Abang Kabupaten PALI lebih memahami dan bertambah pengetahuan mengenai Efektivitas Rapid Test Antibodi dan Rapid Test Antigen dalam Mendeteksi Covid-19. 2. Pelaksanaan Kegiatan Berkoordinasi dengan Kepala Desa di PALI dengan menyampaikan surat ijin, menjelaskan tujuan, prosedur kegiatan, dan memuat kontrak kegiatan. Melakukan penyuluhan dengan membagikan leafleat kepada masyarakat dan menjelaskan tentang manfaat dan efektivitas dari pemeriksaan Rapid test Antibodi dan Antigen dalam mendeteksi Covid-19. Penyuluhan dibantu oleh mahasiswa sebagai anggota pengabdian masyarakat (Via Anjalina Tambunan). Kegiatan penyuluhan pada pukul 09.00 WIB. Langkah pelaksanaannya adalah : 1) Fasilitator membuka acara, menjelaskan tujuan kegiatan, 2) Melakukan pre test kepada peserta, 3) pembagian bahan penyuluhan berupa leafleat dan penjelasan materi mengenai pemeriksaan rapid test antibodi dan antigen dalam mendeteksi 297 covid-19, 4) melakukan post test untuk mengukur kemampuan peserta terhadap materi yang telah diberikan, 5) menyusun laporan akhir serta pendokumentasian kegiatan pengabdian masyarakat. covid-19, 4) melakukan post test untuk mengukur kemampuan peserta terhadap materi yang telah diberikan, 5) menyusun laporan akhir serta pendokumentasian kegiatan pengabdian masyarakat. b. Pembahasan Di tengah pandemi Coronavirus Disease 2019 (Covid-19), banyak pertanyaan tentang apa itu rapid test, swab, dan PCR serta apa perbedaannya. Ketiganya adalah istilah yang berkaitan dengan diagnosis Covid-19. Membekali diri dengan pengetahuan tentang ketiga hal tersebut penting di masa pandemi ini. Tenaga medis mungkin sudah tahu apa itu rapid test, swab, dan PCR. Tapi tidak demikian dengan masyarakat awam. Rapid test, swab, dan PCR adalah istilah dalam metode pemeriksaan medis untuk mengetahui adanya Covid-19 dalam tubuh seseorang. Dokter menegakkan diagnosis Covid-19 setelah melakukan pemeriksaan tersebut. Rapid test juga sering disebut sebagai tes serologis. Dalam hal diagnosis Covid-19, akurasi rapid test bisa mencapai 90 persen. Proses untuk mengetahui hasil tes ini sangat cepat, bisa hanya dalam waktu 30 – 60 menit dan sebaiknya dilakukan di laboratorium oleh petugas yang mempunyai kompetensi. Harga alatnya pun terjangkau. Itu menjadi salah satu kelebihan rapid test sehingga dapat digunakan untuk memeriksa banyak orang sekaligus dalam satu waktu. Rapid tes untuk deteksi virus SARS co-2 saat ini ada yang bisa mendeteksi antibodi dan ada yang bisa antigennya. Tetapi yang dapat mendeteksi antigen banyak laboratorium 298 belum banyak yang melakukan. Rapid tes yang untuk mendeteksi antibodi tidak dapat mendeteksi pada awal sakit, karena mungkin belum terbentuk antibodi atau kadar antibodinya masih rendah. Sehingga bila hasil pemeriksaan non reaktif , harus diulang lagi pada hari 7 – 14 hari kemudian untuk memastikan apakah yang bersangkutan benar tidak mengandung virus dalam tubuhnya. Terutama bila yang bersangkutan ada riwayat terpapar virus SARS co-2. belum banyak yang melakukan. Rapid tes yang untuk mendeteksi antibodi tidak dapat mendeteksi pada awal sakit, karena mungkin belum terbentuk antibodi atau kadar antibodinya masih rendah. Sehingga bila hasil pemeriksaan non reaktif , harus diulang lagi pada hari 7 – 14 hari kemudian untuk memastikan apakah yang bersangkutan benar tidak mengandung virus dalam tubuhnya. Terutama bila yang bersangkutan ada riwayat terpapar virus SARS co-2. Dengan adanya pengabdian masyarakat ini terdapat peningkatan pengetahuan masyarakat mengenai cara yang efektif dalam mendeteksi COVID-19, sehingga masyarakat diharapkan agar tidak ragu memeriksakan diri apabila mengalami gejala dari COVID-19. Dengan adanya deteksi dini diharapkan dapat mencegah penularan virus ini menjadi lebih luas lagi. KESIMPULAN Program pengabdian masyarakat tentang Efektivitas Rapid Tes Antibodi dan Antigen Dalam Mendeteksi Covid-19 ini dapat diselenggarakan dengan baik dan berjalan dengan lancar sesuai dengan rencana kegiatan yang telah disusun. Berdasarkan hasil penyuluhan kesehatan yang dilakukan didapatkan kesimpulan bahwa Masyarakat Desa Pangabuan Kecamatan Tanah Abang Kabupaten PALI Provinsi Sumatera Selatan lebih memahami dan bertambah pengetahuan mengenai pentingnya Pemeriksaan Rapid Test Antibodi dan Rapid Test Antigen dalam Mendeteksi Covid-19 dilihat dari hasil penilaian pre test dan post test yang mengalami peningkatan dari 20 responden yang mengikuti penyuluhan kesehatan tersebut. UCAPAN TERIMA KASIH Terima kasih sebesar-besarnya kepada semua pihak yang telah membantu dan memfasilitasi pelaksanaan kegiatan pengabdian kepada masyarakat ini sehingga terlaksana sesuai dengan rencana yang telah disusun. Alsuliman T, Sulaiman R, Ismail S, Srour M, Alrstom A. 2020. COVID-19 paraclinical diagnostic tools: Updates and future trends. Curr Res Transl Med:1– 9. Ahn D, Shin H, Kim M, Lee S, Kim H, Myoung J, et al. 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https://openalex.org/W2561065388	https://eprints.soton.ac.uk/410728/1/acs.organomet.6b00778.pdf	English	null	Twists and Turns of Platinum-Allene Complexes: NMR Techniques for the Study of the Dynamic Behavior in Solution	Organometallics	2,016	cc-by	12,291	■INTRODUCTION tion of allenes,5 which give acetals or bisindolyl alkanes with double addition of the nucleophile to the terminal or less substituted double bond of the allene, and complete saturation of the second double bond, instead of the most common allyl derivatives obtained under gold catalysis.6 To explain the diﬀerent reactivity of the two metals, diﬀerent mechanisms and metallic intermediates have to be proposed for the two processes.7 The mode of coordination in metal-allene complexes is crucial for the understanding of the reactivity of these important systems.1 η2-Coordination of the metal with one of the double bonds is commonly proposed as the ground state (Scheme 1), Scheme 1. Metal-Allene Coordination Modes Scheme 1. Metal-Allene Coordination Modes As part of our mechanistic studies on these reactions, we sought to gather experimental evidence on the type of coordination of the platinum and the allenes, in order to understand the subtle diﬀerences in reactivities observed in platinum catalysis, and to ascertain the involvement of η1- coordinated allene complexes as reactive species in our catalytic cycle. As the isolation and analysis of PtCl2-allene complexes without stabilizing ligands is very challenging, for this study, we have selected a family of Pt-allene complexes with ancillary nitrogen-containing ligands in a four- or ﬁve-coordinated arrangement,8,9 active catalysts in our dihydroalkoxylation reaction,10 and we have studied them in solid state and in solution using diﬀerent NMR techniques, including our recently reported SSTD NMR method.11 with diﬀerent slippage structures toward the central or terminal carbons involved depending on the metal, the ligands, and the substitution on the allenes. More recently, η1-metal-allene complexes have been proposed as high energy intermediates or transition states in metal-catalyzed reactions involving allenes.2 In this mode, the metal coordinates exclusively to the central carbon of the allene, and several structures have been proposed, detected, and even isolated: zwitterionic carbene, σ-allyl cation, or η1-bent allenes (Scheme 1). Although a lot of attention has been paid to Au-allene complexes,3 studies on Pt-allene complexes are still rare. Twists and Turns of Platinum-Allene Complexes: NMR Techniques for the Study of the Dynamic Behavior in Solution Twists and Turns of Platinum-Allene Complexes: NMR Techniques for the Study of the Dynamic Behavior in Solution Twists and Turns of Platinum-Allene Complexes: NMR Techniques for the Study of the Dynamic Behavior in Solution Twists and Turns of Platinum-Allene Complexes: NMR Techniques for the Study of the Dynamic Behavior in Solution M. Teresa Quirós,†,∥María Paz Muñoz,,†,∥ Jeppe Christensen,‡,§ and Simon J. Coles‡ †School of Chemistry, University of East Anglia, Earlham Road, NR4 7TJ, Norwich, U.K. ‡UK National Crystallography Service, Chemistry, University of Southampton, Highﬁeld Campus, Southampton, SO17 1BJ, U.K. §Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, U.K. S Supporting Information ABSTRACT: Classic (dynamic exchange line-shape analysis) and novel (SSTD NMR) NMR techniques have been applied in order to obtain the kinetic and thermodynamic parameters of the three main processes occurring in the ﬂuxional behavior of Pt-allene complexes with N-containing ligands, in four and ﬁve coordination mode, in solution. Our results show intramolecular helical and rotational movements closely related to each other, conﬁrming η1-staggered structures as possible intermediates. The ligand exchange in these complexes seems to occur via a ligand-independent dissociative mechanism, where coordinating solvents might be involved in the stabilization of the intermediates. The diﬀerences observed in the interaction of allenes with other metals could be the basis to explain the divergent reactivity observed in platinum-catalyzed processes. g g be involved in the stabilization of the intermediates. The diﬀerences observed in the interaction of allenes with other metals could be the basis to explain the divergent reactivity observed i l i l d © XXXX American Chemical Society pubs.acs.org/Organometallics This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. ■INTRODUCTION Three processes are proposed for the behavior of metal- allene complexes in solution: the ligand exchange, the rotational movement (π-face exchange, rotation of the metal around the allene), and the helical movement (the movement of the metal Although platinum exhibits similar reactivity to gold in general, there are increasing examples where, under similar conditions, these two metals get involved in diﬀerent reaction pathways and give diﬀerent products.4 For example, we have reported the Pt-catalyzed dihydroalkoxylation and bisindolyla- Received: October 10, 2016 Received: October 10, 2016 © XXXX American Chemical Society DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX A Article Organometallics Article from one double bond to another, Scheme 2). The mechanism of the last two processes can go via inter- or intramolecular spectrum after saturation of one of the sites undergoing chemical exchange. We deﬁned the ηSSTD (spin saturation transfer diﬀerence parameter) as the measurement of the intensity of the “diﬀerence spectrum” (equilibrium-saturated, M0A −MA) over the intensity of the equilibrium spectrum (M0A), ηSSTD = (M0A −MA)/M0A.9 This method was successfully applied in the most challenging cases to study the π-face exchange, as coalescence of the signals is not needed, so a more ﬂexible temperature range can be used in the study. Further advantages of the method include: signal overlap does not interfere; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly and T1 values are obtained in the same experiment using only one set of experiments; and in contrast to line-shape analysis, the methodology is appropriate for low and high magnetic ﬁelds. We have also applied the method for the ﬁrst time to the study of the ligand exchange process, showing the potential for further application of this methodology to the study of ligand exchange processes in other organometallic systems. Scheme 2. Proposed Fluxional Pathways in Solution for Pt- Allene Complexes Scheme 2. Proposed Fluxional Pathways in Solution for Pt- Allene Complexes Scheme 2. Proposed Fluxional Pathways in Solution for Pt- All C l p Allene Complexes pathways,12 with important implications in catalysis and enantioselective processes involving chirality transfer from the allenes.13 Interestingly, despite the suggested disparity between the ground state and the reactive forms of Pt-allene complexes, experimental data regarding their behavior in solid state and solution are still lacking. Scheme 3. Synthesis of N-Ligand-Pt-Allene Complexes Organometallics Article than to the terminal carbon and also shorter than in the 1,1- DMA (dimethyl allene) complex (Pt−C(central) = 2.071(3) Å in 1, 2.108(5) Å in 2; Pt−C(terminal) = 2.252(3) Å in 1, 2.138(6) Å in 2), which resembles the η1-staggered structures proposed in similar metal-allene complexes and suggested as reactive intermediates or transition states in platinum-catalyzed trans- formations.22 The observed asymmetric binding has been previously attributed to backbonding interactions between the platinum and the π* orbital of the noncoordinated double bond that strengthens the platinum−C(central) bond, and causes the bending.23 which supports the trigonal bypiramidal structure in these complexes (Scheme 3). This trend is also observed in the precursor ethylene complexes (see the SI for details), and it is in accordance with previous data reported for ﬁve-coordinated Pt-oleﬁn complexes.17,18 p Complexes 1 and 2 had been synthesized before,19 but to the best of our knowledge, no crystal structure of the complexes has been reported to date. We were able to obtain crystals of the two pyridine-complexes (1 and 2) by slow diﬀusion of EtOAc into a petroleum ether solution.20 The crystal structure of both compounds showed slipped η2-coordination with a clear deviation from linearity in the CCC bond (159.7(5)° in 2, 153.7(3)° in 1),21 elongation of the CC bond coordinated to the platinum (1.382(8) Å (coordinated) vs 1.293(8) Å (noncoordinated) in 2, 1.371(4) Å vs 1.304(4) Å in 1), and distorted linear conformation (Pt−C1−C2 (central) 72.2(3)° in 2, 36.43(12)° in 1) with a clearly diﬀerent distance of the platinum to the central and terminal carbons of the coordinated double bond, which is in accordance with other reported metal-allene complexes2 (Figures 1 and 2; see the SI Complexes 3 and 4 present a ﬁve-coordinated platinum(II), which is viable thanks to the ﬂuxional behavior of the bidentate nitrogen ligand (DAD = PMP-NCH-CHN-PMP = diazadiene, Scheme 4).24 This has been previously studied in Scheme 4. Fluxional Behaviour of the Nitrogen Ligand in the (DAD)-Pt-Allene Complexes Scheme 4. Fluxional Behaviour of the Nitrogen Ligand in the (DAD)-Pt-Allene Complexes Figure 1. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-TMA complex 1. (DAD)-Pt-oleﬁn complexes,16 and although we observed similar behavior in our experiments (see the SI for VT experiments showing the coalescence temperature), the details are not included here, as we were more interested in the ﬂuxional behavior of the allene ligand. ■RESULTS AND DISCUSSION Synthesis of the Pt-Allene Complexes. The synthesis of the N-ligand-PtCl2-allene complexes was carried out by ligand exchange with the corresponding ethylene analogue, synthe- sized from the Zeise’s salt and the corresponding ligand in good yields.16 The Pt(II)−allene bond in the pyridine complexes has been shown to be stronger than the Pt−ethylene bond due to the relief of strain in the allene system upon coordination,16a with the release of ethylene gas as the driving force of the reaction. We encountered that classic VT experiments and line-shape analysis techniques, commonly used in the analysis of ﬂuxional processes of organometallic complexes in solution, were not suitable to study some of our complexes due to thermal instability and higher coalescence temperatures than the decomposition temperatures. Therefore, a new method was developed and applied to these challenging systems, the SSTD (Spin Saturation Transfer Diﬀerence) NMR method.11 This new NMR protocol combines the Spin Saturation Transfer (SST) method used for small molecules,14 with the Saturation Transfer Diﬀerence (STD) NMR method employed for the study of protein−ligand interactions,15 by measuring transient spin saturation transfer along increasing saturation times (build- up curves) in systems undergoing mutual-site exchange. The method uses diﬀerence spectroscopy, based on the spin saturation transfer experiment, but avoiding the need to ensure steady state conditions and the determination of T1 values. The transient spin saturation transfer is measured as the diﬀerence between the normal 1H NMR spectrum and the 1H NMR Analysis of the complexes in solution by 1H NMR and solid state by X-ray crystallography showed in all cases η2- coordination in the ground state, with the platinum coordinated to the less substituted double bond in the complexes with mono- and 1,1-disubstituted allenes. This was supported by a clear shift of the protons in the terminal double bond to lower chemical shift in the 1H NMR spectra, as reported for other metal-allene complexes.2 Interestingly, ﬁve-coordinated com- plexes 3 and 4 showed an upﬁeld shift (>1 ppm) compared with four-coordinated complexes 1 and 2, and larger J(Pt‑H), Scheme 3. Synthesis of N-Ligand-Pt-Allene Complexes DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX B Scheme 3. Synthesis of N-Ligand-Pt-Allene Complexes DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX B DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics Dissociation of the allene in complex 4 in diﬀerent solvents and correlation of Keq with solvent parameters: DN (kcal mol−1, blue diamonds); dielectric constant (ε, green triangles); dipole moment μ (Debyes, red squares). Figure 3. Dissociation of the allene in complex 4 in diﬀerent solvents and correlation of Keq with solvent parameters: DN (kcal mol−1, blue diamonds); dielectric constant (ε, green triangles); dipole moment μ (Debyes, red squares). Previous studies of Py-Pt(II)-allene complexes of type 1 have shown that the ﬂuxional movement is mononuclear with the 195Pt−CH3 coupling retained while the rate is independent of the concentration of complex.19,28 Besides, the inﬂuence of diﬀerent substituted pyridine ligands showed that the rate of migration decreases with the increase basicity of the pyridines, which is in accordance with the increase in the strength of the Pt-allene π-bond with the σ-donor capacity of the ligands. Data for complex 1 with the unsubstituted pyridine showed a rate constant for the ﬂuxional behavior of k = 30 s−1 at −46 °C in CDCl3.19b This rate was attributed to the helical movement (the Pt moves across the central carbon atom from one double bond to the other, which orbitals are orthogonal to each other), but the combination with the rotational movement was not analyzed in detail in this complex. In those studies, the rotational movement was suggested for the analogous complex with the DMA (2), to explain the same observed chemical shift, even at low temperatures, of the two terminal protons of the coordinated allene. In order to clarify the role of slipped η2-complexes, η1- coordinated species as intermediates or transition states, and the ligand exchange in our platinum-catalyzed transformations, we analyzed the three diﬀerent processes of ﬂuxional behavior (Scheme 2) of the complexes in Scheme 3 in solution, by using a combination of classic and new NMR techniques. q Helical Movement and π-Face Exchange. Some data have been already reported in the study of ﬂuxional behavior in Pt(II)- and Pt(0)-allene complexes using line-shape analysis and SST NMR techniques. The helical movement or 1,2-metal shift has been studied in Pt-cyclic allene complexes (where the rotational movement is not possible due to the rigidity of the system) by Jones et al. using magnetization transfer experi- ments.27 They showed that a cationic Pt(II)-cycloheptatrienyli- dene complex exists preferably as the η1-carbene tropylium ion form in solution, or as a very fast equilibrium between the η2- Pt-allene structures (Scheme 5). Organometallics The stability of the ﬁve-coordinated complex (DAD)PtCl2- (DMA) 3 in solution was poor, which made its isolation diﬃcult. Therefore, this complex was characterized and used in situ once the ethylene gas had been completely excluded from the reaction mixture. Complex 4, however, was more stable and could be isolated in good yield. g y During the synthesis of the (DAD)-PtCl2-allene complexes, we observed that complexation of the allene and displacement of the ethylene ligand were dependent on the solvent used. Moreover, we observed some dissociation of the allene in complex 4 depending on the solvent used to record the 1H NMR spectra, in samples prepared from the isolated Pt-allene complexes. More interestingly, we observed that, excluding the chloroform, there was very good linear correlation between the ratio of free to coordinated allene (Keq in THF < Acetone < CH3CN) with several solvent parameters (Figure 3; see the SI for full details). For example, Keq decreases with higher dipole moment, donicity (DN), and surface tension (solvent parameter values for THF > Acetone > CH3CN) and increases with increasing dielectric constant, solvent polarity, or acceptor number (AN) (solvent parameter values for THF < Acetone < CH3CN). The best solvent to avoid dissociation of the allene seemed to be THF, with a high dipole moment, high donicity (DN = 20),25 and low dielectric constant. The outliner behavior of the system in chloroform could be explained by the acidity and lack of coordinating ability of this solvent (DN is considered 0 for chlorohydrocarbons), which does not oﬀer stabilization of the complex in solution and promotes decoordination and decomposition in the ﬁve-coordinated series. Figure 1. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-TMA complex 1. Figure 1. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-TMA complex 1. Figure 2. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-DMA complex 2. Figure 2. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-DMA complex 2. Figure 2. Ball-and-stick representation of the crystal structure of Pyridine-PtCl2-DMA complex 2. These observations suggest ligand exchange processes and dynamic behavior in solution of these complexes, which is in agreement with previous reports in other metal-allene complexes ﬂuxional behavior.26 for full details). The distorted conformation is more pronounced in the TMA (tetramethyl allene) complex 1 with a distance of the platinum to the central carbon much shorter C C DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics Article Figure 3. DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics However, the Pt(0)-cyclo- heptatrienylidene complex exists only in the η2-form and the helical movement was shown to be intramolecular (possibly via a carbene type intermediate, similar to the carbene tropylium) with a superimposed intermolecular component at higher temperatures. In order to obtain more information and all the thermodynamic parameters for the helical process, in which the platinum migrates between the allene double bonds, and to compare it with the rotational movement of the platinum around the allene axis, we analyzed complex 1 by dynamic exchange line-shape analysis using the DNMR module of TopSpin 3.0 (Bruker).29 This software simulates temperature- dependent experimental spectra, interactively set up according to a number of parameters (e.g., chemical shifts, intensity, line broadening, or rate constants) which can be ﬁxed or allowed to vary, and iteratively reﬁnes the model parameters to get the best ﬁt of the measured and simulated 1D NMR spectra to extract exchange rates. Although the kinetic data obtained are less precise than those obtained using classic line-shape analysis, coalescence of the signals is not needed, which was key in our complexes. Scheme 5. Helical Movement in Pt-Cyclic Allenes as Reported by Jones et al.27 Scheme 5. Helical Movement in Pt-Cyclic Allenes as Reported by Jones et al.27 p 1H NMR spectra of the complex at diﬀerent temperatures were used for the ﬁtting in the DNMR module of the TopSpin. The 1H NMR of complex 1 at room temperature in deuterated D DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 4. 1H NMR of (Py)PtCl2(TMA) 1 at 25 °C (a) and −40 °C (b) in 1,1,2,2-tetrachloroethane-d2. Figure 5. Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent temperatures Organometallics Article Fi 4 1H NMR f (P )P Cl (TMA) 1 25 °C ( ) d 40 °C (b) i 1 1 2 2 hl h d Figure 4. 1H NMR of (Py)PtCl2(TMA) 1 at 25 °C (a) and −40 °C (b) in 1,1,2,2-tetrachloroethane-d2. Figure 5. Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent temperatures. Figure 5. Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent temperatures. Figure 5. Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent temperatures Figure 5. DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics See the SI for full E DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Organometallics Article Table 1. Rate Constants (k) Obtained for Rotational and Helical Movements of (Py)PtCl2(TMA) 1 in 1,1,2,2,- Tetrachloroethane Table 1. Rate Constants (k) Obtained for Rotational and Helical Movements of (Py)PtCl2(TMA) 1 in 1,1,2,2,- Tetrachloroethane simple dissociation−recombination mechanism)12 or in an intramolecular fashion via η1-staggered intermediates (or transition states). These staggered intermediates can be either η1-bent allene structures, if the pathways goes through a Vrieze−Rosenblum mechanism,32 or planar η1-allyl cation structures, in a higher energy process (Scheme 6).26 Recently, studies by Widenhoefer et al. in Au-allene complexes showed that the π-face exchange in those complexes occurs through these staggered η1-intermediates with the 1,2-metal shift (helical movement) occurring as the ﬁrst step of the process (although this intermediate was not directly observed in their experiments).23 T (°C) k1 (s−1)rotational movement k2 (s−1) helical movement ﬁtting overlap (%) −40 1.06 × 10−1 10.40 95 −30 5.62 24.70 94 −20 21.63 75.76 96 −10 39.55 150.68 96 0 130.12 195.64 97 10 177.59 196.72 97 To gather more evidence of the mechanisms in our systems, we repeated the experiments changing the concentration of the platinum complex in the media. A change of the kinetic parameters with the concentration of the complex will be in accordance with an intermolecular mechanism, whereas the opposite will point to an intramolecular mechanism. We found that the rate was not dependent on the concentration at any temperature tested, pointing to an intramolecular process (for full analysis, see the SI). details). The low energy barriers and the small diﬀerence in the activation energy of the two processes (ΔEa ∼2 kcal mol−1) point to a mechanism with similar intermediates or transition states in both processes.31 Interestingly, the process in tetrachloroethane is slower than that in chloroform (k = 30 s−1 at −46 °C in CDCl3).19 The rotational movement of the platinum around the allene was also studied in complex 2. The analysis of this complex has not been possible until now using classic techniques, due to decomposition temperatures higher than the coalescence temperature of the signals, which pointed to a very slow rotation. Organometallics However, with our new SSTD NMR method, we were able to calculate the kinetic parameters of this process in 1,1,2,2-tetrachloroethane-d2, by measuring the magnetization transfer in the methyl groups undergoing chemical exchange (π-face exchange in this case), as the spin saturation transfer diﬀerence parameter (ηSSTD). Plotting this parameter versus the saturation times gave us build-up curves that ﬁtted to the exponential equation allowed us to obtain the values of the rate constants of the process at the diﬀerent temperatures (see the SI for full details). It is worth noting that the temperatures required for these experiments were higher (above room temperature) than the temperatures used in the line-shape analysis of complex 1. The calculated kinetic and thermody- namic parameters (ΔH⧧= 18.6 ± 0.4 kcal mol−1; ΔS⧧= −9 ± 1 Cal mol−1 K−1; Ea (298 K) = 19.2 ± 0.4 kcal mol−1; ΔG⧧ (298 K) = 21.1 ± 0.4 kcal mol−1) were in accordance with a slow process, as previously suggested, although the movement of the platinum is deﬁnitely happening.11 The thermodynamic parameters were also calculated with diﬀerent concentrations of the complex. As shown in Figure 6 (see the SI for full analysis), the values for the Ea in both processes for complexes 1 and 2 suﬀered scarce variation with the change of concentration of the platinum complex, which suggests that both processes occur intramolecularly through η1- staggered Pt-allene complexes as possible high energy intermediates. The similar values obtained for the helical and rotational movements in 1 support this proposal (ΔEa ∼2 kcal mol−1). The lower activation energy for the rotational movement of complex 1 vs 2 (ΔEa = 7.2 kcal mol−1) is also consistent with the η1-staggered intermediates,33 which could be stabilized by an inductive eﬀect in more substituted allenes, lowering the activation barrier for the 1,2-metal shift (helical movement), that could be the ﬁrst step in the rotational movement process. This is also supported by the X-ray structures of the TMA and DMA complexes, where a more pronounced slippage toward the central carbon of the allene is observed in complex 1. p Ligand Exchange. It has been proposed before that, at temperatures > −40 °C, the pyridine ligands in the Py-Pt-allene dissociate, with pyridines substituted with EWG dissociating Helical and rotational movements have been proposed to be intrinsically related and can occur in an intermolecular (by e 6. Organometallics Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)P temperatures. Figure 5. Expansions of the experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent temperatures. experimental 1H NMR (bottom) and ﬁtting performed by DNMR (top) for (Py)PtCl2(TMA) 1 at diﬀerent 1,1,2,2-tetrachloroethane30 shows a broad singlet in the aliphatic region, corresponding to the four methyl groups of the coordinated allene (Figure 4a). However, when the temperature decreases, three signals appear, two corresponding to the methyl groups in the noncoordinated double bond of the allene, Me3 and Me4, and a third signal corresponding to the two methyl groups in the coordinated double bond, Me1 and Me2 (Figure 4b). deﬁned. In this case, as well as the chemical shifts, intensity, and LB value, the equilibria occurring between the exchanging protons were deﬁned (rotational and helical movements). The values of the rate constants in these equilibria were the parameters to ﬁt in this case. The simulated spectra obtained for each temperature along with the experimental ones are shown in Figure 5. The rate constants for both processes at diﬀerent temperatures were obtained by the explained protocol, with the overlap of the ﬁtting of simulated to experimental data being higher than 90% in all the cases (Table 1; see the SI for full details). 19 One spectrum is ﬁtted at a time in the DNMR module. To establish the model, the region to be ﬁtted must be selected and the signals of the protons appearing in the region must be deﬁned (chemical shifts, coupling constants, and intensity). Initially, a region containing no exchanging signals was selected and the protons deﬁned, to ﬁrst determine the appropriate LB (line broadening parameter) of the spectra (since the wrong value of this parameter will aﬀect the values of the rate constants obtained). Once the LB was calculated for the spectra, a second region containing the exchanging protons was As previously speculated by Vrieze et al.,19 the π-face exchange process (rotational movement) showed to be slightly slower than the helical movement for complex 1, as also shown when comparing the thermodynamic parameters for both processes in tetrachloroethane, calculated using the Eyring equation (i.e.: Ea = 12 kcal mol−1 for rotational movement in 1; Ea = 9.9 kcal mol−1 for helical movement in 1. DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics Possible Pathways for the Helical and Rotational Movements of the Platinum around the Allene in a Pt-Allene ex DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX F F F DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 6. Plot of Ea versus [Pt-allene] for the rotational (blue diamonds) and helical (green triangles) movements of 1, and rotational movement (red squares) for 2. Organometallics Article easier. Besides, in the work reported by Vrieze et al., it was the SSTD NMR method has been applied in the study of an Figure 6. Plot of Ea versus [Pt-allene] for the rotational (blue diamonds) and helical (green triangles) movements of 1, and rotational movement (red squares) for 2. Figure 7. 1H NMR spectra of (Py)PtCl2(DMA) 2 at 40 °C in 1,1,2,2-tetrachloroethane-d2. (a) 1H NMR expansion of the region from 1.4 to 5.0 ppm before irradiation. (b) Same region after the irradiation of the methyl group at 2.48 ppm. (c) Diﬀerence spectrum [(a) −(b)]. Figure 6. Plot of Ea versus [Pt-allene] for the rotational (blue diamonds) and helical (green triangles) movements of 1, and rotational movement (red squares) for 2. [Pt-allene] for the rotational (blue diamonds) and helical (green triangles) movements of 1, and rotational movement Figure 7. 1H NMR spectra of (Py)PtCl2(DMA) 2 at 40 °C in 1,1,2,2-tetrachloroethane-d2. (a) 1H NMR expansion of the region from 1.4 to 5.0 ppm before irradiation. (b) Same region after the irradiation of the methyl group at 2.48 ppm. (c) Diﬀerence spectrum [(a) −(b)]. Figure 7. 1H NMR spectra of (Py)PtCl2(DMA) 2 at 40 °C in 1,1,2,2-tetrachloroethane-d2. (a) 1H NMR expansion of the region from 1.4 to 5.0 ppm before irradiation. (b) Same region after the irradiation of the methyl group at 2.48 ppm. (c) Diﬀerence spectrum [(a) −(b)]. Figure 7. 1H NMR spectra of (Py)PtCl2(DMA) 2 at 40 °C in 1,1,2,2-tetrachloroethane-d2. (a) 1H NMR expansion of the region from 1.4 to 5.0 ppm before irradiation. (b) Same region after the irradiation of the methyl group at 2.48 ppm. (c) Diﬀerence spectrum [(a) −(b)]. the SSTD NMR method has been applied in the study of an intermolecular chemical exchange. The good results obtained for our complexes emphasize the relevance of this method and the potential for further application to the study of other ligand exchange processes in organometallic systems. easier. DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics Article metal-allene complexes.35 The allene-dependent mechanism is consistent with an associative mechanism involving direct attack of the free allene at the metal to form a bis(allene) intermediate, followed by rapid allene dissociation, or rapid solvation, followed by rapid associative exchange with free allene (Scheme 7). On the contrary, in the dissociative mechanism, the ﬁrst step would be the dissociation of the allene ligand, and this is normally the rls in the process. Both pathways can be diﬀerentiated measuring the kinetic behavior with diﬀerent concentrations of free allene in the media. The change in the speed of the process by changing the concentration of ligand, and a two-term rate law, would point to an associative mechanism, whereas, if no change in speed is observed with diﬀerent concentrations of the external allene, it would indicate that the process occurs through a dissociative mechanism. the free allene, which indeed shows that ligand exchange is occurring. This process of ligand exchange appeared to be quicker than the internal rotation, and a temperature range of 293−313 K was enough to observe solely the ligand exchange, with no signs of the intramolecular rotational movement happening (Figure 7).34 the free allene, which indeed shows that ligand exchange is occurring. This process of ligand exchange appeared to be quicker than the internal rotation, and a temperature range of 293−313 K was enough to observe solely the ligand exchange, with no signs of the intramolecular rotational movement happening (Figure 7).34 With the right conditions in hand, we calculated the rate constants at diﬀerent temperatures for the ligand exchange process applying the SSTD NMR technique. We obtained build-up curves, which ﬁtting gave us the rate constants at diﬀerent temperatures (Figure 8). This allowed us to calculate Figure 8. Plots of the transfer of magnetization (ηSSTD) versus saturation time (s) at diﬀerent temperatures for the mixture (Py)PtCl2(DMA) 2 (1 equiv) + DMA (1 equiv), and table with the obtained rate constants after ﬁt of the exponential curves. Organometallics Besides, in the work reported by Vrieze et al., it was reported that dissociation of the tetramethylallene did not occur at temperatures below 80 °C, or in the presence of free TMA.19b However, no proper kinetic study of the ligand exchange was carried out. We ﬁrst applied the SSTD NMR method to study the ligand exchange process in a mixture of (Py)PtCl2(DMA) 2 in the presence of free 1,1-dimethylallene (DMA). A preliminary experiment irradiating one of methyl groups of the coordinated allene showed that there was a transfer of saturation between this signal and the signal corresponding to the methyl groups in This process of ligand exchange in Pt-allene complexes is important since it is the starting point of most of the catalytic cycles. Therefore, we decided to study it in more depth with Pt- allene complexes bearing diﬀerent N-containing ligands in the four- and ﬁve-coordinative structures using our SSTD NMR technique. It is worth noting here that this is the ﬁrst time that G DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics When the kinetic and thermodynamic parameters were calculated using diﬀerent concentrations of free 1,1-dimethyl- allene (DMA) in the same system as above, minimum variations were observed in the rate constants and in the thermodynamic parameters (see the SI for full details), suggesting that the dissociative mechanism is occurring with this complex at the temperatures studied.36 p p Dissociative mechanisms for ligand exchange have been observed for d8 complexes when the geometry is distorted from square planar or if the coordination number is higher than four.37 For comparison with the four-coordinated Pt-allene complex, and to conﬁrm the dissociative mechanism, we also studied the ligand exchange process in the ﬁve-coordinated complex (DAD)PtCl2(DMA) 3. As mentioned before, the stability of this complex in chlorinated solvents was poor and the allene ligand exchange process was found to be slow (ηSSTD = 0.0124 at 298 K for a saturation time of 40 s). We found that changing the solvent to THF improved both, stability and speed of the exchange (ηSSTD= 0.4362 at 298 K for a saturation time of 40 s). This complex could not be isolated due to decomposition issues, so for the SSTD NMR study, samples were prepared in situ from a solution in THF-d8 of the ethylene complex and an excess of dimethylallene displacing the ethylene from the media by bubbling nitrogen in the solution during 20 min. Although this methodology made it more challenging to precise the concentration of the complex in the sample, we could still obtain the build-up curves, the rate constants at diﬀerent temperatures, and the thermodynamic Figure 8. Plots of the transfer of magnetization (ηSSTD) versus saturation time (s) at diﬀerent temperatures for the mixture (Py)PtCl2(DMA) 2 (1 equiv) + DMA (1 equiv), and table with the obtained rate constants after ﬁt of the exponential curves. the thermodynamic parameters of the ligand exchange using the Eyring equation: ΔH⧧= 6.4 ± 0.3 kcal mol−1; ΔS⧧= −44.4 ± 1.0 Cal mol−1 K−1; Ea (298 K) = 6.9 ± 0.3 kcal mol−1; ΔG⧧ (298 K) = 19.6 ± 0.3 kcal mol−1 (see the SI for details). The value of the energy of activation for the ligand exchange conﬁrms that this process is much faster than the rotational movement in the complex studied (ΔEa = 12.2 kcal mol−1). DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Scheme 7. Possible Pathways for the Ligand Exchange in Pt-Allene Complexes Organometallics p Competitive ligand-dependent and ligand-independent path- ways are common for coordinatively unsaturated transition- metal complexes and have been proposed before in other Scheme 7. Possible Pathways for the Ligand Exchange in Pt-Allene Complexes DOI 10 1021/acs organom H DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX H DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 9. 1H NMR spectra of (DAD)PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv) at 25 °C in THF-d8. (a) 1H NMR expansion of the region from 2.7 to 5.2 ppm before irradiation. (b) Same region after the irradiation of the protons at 3.08 ppm. (c) Diﬀerence spectrum [(a) −(b)]. Figure 9. 1H NMR spectra of (DAD)PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv) at 25 °C in THF-d8. (a) 1H NMR expansion of the region from 2.7 to 5.2 ppm before irradiation. (b) Same region after the irradiation of the protons at 3.08 ppm. (c) Diﬀerence spectrum [(a) −(b)]. Figure 10. Plots of the transfer of magnetization (ηSSTD) versus saturation time at diﬀerent temperatures for the mixture (DAD)- PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv), and table with the obtained rate constants after ﬁt of the exponential curves. parameters (ΔH⧧= 4.5 ± 0.6 kcal mol−1; ΔS⧧= −48 ± 2 Cal mol−1 K−1; Ea (298 K) = 5.1 ± 0.6 kcal mol−1; ΔG⧧(298 K) = 18.7 ± 0.6 kcal mol−1) for the ligand exchange process of complex 3 and free DMA. Although measured in diﬀerent solvents, these values are in the same range of the ones obtained for the four-coordinated complex 2, suggesting a dissociative mechanism in both cases. The fact that the process is faster in THF when compared to TCE might point to a more complex mechanism with fast solvation of the free platinum after allene dissociation. In order to conﬁrm the dissociative mechanism with a more stable, isolable ﬁve-coordinated complex, we studied the ligand exchange process using the SSTD NMR method with complex 4 and free cyclohexylallene (CHA). Similarly to complex 3, the stability of 4 was better and the speed of ligand exchange faster in THF as solvent when compared to TCE or chloroform. Organometallics This is not surprising as the donicity of THF is much higher than the chlorinated solvents, normally considered nearly 0.25 This is in agreement with the observations of allene dissociation mentioned when describing the synthesis of the complexes and suggests further stabilization by solvation and possible coordination of the THF at some stage in the mechanism. To investigate the ligand exchange process in this complex, a mixture of the platinum-allene complex and free allene was prepared in THD-d8. The signal chosen for irradiation was the signal of the external protons of the coordinated allene, which appears at 3.08 ppm in the 1H NMR (Figure 9). Figure 10 depicts the build-up curves at diﬀerent temperatures along with the obtained rate constants. With the values of the rate constants, the thermodynamic parameters were calculated using the Eyring equation as shown in Figure 11. Figure 10. Plots of the transfer of magnetization (ηSSTD) versus saturation time at diﬀerent temperatures for the mixture (DAD)- PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv), and table with the obtained rate constants after ﬁt of the exponential curves. for the analogous complex with dimethylallene (ΔEa = 11.6 kcal mol−1), which points to a slower ligand exchange process for the cyclohexylallene. This will further support a dissociative mechanism, as 1,1-DMA is sterically more hindered than CHA and, therefore, easier to dissociate from the platinum coordination sphere. Once again, to check the dependence of the ligand exchange process with the concentration of free allene, SSTD NMR experiments were carried out changing the concentration of CHA. The calculated rate constants with diﬀerent amounts of free allene are shown in Figure 12 (for full analysis, see the SI). The value for the activation energy for the diazadiene complex with cyclohexylallene is higher than the one obtained I DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Figure 11. Eyring plot for the mixture (DAD)PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv). Slope and intercept values were used to calculate the enthalpy and entropy of activation, respectively. Organometallics Organometallics Article Figure 11. Eyring plot for the mixture (DAD)PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv). Slope and intercept values were used to calculate the enthalpy and entropy of activation, respectively. Organometallics In summary, we have applied diﬀerent NMR techniques to the study of the ﬂuxional behavior of Pt-allene complexes with N-containing ligands in four and ﬁve coordination mode. Organometallics We have obtained the kinetic and thermodynamic parameters for the three main processes occurring in solution: the ligand exchange, the helical and the rotational movements. Our results show that the helical and rotational movements occur intramolecularly and are closely related to each other, conﬁrming η1-staggered structures as possible intermediates and suggesting that the helical movement could be the ﬁrst step in the rotational pathway in the complexes studied. We have also revealed that the ligand exchange occurs mainly via a ligand-independent dissociative mechanism, where coordinat- ing solvents might be involved in the stabilization of the intermediates. Our results are in agreement with previous speculations on the behavior of platinum complexes, providing the lacking experimental evidence for comparison with other metals. For example, the results presented here, compared with those reported for Au-allene complexes,23 show a divergent coordination in the ground state and behavior of Pt-allene complexes regarding ligand exchange, but related pathways for the rotational movement, although with diﬀerent activation energies, which might explain the divergent reactivity observed with these two metals. Figure 11. Eyring plot for the mixture (DAD)PtCl2(CHA) 4 (1 equiv) + CHA (2 equiv). Slope and intercept values were used to calculate the enthalpy and entropy of activation, respectively. Again, no two-term rate low was observed in this system, and although the variation in the values is slightly bigger than in previous cases, possibly due to the involvement of the solvent in the ligand exchange as mentioned earlier, we can still observe no signiﬁcant change in the kinetic or thermodynamic parameters with concentration of free allene, and therefore, a dissociative mechanism is proposed for this complex too. Although platinum complexes used in catalysis are most commonly free of ligands, the complexes studied in this work have shown to be active catalysts in the dihydroalkoxylation reaction of allenes.10 Therefore, the information obtained with the complexes studied in this work can be extrapolated to real catalytic cycles where the coordination of the platinum to the allenes is generally the ﬁrst step in the cycle. Understanding this coordination is a crucial step forward to the understanding of mechanisms involving Pt-allene systems, which is key to control the processes toward the development of new reactions and more complex structures. ■EXPERIMENTAL SECTION ( ) ( ) ( p ) The analysis of the temperature-dependent 1H NMR of the (Py)PtCl2(TMA) 1 was performed using the DNMR module of the TopSpin. This program simulates the spectra by setting up and reﬁning the model parameters in order to adjust them to the experimentally obtained NMR. In the DNMR line-shape analysis tool, the parameters have to be deﬁned in the parameter panel, starting with the chemical shift range of the spectra to be ﬁtted. Secondly, the spin systems and each of the nuclei included in them must be set (LB, intensity or chemical shifts). The reactions (chemical exchanges) occurring in those systems also have to be deﬁned. Finally, the parameters to be reﬁned can be chosen, and the software then gives the best possible overlap with the measured spectra.40 (Py)PtCl2(ethylene). This compound was prepared from the Zeise’s salt according to reported procedures.14 Pyridine (1 mmol) was added dropwise to a stirred solution of the Zeise’s salt (1.1 mmol) in distilled water (12 mL/mmol). The product precipitated. After stirring for an additional 40 min, the solid was isolated by ﬁltration and washed with water. From 50 mg (0.136 mmol) of the Zeise’s salt, the platinum alkene complex (37.8 mg, 74%) was obtained as a yellow solid. 1H NMR (500 MHz, CDCl3, 25 °C) δ (ppm) = 8.94 (d, J = 3.6 Hz, 2H, Py), 7.95 (dt, J = 7.7, 1.5 Hz, 1H, Py), 7.53 (m, 2H, Py), 4.91 (s, JPt‑H = 60 Hz, 3H, CH2). MP = 107.4−108.9 °C. (Py)PtCl2(TMA) 1. This compound had been previously reported.17 To a suspension of (Py)PtCl2(ethylene) (1 mmol) in methanol (0.37 mL/mmol) was added the tetramethylallene (2 mmol). After 10 min, the platinum-allene complex precipitated and was isolated by centrifugation. From 15 mg (0.0402 mmol) of the platinum-alkene complex, the platinum-allene complex (14.7 mg, 83%) was obtained as a yellow solid. 1H NMR (500 MHz, CDCl2CDCl2, 25 °C) δ (ppm) = 8.86 (d, J = 5.2 Hz, 2H, Py), 7.91 (t, J = 7.7 Hz, 1H, Py), 7.58 (m, 2H, py), 2.11 (br s, 12H, 4CH3). MP = 116.6−117.9 °C. p p p General considerations for the SSTD NMR experiments: A standard 1D STD NMR pulse sequence provided by the manufacturer (“stddiﬀ”, Bruker) was used for the experiments. ■EXPERIMENTAL SECTION They were carried out on a 11.7 T high-resolution Bruker NMR spectrometer (500 MHz) equipped with an inverse triple resonance (H/C/N) z-gradient probe head. The total saturation time for a given experiment was built by a chain of “n” Gaussian pulses of 50 ms length, with a maximum ﬁeld strength γB1 of 104 Hz, providing a saturation time of “0.05·n” seconds. To avoid relaxation artifacts, all the experiments within a build-up curve had total duration constant; i.e., the addition of the relaxation delay plus the saturation time and the acquisition time was kept constant (42 s) along the whole set of experiments. On-resonance frequency was selected at the chemical shift of one of the 1H signals of the exchanging sites, and the oﬀ-resonance (reference NMR spectrum) frequency was set in all the cases to 40 ppm. For all the SSTD NMR experiments, the spectral width was selected to cover the two signals of interest and centered (O1) at the frequency of the irradiated signal. Integration of signals was carried out using TopSpin 3.1.9 (Py)PtCl2(DMA) 2. This compound had been previously reported.17 To a solution of (Py)PtCl2(ethylene) (1 mmol) in DCM (70 mL/mmol) was added the allene (1 mmol). The solution was stirred for 10 min, and then the mixture was ﬁltered through Celite and the solvent was evaporated, aﬀording the platinum-allene complex. From 25 mg (0.067 mmol) of the platinum-alkene complex, the platinum-allene complex (31.2 mg, quantitative) was obtained as a yellow solid. 1H NMR (500 MHz, CDCl2CDCl2, 25 °C) δ (ppm) = 8.94 (d, J = 5.2 Hz, 2H, Py), 7.99 (t, J = 7.7 Hz, 1H, Py), 7.58 (t, J = 6.9 Hz, 2H, Py), 4.17−4.04 (m, JPt‑H = 60 Hz, 2H, ·CH2), 2.46 (s, 3H, CH3 A), 2.21 (s, 3H, CH3 B); 195Pt NMR (107.2 MHz, CDCl3, 25 °C) δ (ppm) = 2254.24. MP = 114.6−115.9 °C. Crystallographic data for complexes 1 and 2 have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1507881 (1) and CCDC 1507882 (2)) and can be obtained free of charge on their Web site (http://www.ccdc.cam.ac.uk). (DAD)PtCl2(ethylene). This compound was prepared from the Zeise’s salt according to reported procedures.22 A solution of the Zeise’s salt (1 mmol) in methanol (10 mL/mmol) was added dropwise to a solution of the α-diimine (1 mmol) in dichloromethane (100 mL/ mmol). The solution was stirred for 10 min. Organometallics Although comparison of the three studied processes has to be done with caution due to the diﬀerent solvent and slightly diﬀerent temperature range used in the analysis of the Py and DAD complexes, we observed a linear compensation eﬀect when plotting values of ΔH⧧versus ΔS⧧for the three processes, which would further support the same type of mechanism along the series (see the SI for details).38 Figure 12. Plot of kobs versus [CHA] for the ligand exchange of 4 at diﬀerent temperatures: 298 K (black squares), 292 K (red dots), and 286 K (green triangles). Figure 12. Plot of kobs versus [CHA] for the ligand exchange of 4 at diﬀerent temperatures: 298 K (black squares), 292 K (red dots), and 286 K (green triangles). DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX J Article ■EXPERIMENTAL SECTION After ﬁltration over Celite, the solvent was evaporated to dryness without heating the sample. From 100 mg (0.271 mmol) of the Zeise’s salt, the platinum alkene complex (139.1 mg, 91%) was obtained as a red solid. 1H NMR (500 MHz, CDCl3, 25 °C) δ (ppm) = 8.91 (s, 2H, CHN), 7.98 (d, J = 9.0 Hz, 4H, PMP), 7.03 (d, J = 9.0 Hz, 4H, PMP), 3.90 (s, 6H, OCH3), 3.89 (s, 4H, CH2CH2). IR = 1599 (CN), 1588 (CC), 1264 (C-O). MP = 282.1−284.0 °C.37 Organometallics Article 2H, CHN), 8.06 (m, 4H, Ar PMP), 7.04 (d, J = 8.8 Hz, 4H, Ar PMP), 5.03 (dt, J = 6.1, 3.0 Hz, 1H, ·CH), 3.86 (s, 6H, 2OCH3), 3.09 (dd, J = 3.0, 1.8 Hz, 2H, ·CH2), 2.35 (m, 1H, CH Cy), 1.73−1.82 (m, 4H, 2CH2 Cy), 1.18−1.84 (m, 6H, 3CH2 Cy). 13C NMR (MHz, THF, 25 °C) δ (ppm) = 162.6 (CN), 158.1 (OCq), 136.4 (Cq), 126,4 (CAr PMP), 115.6 (CAr PMP), 115.4 (·CH), 96.5 (Cq Allene), 56.1 (OCH3), 41,1 (CH Cy), 34.4 (2CH2 Cy), 27.4 (CH2 Cy), 27.1 (2CH2 Cy), 16.1 (CH2). IR = 1593 (CN), 1511 (CC), 1256 (C-O). MP = 350−352 °C (decomposition).39 Besides, mechanisms of ligand exchange are very important not only for these complexes and the speciﬁc allene but for the whole ﬁeld of organometallic chemistry and metal catalysis, and we are convinced that the application of our new SSTD NMR method will contribute to the discovery of new twists and turns in other organometallic systems. * S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organo- met.6b00778. General experimental details, chemical shift comparison in four and ﬁve coordinated complexes, ﬂuxional behavior of the DAD ligand in (DAD)PtCl2(CHA) 4, studies on allene dissociation on the (DAD)PtCl2(CHA) 4 depending on solvent, details on the VT and DNMR line-shape analysis of P(y)PtCl2(TMA) 1, details on experimental setup and analysis of SSTD NMR of the rotational movement and ligand exchange of (Py)- PtCl2(DMA) 2, and ligand exchange of (DAD)- PtCl2(DMA) 3 and (DAD)PtCl2(CHA) 4 with free allenes, enthalpy−entropy compensation eﬀect in the ligand exchange process of complexes 2, 3, and 4, NMR spectra of Pt-allene complexes, and X-ray analysis of complexes 1 and 2 (PDF) General experimental details, chemical shift comparison in four and ﬁve coordinated complexes, ﬂuxional behavior of the DAD ligand in (DAD)PtCl2(CHA) 4, studies on allene dissociation on the (DAD)PtCl2(CHA) 4 depending on solvent, details on the VT and DNMR line-shape analysis of P(y)PtCl2(TMA) 1, details on experimental setup and analysis of SSTD NMR of the rotational movement and ligand exchange of (Py)- PtCl2(DMA) 2, and ligand exchange of (DAD)- PtCl2(DMA) 3 and (DAD)PtCl2(CHA) 4 with free allenes, enthalpy−entropy compensation eﬀect in the ligand exchange process of complexes 2, 3, and 4, NMR spectra of Pt-allene complexes, and X-ray analysis of complexes 1 and 2 (PDF) ( ) (DAD)PtCl2(DMA) 3. This compound was prepared in situ from a mixture of the (DAD)PtCl2(ethylene) (1 mmol) and the dimethy- lallene (1 mmol) in THF-d8 (100 mL/mmol). A nitrogen ﬂow was ﬂuxed though the solution for 10 min to make sure that all the ethylene was removed. 1H NMR (500 MHz, THF, 25 °C) δ (ppm) = 8.92 (m, 2H, CHN), 7.91 (m, 4H, Ar PMP), 7.02 (br s, 4H, Ar PMP), 3.85 (s, 6H, 2OCH3), 3.08 (m, 2H, ·CH2), 2.06 (s, 3H, CH3 A); 1.77 (s, 3H, CH3 B). 13C NMR (MHz, THF, 25 °C) δ (ppm) = 165.9 (CN), 161.6 (OCq), 126.6 (Cq), 123,5 (CAr PMP), 115.5 (CAr PMP), 115,1 (Cq Allene), 94.6 (Cq Allene), 56.0 (OCH3), 24.4 (CH3 A), 21.1 (CH3 B), 19.1 (CH2).37 (DAD)PtCl2(CHA) 4. To a solution of the platinum-alkene complex (1 mmol) in DCM (70 mL/mmol) was added the allene (1 mmol). The solution was stirred for 10 min, and then the mixture was ﬁltered through Celite and the solvent was evaporated, aﬀording the platinum- allene complex. ■ACKNOWLEDGMENTS Funding by the University of East Anglia, the EPSRC (EP/ L012855/1), and the EU (H2020-MSCA-IF-2014-EF-ST- 658172) is gratefully acknowledged (M.T.Q.). The authors thank Dr. C. J. Macdonald, NMR manager of the School of Chemistry at UEA, for his help with the NMR experiments. We thank the EPSRC UK National Crystallography Service at the University of Southampton for the collection of the crystallo- graphic data. The authors thank the EPSRC National Mass Spectrometry Service Centre, Swansea, for MS analysis. ( ) (7) So far, a fully relativistic comparison of gold and platinum catalysis has only been conducted for the activation of alkynes by these metals: Pernpointner, M.; Hashmi, A. S. J. Chem. Theory Comput. 2009, 5, 2717−2725. In this context, also the computational study of σ- versus π-allyl complexes must be noted: Hashmi, A. S.; Schuster, A. M.; Litters, S.; Rominger, F.; Pernpointner, M. Chem. - Eur. J. 2011, 17, 5661−5667. (7) So far, a fully relativistic comparison of gold and platinum catalysis has only been conducted for the activation of alkynes by these metals: Pernpointner, M.; Hashmi, A. S. J. Chem. Theory Comput. 2009, 5, 2717−2725. In this context, also the computational study of σ- versus π-allyl complexes must be noted: Hashmi, A. S.; Schuster, A. M.; Litters, S.; Rominger, F.; Pernpointner, M. Chem. - Eur. J. 2011, 17, 5661−5667. (7) So far, a fully relativistic comparison of gold and platinum catalysis has only been conducted for the activation of alkynes by these metals: Pernpointner, M.; Hashmi, A. S. J. Chem. Theory Comput. 2009, 5, 2717−2725. In this context, also the computational study of σ- versus π-allyl complexes must be noted: Hashmi, A. S.; Schuster, A. M.; Litters, S.; Rominger, F.; Pernpointner, M. Chem. - Eur. J. 2011, 17, 5661−5667. ■REFERENCES (1) For selected reviews on transition-metal-catalyzed reactions of allenes, see: (a) Hashmi, A. S. K. Angew. Chem., Int. Ed. 2000, 39, 3590−3593. (b) Zimmer, R.; Dinesh, C. U.; Nandanan, E.; Khan, F. A. Chem. Rev. 2000, 100, 3067−3125. (c) Alcaide, B.; Almendros, P.; Martínez del Campo, M. T. Chem. - Eur. J. 2010, 16, 5836−5842. (d) López, M.; Mascareñas, J. L. Chem. - Eur. J. 2011, 17, 418−428. (e) Aubert, C.; Fensterbank, L.; Garcia, P.; Malacria, M.; Simonneau, A. Chem. Rev. 2011, 111, 1954−1993. (f) Krause, N.; Winter, C. Chem. Rev. 2011, 111, 1994−2009. (g) Yu, S.; Ma, S. Angew. Chem., Int. Ed. 2012, 51, 3074−3112. (h) Themed issue: Progress in Allene Chemistry (guest editors B. Alcaide and P. Almendros) Chem. Soc. Rev. 2014, 43, 2879−3206. For a review on insertion reactions of allenes with transition-metal complexes, see: (i) Bai, T.; Ma, S.; Jia, G. Coord. Chem. Rev. 2009, 253, 423−448. (8) Phosphine-Pt-allene complexes are unstable and show slow dissociation of the allene in solution, with slow ﬂuxional behavior. For selected examples, see: (a) Yasuoka, N.; Morita, M.; Kai, Y.; Kasai, N. J. Organomet. Chem. 1975, 90, 111−121. (b) Briggs, J. R.; Crocker, C.; McDonald, W. S.; Shaw, B. L. J. Chem. Soc., Dalton Trans. 1981, 121− 125. As phosphine-Pt complexes are not active catalysts in our reactions, we did not include them in this study. (8) Phosphine-Pt-allene complexes are unstable and show slow dissociation of the allene in solution, with slow ﬂuxional behavior. For selected examples, see: (a) Yasuoka, N.; Morita, M.; Kai, Y.; Kasai, N. J. Organomet. Chem. 1975, 90, 111−121. (b) Briggs, J. R.; Crocker, C.; McDonald, W. S.; Shaw, B. L. J. Chem. Soc., Dalton Trans. 1981, 121− 125. As phosphine-Pt complexes are not active catalysts in our reactions, we did not include them in this study. y (9) For selected examples of other Pt-allene complexes, see: (a) Osborn, J. A. Chem. Commun. 1968, 1231−1232. (b) Yasuoka, N.; Morita, M.; Kai, Y.; Kasai, N. J. Organomet. Chem. 1975, 90, 111− 121. (c) Hewitt, T. G.; de Boer, J. J. J. Chem. Soc. A 1971, 817−822. (d) Lentz, D.; Nickelt, N.; Willemsen, S. Chem. - Eur. J. 2002, 8, 1205−1217. y (9) For selected examples of other Pt-allene complexes, see: (a) Osborn, J. A. Chem. Commun. 1968, 1231−1232. (b) Yasuoka, N.; Morita, M.; Kai, Y.; Kasai, N. J. Organomet. Chem. 1975, 90, 111− 121. (c) Hewitt, T. Notes The authors declare no competing ﬁnancial interest. The authors declare no competing ﬁnancial interest. ■AUTHOR INFORMATION (6) For selected reviews on metal-catalyzed hydroalkoxylation of allenes, see: (a) Muñoz, M. P. Org. Biomol. Chem. 2012, 10, 3584− 3594 and references therein. (b) Muñoz, M. P. Chem. Soc. Rev. 2014, 43, 3164−3183. For selected examples on Au-catalyzed hydro- alkoxylation of allenes, see: (c) Hoffmann-Roder, A.; Krause, N. Org. Lett. 2001, 3, 2537−2538. (d) Nishina, N.; Yamamoto, Y. Tetrahedron Lett. 2008, 49, 4908−4911. (e) Zhang, Z.; Widenhoefer, R. A. Org. Lett. 2008, 10, 2079−2081. (f) Zhang, Z.; Lee, S. D.; Fisher, A. S.; Widenhoefer, R. A. Tetrahedron 2009, 65, 1794−1798. (g) Nishina, N.; Yamamoto, Y. Tetrahedron 2009, 65, 1799−1808. (h) Cui, D.-M.; Yu, K. R.; Zhang, C. Synlett 2009, 2009, 1103−1106. (i) Cui, D.-M.; Zheng, Z.-L.; Zhang, C. J. Org. Chem. 2009, 74, 1426−1427. (j) Paton, R. S.; Maseras, F. Org. Lett. 2009, 11, 2237−2240. (k) Hadfield, M. S.; Lee, A.-L. Org. Lett. 2010, 12, 484−487. For selected examples on Au- catalyzed indolylation of allenes, see: (l) Zhang, Z.; Liu, C.; Kinder, R. E.; Han, X.; Qian, H.; Widenhoefer, R. A. J. Am. Chem. Soc. 2006, 128, 9066−9073. (m) Liu, C.; Widenhoefer, R. A. Org. Lett. 2007, 9, 1935− 1938. (n) Widenhoefer, R. A. Chem. - Eur. J. 2008, 14, 5382−5391. (o) Shen, H. C. Tetrahedron 2008, 64, 3885−3903. (4) For selected examples, see: (a) Hashmi, A. S. K.; Kurpejović, E.; Frey, W.; Bats, J. W. Tetrahedron 2007, 63, 5879−5885. (b) Zhang, G.; Catalano, V. J.; Zhang, L. J. Am. Chem. Soc. 2007, 129, 11358−11359. (c) Computational Mechanisms of Au and Pt Catalyzed Reactions; Soriano, E., Marco-Contelles, J. M., Eds.; Topics in Current Chemistry 302; Spriger: Heidleberg, 2011. The first gold-catalyzed reactions of allenes were reported in 2000; see: Hashmi, A. S. K.; Schwarz, L.; Choi, J. H.; Frost, T. M. Angew. Chem., Int. Ed. 2000, 39, 2285−2288. (5) (a) Muñoz, M. P.; de la Torre, M. C.; Sierra, M. A. Adv. Synth. Catal. 2010, 352, 2189−2194. (b) Muñoz, M. P.; de la Torre, M. C.; Sierra, M. A. Chem. - Eur. J. 2012, 18, 4499−4504. Organometallics Article * S Supporting Information From 35 mg (0.062 mmol) of the platinum-alkene complex, the platinum-allene complex (37.2 mg, 91%) was obtained as a red solid. 1H NMR (500 MHz, THF, 25 °C) δ (ppm) = 8.96 (br s, Crystallographic data for complexes 1 and 2 (CIF) Chemical structure for complexes 1 and 2 (XYZ) Crystallographic data for complexes 1 and 2 (CIF) Chemical structure for complexes 1 and 2 (XYZ) K DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Article (3) For studies on coordination of gold to the central carbon of allenes, see: (a) Chenier, J. H. B.; Howard, J. A.; Mile, B. J. Am. Chem. Soc. 1985, 107, 4190−4191. (b) Gandon, V.; Lemière, G.; Hours, A.; Fensterbank, L.; Malacria, M. Angew. Chem., Int. Ed. 2008, 47, 7534− 7538. (c) Dyker, C. A.; Lavallo, V.; Donnadieu, B.; Bertrand, G. Angew. Chem., Int. Ed. 2008, 47, 3206−3209. (d) Kaufhold, O.; Hahn, F. E. Angew. Chem., Int. Ed. 2008, 47, 4057−4061. (e) Fürstner, A.; Alcarazo, M.; Goddard, R.; Lehmann, C. W. Angew. Chem., Int. Ed. 2008, 47, 3210−3214. (f) Esterhuysen, C.; Frenking, G. Chem. - Eur. J. 2011, 17, 9944−9956. ■REFERENCES 1973, 7, 1−10. For a review on gold-η2-coordination to unsaturated compounds, see: (c) Schmidbaur, H.; Schier, A. Organometallics 2010, 29, 2−23. For computational studies on metal-allene complexes: (d) Yang, W.; Hashmi, S. A. K. Chem. Soc. Rev. 2014, 43, 2941−2955. (e) Soriano, E.; Fernández, I. Chem. Soc. Rev. 2014, 43, 3041−3105. (2) For selected reviews on metal-allene complexes, see: Bowden, F. L.; Giles, R. Coord. Chem. Rev. 1976, 20, 81−106. (b) Shaw, B. L.; Stringer, A. J. Inorg. Chim. Acta, Rev. 1973, 7, 1−10. For a review on gold-η2-coordination to unsaturated compounds, see: (c) Schmidbaur, H.; Schier, A. Organometallics 2010, 29, 2−23. For computational studies on metal-allene complexes: (d) Yang, W.; Hashmi, S. A. K. Chem. Soc. Rev. 2014, 43, 2941−2955. (e) Soriano, E.; Fernández, I. Chem. Soc. Rev. 2014, 43, 3041−3105. (3) For studies on coordination of gold to the central carbon of allenes, see: (a) Chenier, J. H. B.; Howard, J. A.; Mile, B. J. Am. Chem. Soc. 1985, 107, 4190−4191. (b) Gandon, V.; Lemière, G.; Hours, A.; Fensterbank, L.; Malacria, M. Angew. Chem., Int. Ed. 2008, 47, 7534− 7538. (c) Dyker, C. A.; Lavallo, V.; Donnadieu, B.; Bertrand, G. Angew. Chem., Int. Ed. 2008, 47, 3206−3209. (d) Kaufhold, O.; Hahn, F. E. Angew. Chem., Int. Ed. 2008, 47, 4057−4061. (e) Fürstner, A.; Alcarazo, M.; Goddard, R.; Lehmann, C. W. Angew. Chem., Int. Ed. 2008, 47, 3210−3214. (f) Esterhuysen, C.; Frenking, G. Chem. - Eur. J. 2011, 17, 9944−9956. (3) For studies on coordination of gold to the central carbon of allenes, see: (a) Chenier, J. H. B.; Howard, J. A.; Mile, B. J. Am. Chem. Soc. 1985, 107, 4190−4191. (b) Gandon, V.; Lemière, G.; Hours, A.; Fensterbank, L.; Malacria, M. Angew. Chem., Int. Ed. 2008, 47, 7534− 7538. (c) Dyker, C. A.; Lavallo, V.; Donnadieu, B.; Bertrand, G. Angew. Chem., Int. Ed. 2008, 47, 3206−3209. (d) Kaufhold, O.; Hahn, F. E. Angew. Chem., Int. Ed. 2008, 47, 4057−4061. (e) Fürstner, A.; Alcarazo, M.; Goddard, R.; Lehmann, C. W. Angew. Chem., Int. Ed. 2008, 47, 3210−3214. (f) Esterhuysen, C.; Frenking, G. Chem. - Eur. J. 2011, 17, 9944−9956. (11) SSTD NMR method: (a) Quirós, M. T.; Angulo, J.; Muñoz, M. P. Chem. Commun. 2015, 51, 10222−10225. (b) Quirós, M. T.; MacDonald, C.; Angulo, J.; Muñoz, M. P. J. Visualized Exp. 2016, 117, e54499. (11) SSTD NMR method: (a) Quirós, M. T.; Angulo, J.; Muñoz, M. P. Chem. Commun. (12) For an example of intra- vs intermolecular pathways in the ﬂuxional behavior of platinum(0) complexes, see: Zenkina, O. V.; Konstantinovski, L.; Freeman, D.; Shimon, L. J. W.; van der Boom, M. E. Inorg. Chem. 2008, 47 (9), 3815−3822. ■REFERENCES G.; de Boer, J. J. J. Chem. Soc. A 1971, 817−822. (d) Lentz, D.; Nickelt, N.; Willemsen, S. Chem. - Eur. J. 2002, 8, 1205−1217. (10) We have tested some of the complexes in our dihydroxylation reaction, and they have shown catalytic activity. For example, reaction of cyclohexylallene with 1,2-propandiol in the presence of catalytic DADPtCl2 (DAD = tBu-NCH-CHN-tBu) (5 mol%)/AgNTf2 (10 mol%) gave the cyclic acetal in 66% when the reaction was carried out in THF at 70 °C for 20 h. DADPtCl2(ethylene) (DAD = PMP- NCH-CHN-PMP) gave the cyclic acetal in 96% in 7 h, and a similar result was obtained with that complex and AgNTf2. PyPtCl2(ethylene) gave the cyclic acetal in 60% in 30 h. Besides, the preformed complex 4 also acts as catalyst in the reaction of allenes with alcohols as nucleophiles. These results and the full mechanistic study of this reaction will be reported in detail in due course. (10) We have tested some of the complexes in our dihydroxylation reaction, and they have shown catalytic activity. For example, reaction of cyclohexylallene with 1,2-propandiol in the presence of catalytic DADPtCl2 (DAD = tBu-NCH-CHN-tBu) (5 mol%)/AgNTf2 (10 mol%) gave the cyclic acetal in 66% when the reaction was carried out in THF at 70 °C for 20 h. DADPtCl2(ethylene) (DAD = PMP- NCH-CHN-PMP) gave the cyclic acetal in 96% in 7 h, and a similar result was obtained with that complex and AgNTf2. PyPtCl2(ethylene) gave the cyclic acetal in 60% in 30 h. Besides, the preformed complex 4 also acts as catalyst in the reaction of allenes with alcohols as nucleophiles. These results and the full mechanistic study of this reaction will be reported in detail in due course. ( ) ( ) (2) For selected reviews on metal-allene complexes, see: Bowden, F. L.; Giles, R. Coord. Chem. Rev. 1976, 20, 81−106. (b) Shaw, B. L.; Stringer, A. J. Inorg. Chim. Acta, Rev. 1973, 7, 1−10. For a review on gold-η2-coordination to unsaturated compounds, see: (c) Schmidbaur, H.; Schier, A. Organometallics 2010, 29, 2−23. For computational studies on metal-allene complexes: (d) Yang, W.; Hashmi, S. A. K. Chem. Soc. Rev. 2014, 43, 2941−2955. (e) Soriano, E.; Fernández, I. Chem. Soc. Rev. 2014, 43, 3041−3105. (2) For selected reviews on metal-allene complexes, see: Bowden, F. L.; Giles, R. Coord. Chem. Rev. 1976, 20, 81−106. (b) Shaw, B. L.; Stringer, A. J. Inorg. Chim. Acta, Rev. Organometallics ( ) (36) Interestingly, the gold complex with the same allene displays an associative mechanism with a two-term rate law with nonzero intercept, suggesting competition between the allene and solvent complexation in the associative mechanism. See ref 23b. However, our k h l h h b d f h h (36) Interestingly, the gold complex with the same allene displays an associative mechanism with a two-term rate law with nonzero intercept, suggesting competition between the allene and solvent complexation in the associative mechanism. See ref 23b. However, our kobs are much slower than the observed for the associative mechanism in the gold-allene complexes, supporting the dissociative mechanism in the Pt-allene complexes. complexation in the associative mechanism. See ref 23b. However, our kobs are much slower than the observed for the associative mechanism in the gold-allene complexes, supporting the dissociative mechanism in the Pt-allene complexes. Mingos, P., Crabtree, R. H., Eds.; Comprehensive Organometallic Chemistry III; Elsevier: Oxford, 2007; Vol. 8.09, pp 611−673. (19) (a) Vrieze, K.; Volger, H. C.; Gronert, M.; Praat, A. P. J. Organomet. Chem. 1969, 16, P19−P22. (b) Vrieze, K.; Volger, M.; Praat, A. P. J. Organomet. Chem. 1970, 21, 467−475. p (37) (a) Herrick, R. S.; Leazer, D. M.; Templeton, J. L. Organometallics 1983, 2, 834−840. (b) Wendt, O. F.; Deeth, R. J.; Elding, L. I. Inorg. Chem. 2000, 39, 5271−5276. (c) Hatrwig, J. Organotransition Metal Chemistry: From Bonding to Catalysis; University Science Books: Sausalito, CA, 2010; Chapters 5.3 and 5.4, pp 230−231. (37) (a) Herrick, R. S.; Leazer, D. M.; Templeton, J. L. Organometallics 1983, 2, 834−840. (b) Wendt, O. F.; Deeth, R. J.; g , , ( ) , ; , J ; Elding, L. I. Inorg. Chem. 2000, 39, 5271−5276. (c) Hatrwig, J. Organotransition Metal Chemistry: From Bonding to Catalysis; University Science Books: Sausalito, CA, 2010; Chapters 5.3 and 5.4, pp 230−231. J g (20) Coles, S. J.; Gale, P. A. Chem. Sci. 2012, 3, 683−689. (21) Malacria et al. have reported DFT calculations showing that η1- Pt(II)-allene complexes adopt a bent conformation with CCC angles of 129−140°. See ref 3b. pp (38) (a) Leffler, J. E. J. Org. Chem. 1955, 20, 1202−1231. (b) Petersen, R. C. J. Org. Chem. 1964, 29, 3133−3135. (c) McGregor, R.; Milicevic, B. Nature 1966, 211, 523−525. (d) Exner, O. Nature 1970, 227, 366−367. (e) Krug, R. R.; Hunter, W. G.; Grieger, R. A. J. Phys. Chem. (30) We choose TCE for our experiments to compare our results with the previously reported in chloroform but with the ﬂexibility of higher temperature range for the studies with complex 2. Both solvents are considered noncoordinating (DN ∼0), so no interactions with the solvent are expected in any case. Organometallics 1976, 80, 2335−2340. (f) Krug, R. R.; Hunter, W. G.; Grieger, R. A. J. Phys. Chem. 1976, 80, 2341−2351. (g) Liu, L.; Guo, Q.-X. Chem. Rev. 2001, 101, 673−695. (h) Bunten, K. A.; Poë, A. New J. Chem. 2006, 30, 1638−1649. (22) For selected examples of proposed η1-Pt-allene as intermediates or transition states, see: (a) Funami, H.; Kusama, H.; Iwasawa, N. Angew. Chem., Int. Ed. 2007, 46, 909−911. (b) Trillo, B.; López, M.; Gulías, M.; Castedo, L.; Mascareñas, J. L. Angew. Chem., Int. Ed. 2008, 47, 951−954. (23) Interestingly, this eﬀect is opposite in Au-allene complexes as reported by Widenhoefer: (a) Brown, T. J.; Sugie, A.; Dickens, M. G.; Widenhoefer, R. A. Organometallics 2010, 29, 4207−4209. (b) Brown, T. J.; Sugie, A.; Leed, M. G. D.; Widenhoefer, R. A. Chem. - Eur. J. 2012, 18, 6959−6971. (39) HRMS of the new complexes could not be obtained due to decomposition. Complex 4 and its precursor with ethylene were analyzed at the EPSRC National Mass Spectrometry Service Centre (39) HRMS of the new complexes could not be obtained due to decomposition. Complex 4 and its precursor with ethylene were analyzed at the EPSRC National Mass Spectrometry Service Centre (Swansea) by APCI(ASAP) and also solvated in DCM and diluted into MeOH with NH4OAc prior to analysis by data of positive nanospray. A number of ions that suggested the presence of Pt and Cl, and fragments from the loss of Cl and N-ligand were identiﬁed but could not be fully assigned. (24) Crociani, B.; Di Bianca, F.; Bertani, R.; Zanotto, L. Inorg. Chim. Acta 1988, 141, 253−261. (Swansea) by APCI(ASAP) and also solvated in DCM and diluted into MeOH with NH4OAc prior to analysis by data of positive nanospray. A number of ions that suggested the presence of Pt and Cl, and fragments from the loss of Cl and N-ligand were identiﬁed but could not be fully assigned. (Swansea) by APCI(ASAP) and also solvated in DCM and diluted into MeOH with NH4OAc prior to analysis by data of positive nanospray. A number of ions that suggested the presence of Pt and Cl, and fragments from the loss of Cl and N-ligand were identiﬁed but could not be fully assigned. (25) Gutmann, V. Coord. Chem. Rev. 1976, 18, 225−255 and references therein. (26) For studies on dynamic behavior on Au-allene complexes, see ref 23. (28) For a selected example of Py-Pt-oleﬁn complexes, see: Rowe, M. P.; Steinecker, W. H.; Zellers, E. T. Anal. Chem. 2007, 79, 1164−1172. (29) TopSpin 3.0 program; Bruker Corp., 2015. http://www.bruker. com/products/mr/nmr/nmr-software/software/topspin/. DOI: 10.1021/acs.organomet.6b00778 Organometallics XXXX, XXX, XXX−XXX Organometallics Article Article (16) (a) Orchin, M.; Schmidt, P. J. Inorg. Chim. Acta, Rev. 1968, 2, 123−135. (b) Rowe, M. P.; Steinecker, W. H.; Zellers, E. T. Anal. Chem. 2007, 79, 1164−1172. (31) It is interesting to note the faster π-face exchange shown by the analogous (IPr)AuTMA complex (ΔG⧧(214 K) = 9.5 kcal mol−1), calculated by Widenhoefer et al. (ref 23) by line-shape analysis of the 13C NMR spectrum of the complex at diﬀerent temperatures. (17) For examples of ﬁve- vs four-coordination in oleﬁn-Pt-N,N- bidentate ligands complexes, see: (a) Maresca, L.; Natile, G.; Calligaris, M.; Delise, P.; Randaccio, L. J. Chem. Soc., Dalton Trans. 1976, 2386− 2390. (b) van der Poel, H.; van Koten, G. Inorg. Chem. 1981, 20, 2941−2950. (c) Albano, V. G.; Demartin, F.; de Renzi, A.; Morelli, G.; Saporito, A. Inorg. Chem. 1985, 24, 2032−2039. (d) de Felice, V.; Giordano, F.; Orabona, I.; Tesauro, D.; Vitagliano, A. J. 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For a review on Pt-CC complexes, see: (d) Forniés, J.; Lalinde, E. Platinum−Carbon π-Bonded Complexes; Michael, D., Mingos, P., Crabtree, R. H., Eds.; Comprehensive Organometallic Chemistry III; Elsevier: Oxford, 2007; Vol. 8.09, pp 611−673. (19) (a) Vrieze, K.; Volger, H. C.; Gronert, M.; Praat, A. P. J. Organomet. Chem. 1969, 16, P19−P22. (b) Vrieze, K.; Volger, M.; Praat, A. P. J. Organomet. Chem. 1970, 21, 467−475. ■REFERENCES 2015, 51, 10222−10225. (b) Quirós, M. T.; MacDonald, C.; Angulo, J.; Muñoz, M. P. J. Visualized Exp. 2016, 117, e54499. (12) For an example of intra- vs intermolecular pathways in the ﬂuxional behavior of platinum(0) complexes, see: Zenkina, O. V.; Konstantinovski, L.; Freeman, D.; Shimon, L. J. W.; van der Boom, M. E. Inorg. Chem. 2008, 47 (9), 3815−3822. (4) For selected examples, see: (a) Hashmi, A. S. K.; Kurpejović, E.; Frey, W.; Bats, J. W. Tetrahedron 2007, 63, 5879−5885. (b) Zhang, G.; Catalano, V. J.; Zhang, L. J. Am. Chem. 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https://openalex.org/W1974475050	https://europepmc.org/articles/pmc4045534?pdf=render	English	null	A Review of Hypothalamic-Pituitary-Adrenal Axis Function in Chronic Fatigue Syndrome	ISRN Neuroscience	2,013	cc-by	6,994	Cara Tomas,1 Julia Newton,1 and Stuart Watson1,2 Cara Tomas,1 Julia Newton,1 and Stuart Watson1,2 1 Newcastle University, Newcastle upon Tyne NE1 7RU, UK 2 Wolfson Research Centre, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK 1 Newcastle University, Newcastle upon Tyne NE1 7RU, UK 2 Wolfson Research Centre, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK 1 Newcastle University, Newcastle upon Tyne NE1 7RU, UK 2 Wolfson Research Centre, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK Correspondence should be addressed to Stuart Watson; stuart.watson@ncl.ac.uk Received 28 June 2013; Accepted 29 August 2013 Academic Editors: C. Bishop, P. de Gortari, G. Forster, and G. Hans Copyright © 2013 Cara Tomas 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. Hypothalamic-pituitary-adrenal (HPA) axis dysfunction has been found in a high proportion of chronic fatigue syndrome (CFS) patients and includes enhanced corticosteroid-induced negative feedback, basal hypocortisolism, attenuated diurnal variation, and a reduced responsivity to challenge. A putative causal role for genetic profile, childhood trauma, and oxidative stress has been considered. In addition, the impact of gender is demonstrated by the increased frequency of HPA axis dysregulation in females. Despite the temporal relationship, it is not yet established whether the endocrine dysregulation is causal, consequent, or an epiphenomenon of the disorder. Nonetheless, given the interindividual variation in the effectiveness of existing biological and psychological treatments, the need for novel treatment strategies such as those which target the HPA axis is clear. Hindawi Publishing Corporation ISRN Neuroscience Volume 2013, Article ID 784520, 8 pages http://dx.doi.org/10.1155/2013/784520 Hindawi Publishing Corporation ISRN Neuroscience Volume 2013, Article ID 784520, 8 pages http://dx.doi.org/10.1155/2013/784520 Hindawi Publishing Corporation ISRN Neuroscience Volume 2013, Article ID 784520, 8 pages http://dx.doi.org/10.1155/2013/784520 1. Introduction Dysregulation of the biological systems which mediate the response to stress potentially has an important role in the aetiopathogenesis of CFS [1, 4, 10]. The neurobiological stress system comprises a range of networks that form intricate pathways; an important part of this is the hypothalamic- pituitary-adrenal (HPA) axis [11–14] which is a self-regulated feedback system which contributes to the maintenance of homeostasis and which is impacted by multiple factors such as time of day and physical and psychological stressors [2, 15]. There are a number of structures within the HPA axis, includ- ing the paraventricular nucleus (PVN) of the hypothalamus which releases corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) which in turn stimulates the pituitary to secrete adrenocorticotropic hormone (ACTH) into the systemic circulation. The ACTH acts at the adrenal gland to stimulate the synthesis and secretion of cortisol. Cortisol is released in a pulsatile fashion and ensures strict regulation of both feedforward and feedback loops involving the HPA axis. Hence circulating cortisol activates mineralo- corticoid and glucocorticoid receptors (MR and GR) and so decreases the secretion of CRH, AVP, and ACTH [16, 17]. This feedback mechanism is shown in Figure 1. Functional capacity of glucocorticoid receptors is considered, by some, Chronic fatigue syndrome (CFS) is a debilitating illness which was classified as a neurological disease in 1993 by the World Health Organisation (WHO) [1]. Symptoms of CFS include persistent fatigue, difficulty with memory and con- centration, a disturbed sleep pattern, and severe muscular- skeletal pain [2]. Post exertional exacerbation of symptoms is common but not invariable [3]. The symptoms displayed vary markedly from patient to patient; some patients remain bedridden for very long periods of time, while others are able to manage their fatigue by staying within their own energy boundaries [4]. Diagnostic reliability is enhanced by the use of operational criteria such the Centre for Disease Control and Prevention Criteria [5], the Oxford Criteria [6] or the International Consensus Criteria [7, 8]. However, the heterogeneous symptom profile and absence of clear biological markers militate against confidence in the validity of CFS as a unitary diagnosis. It is not known, for instance, whether there is a core set of biological processes which underlie all cases of CFS or whether there are multiple processes (and if so, whether or not these potentially disparate processes converge on a final common pathway) [9]. 3. Adrenal Steroid Metabolic Pathways See Figure 2. See Figure 2. Axis Feedback Loops See Figure 1. The hypocortisolaemia, attenuated diurnal variation, and reduced responsivity to challenge seen in these cross- sectional studies may be mediated by upregulation of GR and MR, reduced hormone synthesis, or increased metabolism [8, 35]. The enhanced suppression of cortisol during the dexamethasone [36, 37] and prednisolone suppression test [38] supports the notion that increased functional activity of GR and possibly MR may have pathophysiological signif- icance in CFS. However, as dexamethasone is metabolised via cortisol metabolic pathways, the enhanced cortisol sup- pression during the DST may therefore be caused not by GR upregulation but by reduced dexamethasone metabolism (as a consequence of the enzyme inhibition secondary to persistent hypocortisolaemia [39]).ht 1. Introduction CRH: corticotropin releasing hormone; AVP: arginine vasopressin; ACTH: adrenocorticotropic hormone; MR: mineralocorticoid receptor; GR: glucocorticoid receptor. Figure 2: The metabolic pathway showing the steps involved in the synthesis of cortisol from cholesterol. Cholesterol is converted to pregnenolone by desmolase, which is then converted to proges- terone by 3-B-hydroxysteroid. Progesterone is converted to 17-a- hydroxyprogesterone (17-OH progesterone) by 17-hydroxylase. The 17-OH progesterone is then converted to 11-deoxycortisol by the enzyme 21-hydroxylase. Finally, 11-deoxycortisol is converted into cortisol by 11-hydroxylase. DHEA: dehydroepiandrosterone. nature/magnitude of the stressor engendered by the experi- mental procedure (e.g., the hospital visit or the venepuncture) in patients with CFS compared to comparator subjects [8, 16, 23]. Further, basal studies have also shown an attenuated diurnal variation [8, 24] particularly with a loss of the morning peak of ACTH [8, 20, 21, 25, 26] or cortisol [8, 20, 27] while challenge studies often, but not invariably, show a diminished HPA axis responsivity. This has been assessed using the ACTH, cortisol, and/or 11-deoxycortisol response to pharmacological challenge using, for example, dexametha- sone combined with corticotropin-releasing hormone (CRH) [28], insulin [29], inflammatory cytokines, and metyrapone [30]; to psychological challenge (e.g., using the Trier Social Stress Test [31]), and to physiological challenge (such as wakening) [32–34].h to be the determining factor in the regulation of the HPA axis [18]. The effects of cortisol are both potent and extensive; it affects numerous physiological functions, for instance, in the regulation of the neuroendocrine and sympathetic nervous systems, modulation of the inflammatory response, inhibition of secretion of multiple hormones, and induction of lymphocyte apoptosis [19, 20]. 1. Introduction 2 ISRN Neuroscience Hypothalamus MR Anterior pituitary GR ACTH Cortisol CRH and AVP Adrenal gland −ve −ve −ve Figure 1: The effect of stress on the hypothalamic-pituitary-adrenal axis. The hypothalamus secretes both CRH and AVP from the par- aventricular nucleus which stimulate the production of ACTH in the anterior pituitary. ACTH travels via the blood until it stimulates the adrenal gland to secrete cortisol. Cortisol acts via a negative feedback mechanism mediated by MR and GR in the hypothalamus and GR in the anterior pituitary, which ultimately causes a decrease in secretion of CRH, AVP and ACTH. CRH: corticotropin releasing hormone; AVP: arginine vasopressin; ACTH: adrenocorticotropic hormone; MR: mineralocorticoid receptor; GR: glucocorticoid receptor. Deoxycorticosterone Cortisol Pregnenolone Progesterone 11-Deoxycortisol Corticosterone Cholesterol DHEA Aldosterone 17-OH progesterone Adrenal androgens Figure 2: The metabolic pathway showing the steps involved in the synthesis of cortisol from cholesterol. Cholesterol is converted to pregnenolone by desmolase, which is then converted to proges- terone by 3-B-hydroxysteroid. Progesterone is converted to 17-a- hydroxyprogesterone (17-OH progesterone) by 17-hydroxylase. The 17-OH progesterone is then converted to 11-deoxycortisol by the enzyme 21-hydroxylase. Finally, 11-deoxycortisol is converted into cortisol by 11-hydroxylase. DHEA: dehydroepiandrosterone. Deoxycorticosterone Cortisol Pregnenolone Progesterone 11-Deoxycortisol Corticosterone Cholesterol DHEA Aldosterone 17-OH progesterone Adrenal androgens Hypothalamus MR Anterior pituitary GR ACTH Cortisol CRH and AVP Adrenal gland −ve −ve −ve Figure 1: The effect of stress on the hypothalamic-pituitary-adrenal axis. The hypothalamus secretes both CRH and AVP from the par- aventricular nucleus which stimulate the production of ACTH in the anterior pituitary. ACTH travels via the blood until it stimulates the adrenal gland to secrete cortisol. Cortisol acts via a negative feedback mechanism mediated by MR and GR in the hypothalamus and GR in the anterior pituitary, which ultimately causes a decrease in secretion of CRH, AVP and ACTH. CRH: corticotropin releasing hormone; AVP: arginine vasopressin; ACTH: adrenocorticotropic hormone; MR: mineralocorticoid receptor; GR: glucocorticoid receptor. Figure 1: The effect of stress on the hypothalamic-pituitary-adrenal axis. The hypothalamus secretes both CRH and AVP from the par- aventricular nucleus which stimulate the production of ACTH in the anterior pituitary. ACTH travels via the blood until it stimulates the adrenal gland to secrete cortisol. Cortisol acts via a negative feedback mechanism mediated by MR and GR in the hypothalamus and GR in the anterior pituitary, which ultimately causes a decrease in secretion of CRH, AVP and ACTH. 5. Cause of the HPA Axis Dysregulation in CFS It is valuable to explore the mechanisms which may explain the HPA axis dysregulation seen in adults with CFS. Specific genes (acting on the HPA axis or otherwise) which confer an increased risk of CFS have not been identified. There is, however, evidence of a heritable component to the disorder [45]. In addition, the role of early adversity also warrants consideration, particularly given the evidence of an increased rate of childhood trauma in patients with CFS. Around 50% of patients report at least one type of childhood trauma [46, 47]. It has been estimated that childhood trauma increases the risk of CFS between 6- and 8-fold [33, 48] with a graded relationship between the severity of the trauma and the risk of developing CFS [33, 46, 48]. Furthermore, an increased severity of symptoms has been noted in those who report childhood trauma [48]. It is increasingly established in other disease areas that childhood trauma acting via the HPA axis impacts the risk of disorder in adulthood, but it must also be remembered that early adversity is a broad concept which encompasses much more than childhood physical, emotional, and sexual abuse and neglect. Another potential cause of disruption to HPA axis func- tion is oxidative stress and a decrease in antioxidant capacity in addition to the presence of histone deacetylase (HDAC) [72]. Increased activity of HDAC 2 and 3 coincides with a decrease in plasma cortisol [14]. This theory has been cited as another possible cause of hypocortisolism found in patients. Animal models of early-life stress reveal HPA axis changes which persist into, or became evident in, adulthood [49]. There are a number of potential mechanisms. Variations in maternal care in rodents [50, 51], early life neonatal stress [52, 53], and childhood trauma in man [54–56] have been shown to increase methylation in the CpG-rich regions of a broad range of candidate gene promotors and transcriptional and intragenic sequences [57, 58]. One of the better studied has been the exon 1F promotor region of the GR gene (NR3C1). Increased methylation reduces transcription of the NR3C1 gene and so reduces hippocampal GR expression and increases glucocorticoid secretion in rodents [50, 51] and the cortisol stress response in man [52, 53]. Whilst this picture is not typical of HPA axis function in CFS patients, it nonetheless demonstrates the potential for neonatal and early childhood environment to impact on HPA axis function in adulthood. 4. HPA Axis Function in Patients with CFS Basal hypocortisolism was first reported in CFS patients in 1981 [21]. Cortisol concentrations have since been measured in blood, saliva, and urine in a number of studies with rather varying results (reviewed in [8]), but the notion of a hypocortisolaemic picture in CFS is supported by a meta- analysis [22]. Reduced cortisol levels are more apparent in female patients and also tend to occur during the later stages of the illness [22]. These abnormal cortisol concentrations may reflect differences in the biological mediation of the stress response or may be consequent on the differential The thesis that the hypocortisolaemia is caused by a shift in the balance of the various metabolic pathways (Figure 2) in steroid synthesis is tentatively supported but by no means 3 ISRN Neuroscience sensitivity of the hippocampus, particularly the subiculum, to adversity. This is noteworthy because the subiculum has an important role in regulating the HPA axis; hence, neu- roarchitectural change secondary to early adversity would be expected to alter the dynamics of the HPA axis in adulthood. Stress in adolescence or adulthood, such as that conferred by infection by microbes such as Epstein-Barr virus, Coxiella burnetii, and enteroviruses, is common but on occasion leads to CFS. It has been argued that this may be a result of disease in the central nervous system [63], but it is also worth considering whether preexisting HPA axis function or the nature or magnitude of the HPA axis response to this stressor plays a part in determining whether infection will precipitate CFS. We should be mindful that the adaptive response to the HPA axis changes of such genetic factors or stressors may confer an allostatic load which predisposes to CFS [64]. It has, for instance, been suggested that the allostatic response to persistent cortisol elevation may be a “switch” to hypocortisolaemia [35, 65–67] mediated by a relative increased reliance on AVP rather than CRH to drive [68], or on MR rather than GR to control, the axis [69, 70]. We are still a long way from understanding the evolutionary advantage of the altered regulation of the HPA axis in patients who have, and those who go on to develop, CFS; perhaps CFS is the cost of the cortisol response to challenge (including social challenge) which may be necessary to adapt to the complex dynamics of human social competition [71]. 5. Cause of the HPA Axis Dysregulation in CFS The methylation is measurable in blood lym- phocytes and therefore provides a useful tool to examine the relationship between attachment and subsequent HPA axis function in patients with CFS [55]. FK506 binding protein 5 (FKBP5) impedes GR activation [59, 60]. Polymorphisms in the FKBP5 gene interact with early trauma to predict a number of stress-related psychiatric disorders. An elegant series of experiments by Elizabeth Binder’s group suggests that this interaction is mediated by stress-induced methyla- tion of FKBP5 during critical developmental windows [61].l 4. HPA Axis Function in Patients with CFS proven by studies examining plasma concentrations of dehy- droepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) and by those which calculate the corti- sol : DHEA ratio. DHEA levels have been shown to be normal [29], numerically [40] or significantly [41] increased; DHEAS levels have been reported to be reduced [42, 43], sand the cortisol : DHEA ratio has been shown to be normal [41, 44] or, in the larger study, reduced [40] in patients with CFS. 9. Conclusion and Further Research HPA axis dysregulation appears to be associated with CFS. A credible body of evidence suggests a mechanism by which genetic and environmental factors (and their interaction) may serve to create an endocrine milieu which may impact on immune and vascular processes and thus putatively precipitate and maintain the symptoms experienced by those with a diagnosis of CFS. Nonetheless the abnormalities are subtle, and there is marked variation in basal and challenge tests in CFS patients and a real risk that these so-called abnormalities are simply confounds or epiphenomena.hi 6. Impact of HPA Axis Dysregulation Having argued that altered HPA axis function may have an aetiopathological role in CFS, it remains necessary to consider the link between cortisol and the typical symptoms of CFS. This may be mediated by immune mechanisms as a dysregulated HPA axis, particularly if characterised by hypocortisolaemia, has the potential to reduce the capacity with which HPA axis hormones can restrain the immune system. As a result, relatively minor physical or psychological stressors may be transduced into an inflammatory response by triggering the release of inflammasomes and subsequently proinflammatory cytokines [73, 74]. This process would be expected to evoke a pathological illness with symptoms such as those found in CFS patients [31, 75–77]. Further work is needed to quantify the inflammatory response in CFS patients. Cytokine-mediated inflammation may also explain the prevalent pain and hypersensitivity that affects CFS patients [78]. A vascular aetiology of CFS has also been proposed. This is a current research interest of our group and is exemplified by the relationship and overlap between CFS and postural orthostatic tachycardia syndrome (POTS) [79, 80] which typically presents with fatigue, dizziness, and an inability to Early life stress also influences brain development [33]. Teicher and colleagues [62] have recently demonstrated the 4 ISRN Neuroscience long-term treatment including Cushing’s syndrome, osteo- porosis, extreme mood changes, and seizures cautions against this approach [99]. The HPA axis though remains a potential target for novel treatment strategies in CFS and this has been examined in studies utilizing animal models to examine traditional medicines with a putative HPA axis effect; for example, Shilajit, a traditional Indian medicine, reduced immobility and increased climbing behavior whilst increas- ing adrenal weight and corticosterone levels in the forced swim test in rats [72] and Myelophil, based on compounds used for fatigue in Chinese medicine, increased glucocorti- coid receptor expression in the hypothalamus and hippocam- pus, and altered expression of cytokines such as interleukin (IL-10) and tumour necrosis factor-alpha (TNF-𝛼) using the chronic cold stress and restraint model in mice [100]. exercise. HPA axis dysregulation, particularly hypocortiso- laemia, can cause hypotension and may possibly mediate the fatigue experienced by CFS patients by inducing orthostatic hypotension and hence reducing cerebral perfusion [81]. 7. Relationship between HPA Axis Dysfunction and Symptoms The demonstrated association between the magnitude of HPA axis dysfunction and symptom severity highlights the relationship between the endocrinology and the disorder [8, 82, 83]. This is further emphasised by the demonstration that HPA axis dysregulation is a poor prognostic factor for CFS patients undergoing psychological treatment [22, 84]. The HPA axis dysregulation may have a causal role in CFS; it may be consequent on the disorder or it may be an epiphenomenon. Experimentally induced, or pathological, hypocortisolaemia (such as that seen in Addison’s disease) is associated with symptoms typical of CFS, including fatigue, weakness, and abdominal pain, but it is also associated with a range of other features which are not typical of CFS [19, 85]. Further work to delineate the relationship between HPA axis dysfunction and fatigue in Addison’s disease and other hypocortisolaemic states would be of benefit [86, 87]. One of the most interesting proposals is the switch to a new steady state from chronic hypocortisolaemia to a healthy, reactive state using the model-based predictive control (MPC) solution originally proposed by Gupta and colleagues [35]. 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Opportunities for Novel Therapeutic Strategies in Treatment of HPA Axis Dysfunction in CFS The findings that successful psychological treatments normalise the HPA axis dysregulation together with the reports that HPA axis dysregulation is a poor prognostic factor do give optimism that treatments targeting the HPA axis may have efficacy alone or in the augmentation of more established psychological, behavioural, or pharmacological treatments.h Cognitive behavioural therapy (CBT) and graded exer- cise plans have demonstrated efficacy but with significant interindividual variation [4, 23, 78, 89]. These therapies modify illness perception and allow patients to make adjust- ment to optimise energy expenditure. CBT has been shown to increase cortisol levels by reversing some of the effects induced by low activity levels, depression and stress in early life [8, 82, 90, 91]. In addition, many pharmaco- logic treatments have been investigated for CFS including psychostimulants, corticosteroids, anti-inflammatories, and antidepressants [92–94]. There is currently no evidence to suggest that any of these medications have an advantage to patients though antidepressants are widely prescribed [95]. The recent launch of the UK ME/CFS Research Collabo- rative [101] demonstrates the commitment of the government and associated funding bodies to pursue research into the understanding and treatment of this potentially debilitating disorder. This next decade may see an enhanced understand- ing of individual facets of CFS including its genetic and epigenetic signature, immune and vascular processes, the fine detail of HPA axis regulation, and the symptoms and psychological underpinning of the disorder. These should be examined using a network approach to map the intricate relationships and should allow consideration of whether CFS, as it is currently defined, is a unitary construct or if it represents multiple illnesses with different causes, albeit with similar symptom patterns. In addition, prospective studies may demonstrate vulnerability and trait factors and Low-dose hydrocortisone [96–98] and DHEA [42] have both been used as treatment agents in pilot studies in CFS, and have benefitted some patients. There is an argument for further trials of steroid treatment in patients selected on the basis of adrenal insufficiency, but the potential impact of 5 ISRN Neuroscience [14] L. Jason, M. Sorenson, K. Sebally et al., “Increased HDAC in association with decreased plasma cortisol in older adults with chronic fatigue syndrome,” Brain, Behavior, and Immunity, vol. 25, no. 8, pp. 1544–1547, 2011. help to explain why some patients develop these symptoms. 8. Opportunities for Novel Therapeutic Strategies in Treatment of HPA Axis Dysfunction in CFS Hopefully, we can dispel any lingering Cartesian dualism and translate the psychological and biological understanding into holistic therapeutic programmes and novel treatment strategies. Progress is continuously being made; however, for patients who have had their lives destroyed, the development of a cure cannot come fast enough. [15] B. M. Kudielka, N. C. Schommer, D. H. Hellhammer, and C. Kirschbaum, “Acute HPA axis responses, heart rate, and mood changes to psychosocial stress (TSST) in humans at different times of day,” Psychoneuroendocrinology, vol. 29, no. 8, pp. 983– 992, 2004. Acknowledgment [16] A. J. Cleare, “The neuroendocrinology of chronic fatigue syn- drome,” Endocrine Reviews, vol. 24, no. 2, pp. 236–252, 2003. Funding for C. Tomas was provided by the Wellcome Trust. Funding for C. Tomas was provided by the Wellcome Trust. [17] A. M. Bao, G. Meynen, and D. F. 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https://openalex.org/W4393987320	https://ejournal.itn.ac.id/index.php/jati/article/download/8842/5166	Indonesian	null	ANALISIS SENTIMEN DENGAN ALGORITMA NAÏVE BAYES CLASSIFIER TERHADAP ULASAN APLIKASI MY F&B ID	JATI	2,024	cc-by-sa	3,479	1. PENDAHULUAN sentimen memiliki dua metode yaitu metode machine learning dan knowledge based method. Machine learning yang digunakan dalam penelitian analisis sentimen yaitu Support Vector Machine [3], Fuzzy Neural Networks [4], Naïve Bayes Classifier [5], Particle Swarm Optimization [6] dan lain-lain. Salah satu jenis knowledge based adalah Lexicon Based [7]. Di era teknologi yang semakin canggih mendorong semua aspek bidang kehidupan ikut dalam perkembangannya, salah satunya pada bidang maknaan atau kuliner. Kecanggihan hal tersebut ialah memebuat perusahaan makanan berlomba-lomba untuk membuat aplikasi mobile untuk memberi kemudahan pada masyarakat terhadap pembelian produk yang mereka jual. Salah satu bentuk implementasi nyata dari hal tersebut dengan adanya Aplikasi My F&B ID. Aplikasi My F&B ID adalah aplikasi keluaran PT. Foods Beverages yang menaungi brand kuliner seperti Chatime, Go! Go! Curry, Gindaco dan lain-lain. Aplikasi ini dapat diunduh oleh masyarakat melalui Google Playstore dan AppStore. Penelitian terhadap analisis sentimen ulasan aplikasi Google Playstore pernah dilakukan oleh Muhammad Zaki Hariansyah dan Siswanto yang menganalisis ulasan aplikasi Livin By Mandiri menggunakan Multinomial Naïve Bayes [8]. Hasil akhir pada penelitian tersebut berhasil mendapatkan hasil performansi yang sangat baik, dengan memperoleh nilai accuracy sebesar 93%, precision 90%, recall% dan f1-scrore 91% serta diperoleh juga bahwa mayoritas masyarakat mengeluarkan ulasan positif terhadap apliaksi tersebut. Pada Google Play Store selain dapat mengunduh aplikasi, masyarakat juga dapat memberi pendapat terhadap aplikasi tersebut. Pendapat yang berupa memberi ulasan masukkan, kritik, pujian, keluh kesah terhadap aplikasi, layanan, tampilan dan menu dari aplikasi yang digunakan. Hal tersebut dapat dijadikan pertimbangan dalam mengembangkan, menganalisis serta memperbaiki aplikasi [1]. Ulasan Aplikasi dapat diberi score rating 1 hingga 5 oleh masayarakat untuk mengetahui kepuasannya atas penggunaan aplikasi tersebut. Namun, jika hanya dilihat melalui score rating, tingkat kepuasaan dirasa masih kurang tepat. Penelitian lain yang membahas penggunaan Naïve Bayes sebagai metode yang digunakan dalam melakukan analisis sentimen terhadap aplikasi Tokopedia pada tahun 2019. Hasil yang didapatkan dari penelitian ini adalah nilai accuracy sebesar 97,13%, nilai precision 1, recall sebesar 95,49% [9]. Penggunaan Naïve Bayes dalam penelitian sangat membantu proses klasifikasi terhadap ulasan aplikasi. Hal ini mendorong peneliti untuk melakukan penelitian dalam menganalisis sentimen ulasan masyarakat mengenai aplikasi My F&B ID. Ulasan aplikasi akan dikategorikan ke dalam kelas sentimen positif dan negatif. Data yang digunakan dalam penelitian ini berasal dari data ulasan aplikasi My F&B ID di Google Play Store yang berjumlah 3000 ulasan Analisis sentimen dapat digunakan pada opini atau pendapat dari berbagai sumber. ANALISIS SENTIMEN DENGAN ALGORITMA NAÏVE BAYES CLASSIFIER TERHADAP ULASAN APLIKASI MY F&B ID Nabila Ayu Puspita, Fetty Tri Anggraeny, Agung Mustika Rizki Teknik Informatika, Universitas Pembangunan Nasional Veteran Jawa Timur Jl. Rungkut Madya No.1, Gn. Anyar, Kec. Gn. Anyar, Surabaya, Jawa Timur 19081010006@student.upnjatim.ac.id JATI (Jurnal Mahasiswa Teknik Informatika) JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 ABSTRAK Di era teknologi yang semakin canggih mendorong semua aspek bidang kehidupan ikut dalam perkembangannya, salah satunya pada bidang maknaan atau kuliner. Kecanggihan hal tersebut ialah memebuat perusahaan makanan berlomba-lomba untuk membuat aplikasi mobile untuk memberi kemudahan pada masyarakat terhadap pembelian produk yang mereka jual. Salah satu bentuk implementasi nyata dari hal tersebut dengan adanya Aplikasi My F&B ID. Pada Google Play Store selain dapat mengunduh aplikasi, masyarakat juga dapat memberi pendapat terhadap aplikasi tersebut. Pendapat yang berupa memberi ulasan masukkan, kritik, pujian, keluh kesah terhadap aplikasi yang dapat meningkatkan kualitas dan pelayanan yang ditawarkan aplikasi. Maka dalam penelitian ini akan dilakukan analisis sentimen terhadap ulasan aplikasi My F&B ID pada Google Playstore menggunakan algoritma Naïve Bayes Classifier untuk mengetahui permasalahan tersebut serta performansi dari klasifikasi algoritma tersebut. Penelitian ini menggunakan perbandingan dataset untuk data latih dan data uji sebesar 80:20, 70:30, 60:40 untuk membandingkan performansi nilai accuracy yang dihasilkan. Perbandingan dataset dilakukan untuk mengetahui nilai accuracy terbaik dari ketiganya. Hasil dari penelitian diperoleh nilai accuracy tertinggi didapatkan klasifikasi Multinomial Naïve Bayes Classifier dengan perbandingan dataset 80:20 sebesar 85%. 2. TINJAUAN PUSTAKA Pada bagian ini, peneliti akan menjabarkan dan menjelaskan tentang teori atau metode yang digunakan dalam penelitian. 1. PENDAHULUAN Selain itu, analisis sentimen dapat digunakan untuk opini sosial media seperti Twitter, Instagram, Facebook dan lain-lain. Opini dapat diklasifikasikan ke dalam kelas sentimen posititf dan negatif [2]. Dalam penerapannya, analisis 1090 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 d. Normalisasi Normalisasi digunakan untuk mengubah kata yang tidak baku menjadi kata baku. berbahasa Indonesia. Dalam analisis sentimen yang dilakukan bertujuan untuk mengetahui kecondongan masyarakat terhadap aplikasi tersebut dan juga mengetahui hasil performansi nilai accuracy dari model klasifikasi yang dihasilkan dengan jumlah perbandingan dataset yang berbeda. e. Stopwords Removal Menghilangkan kata-kata yang tidak memiliki makna seperti kata hubung dan lain-lain. p g f. Stemming Tahapan stemming akan mengubah potongan kata yang berimbuhan menjadi kata dasar. 2.1. Analisis Sentimen Pembobotan yang dilakukan dengan cara perhitungan scoring dengan kamus yang telah berisi score yang telah dibuat dan dicocokkan dengan kata pada dokumen [14]. TF-IDF untuk pelabelan data yang didapatkan menurut setiap term/kata yang ada dalam kalimat atau dokumen. Bobot kata semakin besar jika sering muncul pada suatu kalimat [15]. Analisis sentimen merupakan bentuk pemrosesan bahasa lama yang berfungsi dalam mencari sentimen masyarakat menurut produk atau suatu topik [10]. Analisis sentimen adalah salah satu bidang yang berada diantara berbagai bidang seperti Data Mining, Natural Language Processing dan Machine Learning yang berfokus untuk ektraksi sentimen sebuah teks [11]. Analisis sentimen bagaikan tambang pendapat yang terlibat dalam membangun sistem sebagai tempat mengumpulkan pendapat atau ulasan tentang topik atau produk yang dibangun [12]. g p Term frequency (TF) menghitung jumlah kemunculan kata pada dataset. Perhitungan tersebut menggunakan persamaan di bawah : 𝑇𝐹= 𝑡𝑓 max⁡(𝑓)⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡⁡(1) Keterangan : Keterangan : 2.2. My F&B ID tf = jumlah kata t yang muncul pada dokumen (term frquency). My F&B ID merupakan aplikasi perkembangan dari aplikasi sebelumnya yaitu “Chatime Indoneisa”. Aplikasi ini dikeluarkan oleh PT. Food and Beverages Indonesia yang bergerak di bidang makanan dan minuman. Brand yang dijual atau yang dapat dibeli masyarakat pada aplikasi ini antara lain adalah Chatime, Cupbop, Gindaco dan lain-lain. Dengan adanya aplikasi My F&B ID, masyarakat dapat memesan secara pesan antar atau dengan memesan terlebih dahulu lalu mengambil sendiri tanpa antri dan tanpa driver. Pada aplikasi My F&B ID, pelanggan bukan hanya dapat memesan produk Chatime saja tetapi juga dapat memberi penilaian terhadap aplikasi. max(f) = panjang kata pada sebuah dokumen. Berdasarkan persamaan Term Frequency (TF) di atas, cara menghitung jumlah kemunculan kata t di dataset adalah membagi jumlah kata t yang muncul pada dokumen (tf) dengan panjang kata pada dokumen tersebut (max(f)). Inverse Document Frequency (IDF) merupakan jumlah dokumen yang berisi term/kata yang akan dicari dalam dokumen dataset menggunakan persamaan di bawah : 𝑖𝑑𝑓= 𝑙𝑜𝑔 𝐷 𝑑𝑓 (2) (2) \|𝑉\| = total kata unik dalam seluruh kategori \|𝑉\| = total kata unik dalam seluruh kategori Berdasarkan persamaan di atas, cara menghitung peluang kata i kategori j (𝑃(𝑤𝑖\|𝑐𝑗)) yaitu total kata i dalam kelas kategori j (𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗)) dan alpha=1 terhadap total kata dalam kategori j (∑𝑤⁡𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗)) dan total kata unik dalam seluruh kategori (\|V\|). Gambar 1. Alur metode penelitian Maka, proses klasifikasi pada data uji dapat dilakukan menggunakan persamaan di bawah ini. 𝑃(𝑑\|𝑐𝑗) = 𝑃(𝑐𝑗)𝑥∏ 𝑃(𝑤\|𝑐𝑗) 1≤𝑖≤𝑐𝑜𝑢𝑛𝑡(𝑣,𝑑) (7) Keterangan : 𝑐𝑜𝑢𝑛𝑡(𝑣, 𝑑) = total kata unik dalam dokumen 𝑃(𝑑\|𝑐𝑗) = 𝑃(𝑐𝑗)𝑥∏ 𝑃(𝑤\|𝑐𝑗) 1≤𝑖≤𝑐𝑜𝑢𝑛𝑡(𝑣,𝑑) (7) Keterangan : 𝑐𝑜𝑢𝑛𝑡(𝑣, 𝑑) = total kata unik dalam dokumen 𝑃(𝑑\|𝑐𝑗) = 𝑃(𝑐𝑗)𝑥∏ 𝑃(𝑤\|𝑐𝑗) 1≤𝑖≤𝑐𝑜𝑢𝑛𝑡(𝑣,𝑑) (7) Keterangan : 𝑐𝑜𝑢𝑛𝑡(𝑣, 𝑑) = total kata unik dalam dokumen 𝑐𝑜𝑢𝑛𝑡(𝑣, 𝑑) = total kata unik dalam dokumen Berdasarkan persamaan di atas, cara klasifikasi terhadap data uji dalam kategori j adalah peluang kategori j dan peluang kata pada kategori j. D = jumlah kalimat atau dokumen f( ) = jumlah dokumen yang di dalamnya terdapat kata t D = jumlah kalimat atau dokumen D = jumlah kalimat atau dokumen D = jumlah kalimat atau dokumen f( ) = jumlah dokumen yang di dalamnya terdapat kata t agar hasil dari peluang mustahil 0, ini disebut dengan laplace smooting. 𝑃(𝑤𝑖\|𝑐𝑗) = 𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗)+𝑎𝑙𝑝ℎ𝑎 ∑𝑤⁡𝑐𝑜𝑢𝑛𝑡(𝑤\|𝑐𝑗)+\|𝑉\| (6) 𝑃(𝑤𝑖\|𝑐𝑗) = 𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗)+𝑎𝑙𝑝ℎ𝑎 ∑𝑤⁡𝑐𝑜𝑢𝑛𝑡(𝑤\|𝑐𝑗)+\|𝑉\| (6) Keterangan : Berdasarkan perhitungan kata (TF-IDF) di atas, cara menghitung pembobotan suatu kata pada dokumen yaitu mengalikan jumlah suatu kata pada suatu dokumen (TF) dengan jumlah dokumen yang mengandung kata tersebut (IDF). Keterangan : 𝑃(𝑤𝑖\|𝑐𝑗)⁡= Peluang sebuah kata i masuk ke kategori 𝑃(𝑤𝑖\|𝑐𝑗)⁡= Peluang sebuah kata i masuk ke kategori j 𝑃(𝑤𝑖\|𝑐𝑗)⁡= Peluang sebuah kata i masuk ke kategori j 𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗) = total sebuah kata i dalam kategori j ∑𝑤⁡𝑐𝑜𝑢𝑛𝑡(𝑤\|𝑐𝑗) = total semua kata dalam kategori j \|𝑉\| = total kata unik dalam seluruh kategori 𝑐𝑜𝑢𝑛𝑡(𝑤𝑖\|𝑐𝑗) = total sebuah kata i dalam kategori j ∑𝑤⁡𝑐𝑜𝑢𝑛𝑡(𝑤\|𝑐𝑗) = total semua kata dalam kategori j 2.5. Naïve Bayes Classifier Naïve Bayes Classifier menjadi salah satu algoritma yang digunakan dalam analisis sentimen. Konsep dasar metode klasifikasi ini menggunakan Teorema Bayes yang pertama kali dinyatakan oleh Thomas Bayes [16]. Klasifikasi ini menggunakan perhitungan probabilitas terhadap kelas, fitur kemunculan fitur pada kelas serta probabilitas berdasarkan kategori pada data latih [17]. 2.3. Preprocessing Text Keterangan : Keterangan : Preprocessing adalah tahapan untuk mempersiapkan dataset mentah untuk siap diproses [13]. Tahapan pada preprocessing meliputi sebagai berikut. log = logaritma natural. D = jumlah semua dokumen. df = jumlah term/kata dari dokumen. log = logaritma natural. = jumlah term/kata dari dokumen. a. Case Folding Pada tahap case folding, kalimat ulasan dari dataset yang memiliki huruf kapital akan diubah menjadi huruf kecil semua agar terdeteksi dengan setara atau arti yang sama. Berdasarkan persamaan Inverse Document Frequency (IDF) di atas, cara menghitung jumlah dokumen yang mengandung suatu kata yaitu logaritma dari rasio total dokumen (D) terhadap jumlah kata dari dokumen (df). b. Cleaning Pada proses cleaning dilakukan penghilangan atau penghapusan tanda baca yang tidak diperlukan. Selain itu, pada tahap ini karakter yang tidak relevan. TF-IDF dapat diuraikan seperti persamaan di bawah ini: 𝑤= 𝑇𝐹⁡ × 𝐼𝐷𝐹 (3) 𝑤= 𝑓𝑡, 𝑑⁡ × 𝑙𝑜𝑔 𝐷 𝑑𝑓(𝑡) (4) 𝑤= 𝑇𝐹⁡ × 𝐼𝐷𝐹 (3) 𝑤= 𝑓𝑡, 𝑑⁡ × 𝑙𝑜𝑔 𝐷 𝑑𝑓(𝑡) (4) Keterangaan : W = bobot c. Tokenization c. Tokenization Tokenization dilakukan dengan mengubah kalimat menjadi potongan-potongan kata untuk diproses lebih lanjut. 𝑇𝐹 = 𝑓𝑡,d = jumlah kata t yang muncul pada dokumen d (term frquency). IDF = nilai inverse document frequency 1091 JATI (Jurnal Mahasiswa Teknik Informatika) Vol. 8 No. 1, Februari 2024 2.6. Confusion Matrix C f i i Pada kamus tersebut terdapat 3.609 kata positif dan 6.609 kata negative dan tiap kata memiliki bobot yang berbeda mulai dari -5 hingga +5[19]. Tiap ulasan akan dimasukkan ke dalam 2 kelas sentimen yaitu kelas positif dan negatif. Rahmaningtyas. Pada kamus tersebut terdapat 3.609 kata positif dan 6.609 kata negative dan tiap kata memiliki bobot yang berbeda mulai dari -5 hingga +5[19]. Tiap ulasan akan dimasukkan ke dalam 2 kelas sentimen yaitu kelas positif dan negatif. Rahmaningtyas. Pada kamus tersebut terdapat 3.609 kata positif dan 6.609 kata negative dan tiap kata memiliki bobot yang berbeda mulai dari -5 hingga +5[19]. Tiap ulasan akan dimasukkan ke dalam 2 kelas sentimen yaitu kelas positif dan negatif. g 𝑅𝑒𝑐𝑎𝑙𝑙=⁡ 𝑇𝑃 𝑇𝑃+𝑇𝑁 (10) g 𝑅𝑒𝑐𝑎𝑙𝑙=⁡ 𝑇𝑃 𝑇𝑃+𝑇𝑁 (10) g 𝑅𝑒𝑐𝑎𝑙𝑙=⁡ 𝑇𝑃 𝑇𝑃+𝑇𝑁 (10) (10) 4. F1-score adalah hasil perbandingan dari rata- rata nilai presicion dan recall dari hasil pengujian. 4. F1-score adalah hasil perbandingan dari rata- rata nilai presicion dan recall dari hasil pengujian. g 5. Tahap TF-IDF membobotkan tiap kata pada suatu teks yang sebanding dengan kemunculannya pada teks tersebut, namun berbanding terbalik dengan teks lain. p p g j 𝐹1 −𝑠𝑐𝑜𝑟𝑒= ⁡2 × 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛⁡×𝑅𝑒𝑐𝑎𝑙𝑙 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑅𝑒𝑐𝑎𝑙𝑙 (11) 3. METODE PENELITIAN Pada penelitian ini menggunakan data ulasan aplikasi My FB ID dari Google Playstore. Tahapan penelitian yang akan diproses seperti bawah berikut. 6. Klasifikasi model dengan pembagian dataset menjadi data latih dan data uji (80:20, 70:30, 60:40) yang digunakan pada penelitian adalah model klasifikasi Multinomial Naïve Bayes. Gambar 2. Alur metode penelitian y 7. Pengujian sistem dan evaluasi dengan confussion matrix dan classificataion report untuk melihat hasil performansi accuracy. 7. Pengujian sistem dan evaluasi dengan confussion matrix dan classificataion report untuk melihat hasil performansi accuracy. 2.6. Confusion Matrix C f i i Confusion matrix merupakan tabel klasifikasi kelas-kelas yang telah melalui proses algoritma yang digunakan. Bagian baris mempresentasikan jumlah data kelas actual sedangkan kolom mempresentasikan jumlah data prediksi. Gambar 1. Alur metode penelitian Berdasarkan Gambar 1. Alur perhitungan algoritma Naïve Bayes yaitu dengan menghitung peluang suatu kategori pada dataset lalu menghitung peluang kata pada kategori dan selanjutnya mengklasifikasi data uji mengalikasi peluang kategori dan peluang kata pada kategori untuk didapatkan jenis kategori dari data uji. Untuk menghitung kemunculan dari peluang suatu kategori pada seluruh dokumen dapat dengan persamaan berikut [18]. 1. True Positive (TP) = jumlah data yang diprediksi positif dan data aktualnya pun positif. 2. False Positive (FP) = jumlah data yang diprediksi positif namun data aktualnya negatif. 3. False Negative (FN) = jumlah data yang diprediksi negatif namun data aktualnya positif. 4. True Negative (TN) = jumlah data yang 3. False Negative (FN) = jumlah data yang diprediksi negatif namun data aktualnya positif. 𝑃(𝑐𝑗) = 𝑁𝑐𝑗 𝑁 (5) Keterangan: 𝑃(𝑐𝑗) = Peluang dari kategori j 𝑁𝑐𝑗 = Dokumen kategori j N = Jumlah data latih yang digunakan 𝑃(𝑐𝑗) = 𝑁𝑐𝑗 𝑁 (5) Keterangan: diprediksi negatif dan data aktualnya negatif. Lalu pada confusion matrix menghitung accuracy, i i ll d f1 g 𝑃(𝑐𝑗) = Peluang dari kategori j 𝑁𝑐𝑗 = Dokumen kategori j g 𝑃(𝑐𝑗) = Peluang dari kategori j 𝑁𝑐𝑗 = Dokumen kategori j g 𝑃(𝑐𝑗) = Peluang dari kategori j 𝑁𝑐𝑗 = Dokumen kategori j 1. Accuracy adalah digunakan untuk mengukur tingkat keakuratan model algoritma yang digunakan dalam membuat ketetapan hasil klasifikasi. N = Jumlah data latih yang digunakan 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦=⁡ 𝑇𝑃+𝑇𝑁 𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁 (8) 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦=⁡ 𝑇𝑃+𝑇𝑁 𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁 (8) 𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦=⁡ 𝑇𝑃+𝑇𝑁 𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁 (8) Berdasarkan persamaan di atas, cara menghitung peluang kategori j (𝑃(𝑐𝑗)) yaitu jumlah dokumen kategori j (𝑁𝑐𝑗) terhadap jumlah data latih (N). (8) 2. Precision merupakan visualisasi untuk persentase keakuratan hasil perkiraan oleh metode yang digunakan. 2. Precision merupakan visualisasi untuk persentase keakuratan hasil perkiraan oleh metode yang digunakan. Selanjutnya, menghitung peluang kata dalam suatu kategori. Dalam perhitungan peluang ini dibantu menggunakan konstanta alpha. Hal ini bertujuan untuk y g g 𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛= ⁡ 𝑇𝑃 𝑇𝑃+𝐹𝑃 (9) (9) 1092 Vol. 8 No. 1, Februari 2024 JATI (Jurnal Mahasiswa Teknik Informatika) 3. Recall adalah visualisasi dari kesesuaian metode dalam mencari ulang sebuah informasi. 3. Recall adalah visualisasi dari kesesuaian metode dalam mencari ulang sebuah informasi. Rahmaningtyas. JATI (Jurnal Mahasiswa Teknik Informatika) Bayes perbanding dataset sebesar 80:20 senilai 85%. Sedangkan nilai performansi accuracy terendah diperoleh klasifikasi Multinomial Naïve Bayes perbanding dataset sebesar 60:40 senilai 83%. [7] Kaswidjanti, W., Himawan, H., & Silitonga, P. D. (n.d.). The Accuracy Comparison of Social Media Sentiment Analysis Using Lexicon Based and Support Vector Machine on Souvenir Recommendations. http://bsd.pendukasi.id. 5. KESIMPULAN DAN SARAN p p [8] Zaki Hariansyah, M. (2022). Implementasi Metode Multinomial Naive Bayes pada Analisis Sentimen Terhadap Layanan Aplikasi Livin by Mandiri Implementation of Naive Bayes Multinomial Method on Sentiment Analysis of Livin by Mandiri Application Services. In Seminar Nasional Mahasiswa Fakultas Teknologi Informasi (SENAFTI) Jakarta- Indonesia. https://senafti.budiluhur.ac.id/index.php Hasil klasifikasi analisis sentimen ulasan aplikasi My F&B ID dengan 3 jenis pembagian dataset untuk pengujian menghasilkan performansi yang cukup baik. Dan dapat dilihat pada pembagian data tes dan data uji yang dilakukan, semakin kecil nilai data uji dan semakin besar nilai data latih maka semakin baik nilai accuracy yang didapatkan. Nilai accuracy yang diperoleh pada klasifikasai penelitian berdasarkan perbandingan data latih dan data uji, 80:20 mendapatkan 85%, 70:30 mendapatkan 84% dan 60:40 mendapatkan 83%. Untuk klasifikasi yang mendapatkan nilai accuracy tertinggi diperoleh klasifikasi Multinomial Naïve Bayes dengan perbandingan dataset 80:20 (2329 data latih dan 583 data uji) sebesar 85%. Lalu untuk penelitian selanjutnya diharapkan untuk dapat menggunakan jenis algoritma lain untuk analisis sentimen ulasan aplikasi atau mencoba menggunakan seleksi fitur dalam penelitian untuk menunjang model klasifikasai yang digunakan dalam penelitian. [9] [9] Apriani, R., Gustian, D., Program, S., Sistem, I., Putra, U. N., Indonesia, S., Raya, J., Kaaler, C., 21, N., & Sukabumi, K (2019). ANALISIS SENTIMEN DENGAN NAÏVE BAYES TERHADAP KOMENTAR APLIKASI TOKOPEDIA. In Jurnal Rekayasa Teknologi Nusa Putra (Vol 6, Issue 1). [10] Nuris, N., Rini Yulia, E., Solecha, K., Bina, U., Infromatika, S., & Mandiri, U. N. (2021). Implementasi Particle Swarm Optimization (PSO) Pada Analysis Sentiment Review Aplikasi Halodoc Menggunakan Algoritma Naïve Bayes. Jurnal Teknologi Informasi, 7. 4. HASIL DAN PEMBAHASAN Dalam penelitian ini, dataset yang digunakan berasal dari aplikasi My F&B ID di Playstore. Pengumpulan dataset mentah berjumlah 3000 ulasan. Dalam penelitian ini, dataset yang digunakan berasal dari aplikasi My F&B ID di Playstore. Pengumpulan dataset mentah berjumlah 3000 ulasan. Lalu setelah proses preprocessing dan filtering tambahan berupa penghapusan dokumen kosong pada dataset, data ulasan yang akan diproses untuk pelabelan terdapat 2912 ulasan. Dalam proses labelisasi menggunakan InSet tiap ulasan akan dimasukkan ke dalam kelas sentimen dan diperoleh hasil ulasan kelas positif sebanyak 1385 data dan ulasan kelas negatif sebanyak 1527 ulasan. Hasil pengujian dari model klasifikasi Multinomial Naïve Bayes Classifier dengan 3 jenis jumlah perbandingan dataset yang telah dijelaskan sebelumnya pada bagian medologi penelitian. 1. Skema pengujian perbandingan dataset 80:20 menghasilkan nilai accuracy sebesar 85%, nilai precision sebesar 86%, nilai recall sebesar 84% dan nilai F1-score sebesar 84%. 2. Skema pengujian perbandingan dataset 70:30 menghasilkan nilai accuracy sebesar 84%, nilai precision sebesar 86%, nilai recall sebesar 83% dan nilai F1-score sebesar 83%. Gambar 2. Alur metode penelitian Berdasarkan gambar 2. alur metode penelitian dijabarkan sebagai berikut. 3. Skema pengujian perbandingan dataset 60:40 menghasilka nilai accuracy sebesar 83%, nilai precision sebesar 85%, nilai recall sebesar 83% dan nilai F1-score sebesar 83%. 1. Mengumpulkan sumber bacaan terdahulu untuk dipelajari dan dijadikan acuan dalam penelitian yan dilakukan serta mencari teori dan metode yang dapat digunakan. Perbandingan nilai accuracy tiap pengujian dapat dilihat pada Tabel 2. 2. Mengumpulkan dataset yang berasal dari ulasan aplikasi My F&B ID dari Google Playstore sebanyak 3000 ulasan. Tabel 2. Perbandingan Nilai Accuracy Pada Klasifikasi Perbandingan Dataset Klasifikasi Multinomial NBC tanpa Seleksi Fitur 80:20 85% 70:30 84% 60:40 83% 3. Pada tahap preprocessing, dataset yang telah dilabelisasi akan dilakukan pembersihan data agar siap menjadi dataset yang bersih untuk diklasifikasi. 6 tahapan preprocessing yang digunakan dalam penelitian ini adalah case folding, cleaning, tokenization, normalisasi, stopwords removal, stemming. Berdasarkan tabel 2. hasil performansi accuracy tertinggi diperoleh klasifikasi Multinomial Naïve 4. Labelisasi data dilakukan dengan kamus InSet (Indonesian Lexicon Sentiment) yang pada tiap kata dibuat oleh Fajri Koto dan Gemala Y. 1093 Vol. 8 No. 1, Februari 2024 JATI (Jurnal Mahasiswa Teknik Informatika) [18] irbookonlinereading. (n.d.). DAFTAR PUSTAKA [1] Putri Nirwandani, E., & Cahya Wihandika, R. (2021). Analisis Sentimen Pada Ulasan Pengguna Aplikasi Mandiri Online Menggunakan Metode Modified Term Frequency Scheme Dan Naïve Bayes (Vol. 5, Issue 3). http://j-ptiik.ub.ac.id [11] A. A. K. Y. J. M. N.-K. Mahmoud Al-Ayyoub, "A comprehensive survey of arabic sentiment analysis," Information Processing and Management”, vol. 56, no. 2, pp. 320-342, 2019. [12] Nuris, N., Rini Yulia, E., Solecha, K., Bina, U., Infromatika, S., & Mandiri, U. N. (2021). Implementasi Particle Swarm Optimization (PSO) Pada Analysis Sentiment Review Aplikasi Halodoc Menggunakan Algoritma Naïve Bayes. Jurnal Teknologi Informasi, 7. [2] Ikegami, A., Dewa, I., Bayu, M., & Darmawan, A. (2022). Analisis Sentimen dan Pemodelan Topik Ulasan Aplikasi Noice Menggunakan XGBoost dan LDA. In JNATIA (Vol. 1, Issue 1). [3] Diandra Audiansyah, D., Eka Ratnawati, D., & Trias Hanggara, B. (2022). Analisis Sentimen Aplikasi MyXL menggunakan Metode Support Vector Machine berdasarkan Ulasan Pengguna di Google Play Store (Vol. 6, Issue 8). http://j-ptiik.ub.ac.id [13] Petty Wahyuningtyas, N., Eka Ratnawati, D., & Yudi Setiawan, N. (2023). Root Cause Analysis (RCA) berbasis Sentimen menggunakan Metode K-Nearest Neighbor (K-NN) (Studi Kasus: Pengunjung Kolam Renang Brawijaya) (Vol. 7, Issue 5). [4] Anggraeny, F. T., Purbasari, I. Y., & Hatta, R. H. (2011). PROBABILISTIC FUZZY NEURAL NETWORK UNTUK DETEKSI DINI PENYAKIT JANTUNG KORONER. In Jurnal Informatika Mulawarman (Vol. 6, No. 2). [14] Darwis, D., Shintya Pratiwi, E., Ferico, A., & Pasaribu, O. (2020). PENERAPAN ALGORITMA SVM UNTUK ANALISIS SENTIMEN PADA TWITTER KOMISI PEMBERANTASAN KORUPSI REPUBLIK INDONESIA. In IJurnal Ilmiah Edutic (Vol, 7, Issu 1). [5] Darwis, D., Siskawati, N., & Abidin, Z. (n.d.). Penerapan Algoritma Naive Bayes untuk Analisis Sentimen Review Data Twitter BMKG Nasional. 15(1). [15] Fremmuzar, P., & Baita, A. (2023). Uji Kernel SVM dalam Analisis Sentimen Terhadap Layanan Telkomsel di Media Sosial Twitter. Komputika : Jurnal Sistem Komputer, 12(2), 57– 66. [6] Rizki, A. M., & Nurlaili, A. L. (2021). Algoritme Particle Swarm Optimization (PSO) untuk Optimasi Perencanaan Produksi Agregat Multi- Site pada Industri Tekstil Rumahan. Journal of Computer, Electronic, and Telecommunication, 1(2). https://doi.org/10.52435/complete.v1i2.73 [16] Aldrich, J. (2008). A. Fisher on Bayes and Bayes’ Theorem. In Bayesian Analysis (Vol. 3, Issue 1). [17] Gunawan, F., Fauzi, M. A., & Adikara, P. P. (2017). Analisis Sentimen Pada Ulasan Aplikasi 1094 Vol. 8 No. 1, Februari 2024 JATI (Jurnal Mahasiswa Teknik Informatika) Mobile Menggunakan Naive Bayes dan Normalisasi Kata Berbasis Levenshtein Distance (Studi Kasus Aplikasi BCA Mobile) (Vol. DAFTAR PUSTAKA 1, Issue 10). [18] irbookonlinereading. (n.d.). [19] Fajri Koto, and Gemala Y. Rahmaningtyas "InSet Lexicon: Evaluation of a Word List for Indonesian Sentiment Analysis in Microblogs". IEEE in the 21st International Conference on Asian Language Processing (IALP), Singapore, December 2017. 1095
https://openalex.org/W2314930765	https://zenodo.org/record/1175318/files/7116ijasuc01.pdf	English	null	Review Over Hole Healing Techniques in Wireless Sensor Networks	International journal of ad hoc, sensor and ubiquitous computing	2,016	cc-by	9,034	International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 KEYWORDS Connectivity, Coverage, Coverage Holes, Hole Healing, Wireless Sensor Network. REVIEW OVER HOLE HEALING TECHNIQUES IN WIRELESS SENSOR NETWORKS Latesh Mehta and Manik Gupta Department of Computer Science, Chitkara University, Himachal Pradesh, India ABSTRACT Improving coverage and connectivity is a very important issue in wireless sensor networks. The unattended or uncovered region in a sensing field is called a coverage hole. A coverage hole impacts the performance of the wireless sensor network, disconnects the network topology and causes delay in data transmission. These coverage holes can be healed randomly or each hole can be assigned a priority value for healing. In this paper we will discuss about various hole healing strategies and their mechanisms. 1. INTRODUCTION To improve the coverage and maintain the network connectivity various strategies are International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 proposed in recent years. In energy-constrained sensor networks, these solutions must be implemented in such a way that they do not incur excessive energy overheads. In [9] coverage problem is classified into three categories i.e. area coverage, point coverage and barrier coverage. In PEAS mechanism[11], based on node density detection, duty cycling is used where some nodes are allowed to sleep and nodes are awakened at random time interval. In [12] unnecessary sensing work is avoided by detecting redundant nodes and sending them to a sleep state. The work in [13] compares energy efficiency of the sensor network with fixed sensing range and the network consisting of nodes with adjustable sensing range. G-MSC [14], a target coverage algorithm, constructs continuous node sets to cover as many targets in the network. The coverage algorithm in [15] relies only on 3 hop neighbors for its information. In[17], holes are classified into 5 types, namely, coverage hole, routing hole, jamming hole, sink/black hole and worm hole. The study in[17] also discuss detection of hole using voronoi and triangular diagram approach. All these studies focuses mainly on the hole detection, but we must not forget after hole detection the next phase i.e. hole healing is also very important. Healing a coverage hole not only improves coverage but it also improves the connectivity of the network. The complete hole healing process can be divided into two parts:Hole Selection and Healing Process. Figure 2. Classification of Hole Selection and Healing Process. Hole Healing Hole Selection Healing Process Voronoi Diagram Based Approach Random Weighted Method Travelling Salesman Problem Method Maximum Size Equally Divided Path Selection Delaunay’s Triangulation Triangular Diagram Based Approach Direction Adjustment Sensor Movement with Direction Adjustment Nearest Distance Figure 2. Classification of Hole Selection and Healing Process. The hole selection process decides which hole should be selected first for healing and the healing process tells how the selected hole should be healed to ensure maximum coverage and connectivity. Fig. 2 shows various types of hole selection methods and healing process. 1. INTRODUCTION In the rest of the paper section 2 will give details about various hole selection methods, section 3 will discuss about healing techniques and section 4 will conclude the paper. 1. INTRODUCTION A wireless sensor consists of a sensing component, on-board processing, communication, and storage unit. Many sensors cooperatively monitoring a large physical environment, form a wireless sensor network (WSN)[7]. The wireless sensor networks have different usage in various fields and applications like monitor and protect civil infrastructure, underwater monitoring[16], monitor large geographic areas, volcano monitoring, collecting structural health information etc. In [10] these applications are classified into monitoring and tracking applications e.g. health and wellness monitoring, power monitoring, inventory location monitoring, tracking objects, animals, humans, and vehicles. Sometimes, sensors are deployed in very remote and inaccessible areas; therefore the task of maintaining the coverage and connectivity becomes a major issue in WSN. The uncovered region in the target sensing field is considered as a coverage hole. Coverage holes can be introduced as a result of a node failure or initial deployment. Fig.1(a) shows a 2- dimentional image of a field which shows coverage holes introduced as a result of initial deployment. Fig. 1(b) shows coverage holes in a field introduced as a result of node failure. (a) (b) Figure 1. An example of coverage holes in a target sensing field. (a) 2-dimentional image showing coverage holes as a result of initial deployment. The white area in the graph shows the uncovered region in the field.[4] (b) Coverage holes as a result of node failure. Hi represents coverage holes in the field.[6] Sensor nodes must be self-managing[7] and should discover, identify, and react to network disruptions. To improve the coverage and maintain the network connectivity various strategies are (b) (a) (a) (b) (a) Figure 1. An example of coverage holes in a target sensing field. (a) 2-dimentional image showing overage holes as a result of initial deployment. The white area in the graph shows the uncovered region in the field.[4] (b) Coverage holes as a result of node failure. Hi represents coverage holes in the field.[6] DOI : 10.5121/ijasuc.2016.7101 1 Sensor nodes must be self-managing[7] and should discover, identify, and react to network disruptions. To improve the coverage and maintain the network connectivity various strategies are DOI : 10.5121/ijasuc.2016.7101 1 Sensor nodes must be self-managing[7] and should discover, identify, and react to network disruptions. 2.3. Nearest Distance In a wireless sensor network, nodes communicate with the sink through a multihop network. Each node passes message coming from other node towards the sink. Fig. 3 shows data flow towards the sink in a wireless sensor network. Here, nodes closer to the sink will have more data to relay towards the sink. Hence they are more likely to lose their energy as compared to other nodes in the network and if the hole occurs near the sink due to node energy depletion it will induce a delay in data transmission because the messages will have to re-route now to reach the sink. Therefore, the nearest distance method selects holes closer to the sink for healing, to balance the traffic load and to reduce the delay in data transmission. Figure 3. The multi-to-one data flow concept.[6] Figure 3. The multi-to-one data flow concept.[6] 2. HOLE SELECTION METHODS The hole selection process is very important during the network maintenance process. Coverage holes in a network are of different shape and size. The effect of their presence in the network is also different and it depends on various factors such as shape and size of the hole, shape and size of the target sensing field, location of the hole and the sink etc. Based on these factors some holes affect the coverage, some affects the connectivity and most of them affect both. The hole which is disturbing network the most, should be selected for healing at the first place. Based on their effect on coverage and connectivity, holes should be assigned a priority value for healing. In recent years, researchers have adopted different methods for hole selection. These methods are as follows: 2 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 2 1 R d International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 2.4. Travelling Salesman Problem There can be different number of holes at different position of time in a wireless sensor network. The hole healing strategy always aim to cover all the holes present in the network for a given interval of time. Therefore, in some hole healing techniques the hole selection procedure is transformed into travelling salesman problem[4]. If the network is using an external source to deploy nodes such as mobile robot, unmanned aerial vehicle(UAV) or other kind of resource, then its path is computed using travelling salesman problem in which a shortest path is computed where every hole is covered only once starting from an origin and returns to its initial position at last. Fig. 4(a) shows the center coordinates of coverage holes in a target sensing field and fig. 4(b) shows the optimal path obtained. This method ensures that every hole is covered once within less time. (a) Center Coordinates of hole[4] (b) Optimal Path Trajectory[4] Figure 4. Hole Selection Procedure converted into Travelling Salesman Problem.[4] (a) Center Coordinates of hole[4] (b) Optimal Path Trajectory[4] (a) Center Coordinates of hole[4] (a) Center Coordinates of hole[4] (b) Optimal Path Trajectory[4] Figure 4. Hole Selection Procedure converted into Travelling Salesman Problem.[4] 2.2. Maximum Size In this method coverage holes are selected based on the size of the hole. The hole larger in size will have maximum effect on the coverage of the network. Therefore hole with maximum size is selected for healing process. This method improves coverage but has better impact on connectivity than random method. 2.1. Random The coverage holes are selected on a random basis. This method increases the coverage but it does not optimize the results. It is not a good method for hole selection. 2.5. Weighted Method This is the most effective hole selection methods available so far. This method not only improves coverage but it also improves the connectivity. Holes present in the network are given some 3 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 priority for healing and their priority is based on three properties of the coverage hole. These three properties are as follows[6]: • Hole Angle Metric: Hole angle tells about the width of the hole. In a wireless sensor network data is relayed from all sides towards the sink as shown earlier in fig. 3. A wider hole will hinder data flow towards the sink. The effect on the data transmission is directly proportional to the width of the hole. Therefore, a hole with greater hole angle will have more effect on data transmission. Fig. 5 shows how to calculate hole angle. In fig. 5 Hi represents hole, Bj represents the hole border node, Lj is a ray starting at sink and extends towards Bj. The ray Lj is obtained for every hole border node Bj and its slope is calculated. The hole border node Bj with maximum slope is represented by Bu and Bj with minimum slope in represented by Bd. The angle between these two rays [SINK,Bu) and [Sink,Bd), represented by Θ(Hi) is called the hole angle and it is calculated as[6]: Here, [SINK,Hi) represents ray starting at sink and extends towards the center of hole. Here, [SINK,Hi) represents ray starting at sink and extends towards the center of hole. Figure 5. Calculation of Hole Angle.[6] Now, the metric of hole angle is given by[6]: Figure 5. Calculation of Hole Angle.[6] Figure 5. Calculation of Hole Angle.[6] Figure 5. Calculation of Hole Angle.[6] Now, the metric of hole angle is given by[6]: Now, the metric of hole angle is given by[6]: W(Θ(Hi)) = Θ(Hi) / 180o ; The calculation for Bnear, Bfar and the depth metric W(deep(Hi)) is given as[6]: Here, Bj represents set of hole boundary nodes, Hi represents the hole, R is the transmiss radius of sensor nodes, d(a,b) gives the distance between a and b. • These three properties are combined to assign weight to every hole as[6]: • These three properties are combined to assign weight to every hole as[6]: W(Hi) = αW(Θ(Hi)) * βW(len(Hi)) * γW(deep(Hi)) ; where α, β, γ are positive harmonic coefficients and their value may vary according to the requirement of the application. These coefficients are defined as: α + β + γ = 1; Hole with maximum value for weight i.e. W(Hi) will be selected for healing process. where α, β, γ are positive harmonic coefficients and their value may vary according to the requirement of the application. These coefficients are defined as: α + β + γ = 1; Hole with maximum value for weight i.e. W(Hi) will be selected for healing process. W(Θ(Hi)) = Θ(Hi) / 180o ; W(Θ(Hi)) = Θ(Hi) / 180o ; • Distance Metric: This property is similar to the nearest distance method. As discussed in nearest distance method, nodes closer to the sink have more data to relay. Hence hole closer to the sink will have greater influence on the data transmission than the same hole located far away from the sink. Healing a hole located at a greater distance or located at the edge of the field will only improve coverage and will have very less effect on the connectivity. Therefore, we can say that the influence on the data transmission is inversely proportional to the distance of the hole from the sink. The distance of hole from sink is calculated as[6]: Where di is the distance of the hole from the sink, Hi.x and Hi.y are the x and y coordinates of the center of the hole, S0.x and S0.y are the x and y coordinates of the sink. Fig. 6 shows different holes with their distance from the sink. 4 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Figure 6. Distance between hole and the sink.[6] Figure 6. Distance between hole and the sink.[6] Now the distance metric is given by[6]: W(len(Hi)) = 1 / di ; W(len(Hi)) = 1 / di ; • Depth Metric: If a hole area is very large, it will greatly reduce the coverage ratio and might result in network topology disconnection. To avoid this, the depth metric is considered. It is defined as the distance between nearest hole boundary node Bnear and farthest hole boundary node Bfar, as shown in fig. 7. Bfar, as shown in fig. 7. Figure 7. Calculation of Depth.[6] Figure 7. Calculation of Depth.[6] To calculate Bnear and Bfar an angle bisector Lc of the hole angle Θ(Hi) is obtained. This angle bisector Lc makes contact with the hole at two hole boundary nodes i.e. it passes through the transmission range of these two nodes. Out of these two nodes, the node near to the sink is called Bnear and the node far from the sink is called Bfar. 2.6. Comparative Study 5 The experimental results for Random, Maximum Size, Nearest Distance and Weighted method is given in fig 8, which shows that weighted method performs better than others in terms of average delivery hops. Note that in this graph the results are obtained when weighted method is implemented with equally divided path selection method[6] which will be discussed in section 3 i.e. healing process section. Average Delivery hops is defined as the average number of hops a message has to go through to reach the sink in a wireless sensor network. A decrease in average 5 5 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 delivery hops means better connectivity, message will take less time to reach sink and it will also reduce energy consumption of the network. Figure 8. Graph for average delivery hops.[6] Figure 8. Graph for average delivery hops.[6] The performance of travelling salesman method is evaluated on the basis of coverage rate[4]. Coverage rate means the percentage of area covered when additional nodes are deployed to cover holes as shown in fig 9. We can see that as we increase the value of additional nodes used for redeployment, the coverage almost reaches 100%. Here[4], Travelling Salesman method is implemented with Delaunay’s triangulation method which will be discussed in section 3 i.e. healing process section. Figure 9. Graph of increase in coverage rate for Travelling Salesman method implemented with D l ’ i l i [4] Figure 9. Graph of increase in coverage rate for Travelling Salesman method implemented with Delaunay’s triangulation.[4] Figure 9. Graph of increase in coverage rate for Travelling Salesman method implemented with Delaunay’s triangulation.[4] The Hole Selection Methods discussed above are compared in table 1 based on their mechanisms and results. Table 1. Comparative study for hole selection methods. Table 1. Comparative study for hole selection methods. Hole Selection Method Mechanism Result Random Hole is selected randomly for healing Does not optimizes results Maximum Size Hole with maximum size is selected first Improves coverage Nearest Distance Hole closer to the sink is selected first Improves connectivity Travelling Salesman Problem Method Hole selection is converted into travelling salesman problem Covers each and every hole once within less time. Weighted Method Hole is selected based on its three properties i.e. hole angle, distance and depth. Improves both connectivity and coverage. 3. 3.1. Voronoi Diagram Based Approach In wireless sensor networks, voronoi diagram[8][18] is mainly used to detect coverage holes. In this section we will discuss the healing technique using voronoi diagrams for directional sensor networks. A voronoi diagram is constructed by the perpendicular bisectors of the line joining adjacent sensor nodes. These perpendicular bisectors intersect with each other to form a voronoi cell, see fig 10. The voronoi diagram has some properties: • One voronoi cell will contain exactly one sensor node. • One voronoi cell will contain exactly one sensor node. • Any point inside a voronoi cell will be closer to the sensor node of the given voronoi cell than any other sensor node in the network, see fig 10, p is a point inside the voronoi cell of sensor s1 and the distance between p and s1 is shorter than the distance of the point from any other sensor node. • Any point inside a voronoi cell will be closer to the sensor node of the given voronoi cell than any other sensor node in the network, see fig 10, p is a point inside the voronoi cell of sensor s1 and the distance between p and s1 is shorter than the distance of the point from any other sensor node. • For a given set of sensor nodes the voronoi diagram generated will be unique[20]. • For a given set of sensor nodes the voronoi diagram generated will be unique[20]. Figure 10. Voronoi Diagram.[2] Figure 10. Voronoi Diagram.[2] Figure 10. Voronoi Diagram.[2] Based on these properties, we can say that, for homogeneous sensor nodes with equal sensing range, if a point inside a voronoi cell is not covered by the sensor node of the cell than this point cannot be covered by any other sensor node in the network. Hence, voronoi diagram is also used to detect coverage holes. In [1][2], the redeployment strategy for homogeneous directional sensor nodes using voronoi diagram, to improve the coverage ratio is proposed. The sensing model of directional sensors is shown in fig 11. Figure 11. Sensing model of directional sensor.[2] Figure 11. 2.6. Comparative Study HEALING PROCESS After hole selection, next step is to carry out the healing process. Healing process should aim to maximize the coverage as well as maintain the network topology and connectivity. To heal a 6 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 20 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 coverage hole, failed nodes are patched or new nodes are redeployed at the location where coverage is lost. Some healing techniques also aim to maintain the network connectivity by deploying nodes in such a way that the average delivery hops for the messages travelling in the network can be reduced. These healing techniques are discussed as follows: 3.1. Voronoi Diagram Based Approach Sensing model of directional sensor.[2] Here, s(xs,ys) is the sensor, Fs(Field of View, FoV) is the area covered by the sensor, α is called angle of view of the sensor, θ is the working direction of the sensor and it is measured relative to positive x-axis, r is the sensing radius, ^w is the unit vector for working direction of the sensor, p(x,y) is a point covered by the sensor ‘s’. δ is the angle between the vector sp-> and unit vector ^w. 7 7 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Based on this sensing model, to cover the point p(x,y), the sensor s(xs,ys) has to satisfy the following conditions: • Distance between ‘p’ and ‘s’ should be less than or equals to ‘r’. • Distance between ‘p’ and ‘s’ should be less than or equals to ‘r’. • Distance between ‘p’ and ‘s’ should be less than or equals to ‘r’. i.e. √( (x-xs)2 + (y-ys)2 ) ≤ r; (1) (1) • The angle ‘δ’ should be less than or equals to half of ‘α’. i.e. δ ≤ α/2; Considering: ⃗ ⋅ ^ = \|\|⃗\|\|.ǁ^ǁ cos ; ⇒ (−) cos + ( − ) sin ≥ √( − )2 + ( − )2 cos (/2) ; (2) (2) So far we have discussed the voronoi diagram and the sensing model for directional sensors. Due to the limited sensing range of the directional sensors and random deployment, the target sensing field is not fully covered and it leaves coverage holes in the field as shown in fig 12. Now we will discuss the hole healing techniques to improve the coverage ratio for directional sensors using voronoi diagram. Figure 12. Random deployment of sensor nodes with their voronoi cells.[1] Figure 12. Random deployment of sensor nodes with their voronoi cells.[1] As = (α/2)r2 ; where As is called Intra-Cell Coverage Area. 3.1.1. Direction Adjustment The area of these triangles is summed up to get the final result. Now the intra-cell coverage area for other cases where field of view of the sensor extends through the cell edges of the voronoi cell as shown in fig 14b,14c,14d, is calculated by dividing this area into several triangles. The area of these triangles is summed up to get the final result. Figure 14. Different cases of Intra-Cell Coverage Area.[1] Figure 14. Different cases of Intra-Cell Coverage Area.[1] To calculate the area of different triangles, first the vertices of triangles are determined. The vertices of triangles include those vertices of voronoi cell which comes inside the range or field of view of the sensor node i.e. those voronoi cell vertices which satisfies eq1 and eq2. Also it includes those points where voronoi cell edges intersect with the sector sideline or sector arc of the field of view of sensor. The equation for the 2 sector sidelines is given as[1]: (x-xs) cos θ + (y-ys) sin θ = √(x-xs)2 + (y-ys)2 cos (α/2) ; where √(x-xs)2 + (y-ys)2 ≤ r ; (x-xs) cos θ + (y-ys) sin θ = √(x-xs)2 + (y-ys)2 cos (α/2) ; The equation for the sector arc is given as[1]: √(x-xs)2 + (y-ys)2 = r ; √(x-xs)2 + (y-ys)2 = r ; where x∈ [r cos (θ + α/2) + xs , r cos (θ – α/2) + xs] and y∈ [r cos (θ – α/2) + ys , r cos (θ + α/2) + ys] ; √(x-xs) + (y-ys) = r ; where x∈ [r cos (θ + α/2) + xs , r cos (θ – α/2) + xs] and y∈ [r cos (θ – α/2) + ys , r cos (θ + α/2) + ys] ; where x∈ [r cos (θ + α/2) + xs , r cos (θ – α/2) + xs] and y∈ [r cos (θ – α/2) + ys , r cos (θ + α/2 ys] ; The equation for the voronoi cell edge is given as[1]: x(ya - yb) = y(xa - xb); √(x - xa)2 + (y - ya)2 + √(x - xb)2 + (y - yb)2 = √(xa - xb)2 + (ya - yb)2 ; where (xa,ya) and (xb,yb) are two vertices of the voronoi cell edge. 3.1.1. Direction Adjustment This technique is used for direction rotatable sensors. This means the directional sensors can rotate 360o and their working direction θ can be changed. In [1], three algorithms are proposed to adjust the working direction of the sensors such that the overall coverage ratio of the network is improved. Initially the sensor nodes are deployed randomly as shown in fig 12, then each node starts constructing its voronoi cell with the information of its neighbor nodes. After the construction of voronoi cell the three step algorithm works as follows: • The sensor nodes calculate the area covered by them within their voronoi cell, when they are allowed to face each vertex of the voronoi cell as shown in fig 13. The vertex with maximum coverage area is selected and the working direction of the sensor is rotated towards that vertex. Figure 13. Result of different vertex selection.[1] Figure 13. Result of different vertex selection.[1] During the calculation of the covered area, cases shown in fig 14 can arise. In fig 14a the covered area is equal to the field of view of the sensor and can be calculated as: As = (α/2)r2 ; where As is called Intra-Cell Coverage Area. Figure 13. Result of different vertex selection.[1] During the calculation of the covered area, cases shown in fig 14 can arise. In fig 14a the covered area is equal to the field of view of the sensor and can be calculated as: During the calculation of the covered area, cases shown in fig 14 can arise. In fig 14a the covered area is equal to the field of view of the sensor and can be calculated as: As = (α/2)r2 ; where As is called Intra-Cell Coverage Area. As = (α/2)r2 ; where As is called Intra-Cell Coverage Area. As = (α/2)r2 ; where As is called Intra-Cell Coverage Area. 8 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 rnational Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Now the intra-cell coverage area for other cases where field of view of the sensor extends through the cell edges of the voronoi cell as shown in fig 14b,14c,14d, is calculated by dividing this area into several triangles. 3.1.1. Direction Adjustment x(ya - yb) = y(xa - xb); √(x - xa)2 + (y - ya)2 + √(x - xb)2 + (y - yb)2 = √(xa - xb)2 + (ya - yb)2 ; where (xa,ya) and (xb,yb) are two vertices of the voronoi cell edge. where (xa,ya) and (xb,yb) are two vertices of the voronoi cell edge. From these equations the intersecting points are obtained. After getting the vertices for triangles, length of the three sides of each triangle is obtained and area of each triangle is calculated as[1]: d=(e1 + e2 + e3)/2 ; d=(e1 + e2 + e3)/2 ; Area of triangle = √d(d-e1) (d-e2) (d-e3) ; where e1,e2,e3 are the three sides of the triangle. Now this area for each triangle is summed up to get the intra-cell coverage area when a given voronoi cell vertex is selected. The vertex with maximum value of intra-cell coverage area is selected as the working direction of the sensor. This can be written mathematically as[1]: Here ev ij represents the jth side of the ith triangle when vertex v of the voronoi cell is selected, kv is the total number of triangles for the vertex v, Vs is the set of vertices of the given voronoi cell and vD is the vertex with maximum intra-cell coverage area. The working direction θ is calculated as[1]: Here ev ij represents the jth side of the ith triangle when vertex v of the voronoi cell is selected, kv is the total number of triangles for the vertex v, Vs is the set of vertices of the given voronoi cell and vD is the vertex with maximum intra-cell coverage area. The working direction θ is calculated as[1]: 9 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 • The next step is to avoid coverage overlap, which can happen if multiple neighboring sensor nodes selects the same common vertex as their working direction as shown in fig 15. Here, sensor node with smaller intra cell coverage area can change its working direction. 3.1.1. Direction Adjustment The condition for this is given as[1]: Here, Ns is the set of neighboring nodes who are adjacent to the voronoi cell of node s, vs D is the vertex selected as working direction for s, RvD s is the coverage area or field of view of s when vertex v is selected and AvD s is the intra-cell coverage area of s when vertex v is selected. Now a new set Us of vertices is defined as[1]: Us = Vs – {vs D} ; and to avoid overlap the new vertex selected as working direction for s is given as[1]: v’ D = argmaxvϵUs Av s ; v’ D = argmaxvϵUs Av s ; Figure 15. Change in working direction to avoid common vertex and coverage overlap.[1] Figure 15. Change in working direction to avoid common vertex and coverage overlap.[1] In fig 15, we can see that sensor sj changes its working direction from v1 to v2 but still there is some overlap. To avoid this a new set of vertices U` s is defined as[1]: In fig 15, we can see that sensor sj changes its working direction from v1 to v2 but still ther some overlap. To avoid this a new set of vertices U` s is defined as[1]: This set of vertices includes those vertices which have smaller overlap than vD. Here, SvD is the set of those sensor nodes except s whose working direction is vD and UsiϵSvD RvD si is the union of their coverage area or field of view. Now from set U` s overlapped region is subtracted from intra- cell coverage area of each vertex and the vertex with maximum value is selected for working direction as[1]: Here, Cv s is the intra cell coverage region. As shown in fig 15 working direction is again turned to vertex v6 to avoid the coverage overlap. If every vertex of the voronoi cell is selected by different sensors as working direction, then the vertex with maximum coverage and minimum overlap is selected as[1]: Here sv is the other node which has selected vertex v as working direction. Here sv is the other node which has selected vertex v as working direction. • The third and last step is to avoid nodes from sensing outside the boundary of the field. 3.1.1. Direction Adjustment Nodes close to the boundary of the field may face towards the boundary which should be avoided. 10 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 20 If the distance between the boundary of the field and the sensor τ is less than ωr where ω is the coefficient between 0 to 1 then the working direction of the sensor is controlled between the two limits θ1 and θ2 as shown in fig 16. i.e. if τ < ωr; then 0≤θ≤θ1 and θ2≤θ≤2π ; where θ1 and θ2 is calculated as[1]: Hence in fig 16 the choice for the working direction will be vertex vb and vc. Figure 16. Avoiding coverage outside the boundary.[1] Figure 16. Avoiding coverage outside the boundary.[1] 3.1.2. Sensor Movement with Direction Adjustment As the name suggest this technique is meant for the directional sensor nodes which have been provided mobility. The sensor nodes can change their position and choose an optimal location, from where coverage can be maximized. After getting the right location for movement, the direction of the sensor can be adjusted. Initially sensor nodes are deployed randomly and they start constructing their voronoi cell. Then the sensor node selects one of the voronoi cell vertex as its new location as shown in fig 17. Figure 17. Result of choosing different vertex as location.[2] Figure 17. Result of choosing different vertex as location.[2] The vertex whose angle is greater than angle of view of the sensor and whose edges are greater than sensing radius of the sensor is selected as new location for sensor movement. The angle β of the vertex v is calculated by using the information about its left adjacent vertex vL and right adjacent vertex vR as shown in fig 18. The coordinates of these adjacent vertices are (xL,yL) and (xR,xR) respectively and angle β is calculated as[2]: 11 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Figure 18. Calculation of the angle β.[2] Figure 18. Calculation of the angle β.[2] Figure 18. Calculation of the angle β.[2] Figure 18. Calculation of the angle β.[2] Now a set of vertices T, which contains all those vertices of the cell whose angle β is greater than or equals to angle of view α and whose edge length is also greater than or equals to sensing radius r, is defined as[2]: From this set T, a target vertex t is selected with maximum value for β as[2]: From this set T, a target vertex t is selected with maximum value for β as[2]: If the set T is empty, then the target vertex is selected among all those vertices having β greater than or equals to α and only one edge length greater than or equals to r. If we cannot find any vertex satisfying these conditions then the vertex with largest value for β is selected. Once the sensor is moved to the selected vertex, the working direction θ of the sensor is adjusted within the limits θ1 and θ2 as shown in fig 19. Figure 19. 3.1.2. Sensor Movement with Direction Adjustment Calculation of the limits θ1 and θ2.[2] Figure 19. Calculation of the limits θ1 and θ2.[2] Figure 19. Calculation of the limits θ1 and θ2.[2] Figure 19. Calculation of the limits θ1 and θ2.[2] To decide these limits θL and θR [2] are calculated which are defined as the angle of the vector vvL -> and vvR -> with the positive x-axis respectively. θ1, θ2, θL, θR, [2] all lies between 0 and +-π. 12 rnational Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 If the sensor is rotated towards the right side edge eR of the vertex v then θ1 will be the working direction of the sensor and if sensor is rotated towards left side edge eL of the vertex v then θ2 will be the working direction. To improve the coverage ratio, sensor will be rotated towards the longest edge as shown in fig 20. Figure 20. Cases of rotation of sensor.[2] Figure 20. Cases of rotation of sensor.[2] Let θ0 is the initial working direction of the sensor when it is moved to the vertex v, then the extent or range φ to which θ0 should be rotated in either clockwise or anticlockwise direction within the limits θ1 and θ2 is given as[2]: 3.2. Triangular Diagram Based Approach In this technique every hole is healed by a single sensor node. A new node is deployed in the region where hole is detected. In this process first the hole is detected then area of that hole is calculated and thereafter the healing process starts. To heal a hole the area of the hole is required. Area of the hole is calculated with the help of triangular diagram. This diagram is obtained by joining the center points of every adjacent sensor node which as a result forms a triangle for every three adjacent nodes in the network, see fig 21. Figure 21. Triangular structure.[3] Figure 21. Triangular structure.[3] Depending on the location of adjacent sensor nodes and distance between them several cases may arise as shown in fig 22. Here, we can see the location of hole and intersection between adjacent sensor nodes. s1,s2 and s3 is the area of intersection between the sensor’s sensing region and the triangle. 13 rnational Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Figure 22. All cases for three adjacent sensors.[3] Figure 22. All cases for three adjacent sensors.[3] In order to calculate the area of the hole, we have to do some calculations first. Let a,b,c be the three sides of the triangles shown in different cases in fig 22. Then the area of these triangles represented by s∆ is calculated as[3]: s∆ = (1/4) √(a+b+c)(a-b+c)(b-a+c)(c-a+b) ; s∆ = (1/4) √(a+b+c)(a-b+c)(b-a+c)(c-a+b) ; s∆ = (1/4) √(a+b+c)(a-b+c)(b-a+c)(c-a+b) ; and the relationship between region s1, s2 and s3 is given as[3]: and the relationship between region s1, s2 and s3 is given as[3]: s1+ s2+ s3= (1/2)πR2; where R is the sensing radius. s1+ s2+ s3= (1/2)πR2; where R is the sensing radius. Now the area of the intersection of two circles or nodes represented by sintersect is calculated as shown in fig 23. Here, two circles with different radius R, r and location (0,0), (d,0) respectively are considered. Figure 23. Calculation for the area of intersection of two circles.[3] Figure 23. Calculation for the area of intersection of two circles.[3] Figure 23. Calculation for the area of intersection of two circles.[3] From fig 23, area of the intersection represented by A is given as[3]: From fig 23, area of the intersection represented by A is given as[3]: where A(r,d2) is the area of left portion of the intersection divided by line segment ‘a’ and A(R, d1) is for the right portion. d1 and d2 is given as[3]: Now in fig 22 sensor nodes are homogeneous, therefore here r=R and area of hole sh for all these cases is thus calculated as[3]: • For fig 22a,22b,22c,22h area is given by[3]: sh = s∆ – (s1 + s2 + s3) ; • For fig 22a,22b,22c,22h area is given by[3]: sh = s∆ – (s1 + s2 + s3) ; • For fig 22a,22b,22c,22h area is given by[3]: sh = s∆ – (s1 + s2 + s3) ; sh = s∆ – (s1 + s2 + s3) ; 14 rnational Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 • For fig 22d,22g area is given by[3]: • For fig 22d,22g area is given by[3]: • For fig 22d,22g area is given by[3 sh = s∆ – (s1 + s2 + s3) + (1/2)sintersect; and sh = s∆ – (s1 + s2 + s3) + (1/2)sintersect; and • For fig 22e area is given by[3]: • In fig 22f , hole doesn’t exist. • For fig 22i area is given by[3]: • For fig 22i area is given by[3]: After calculating area of the hole, healing process starts. Each hole is healed by a single sensor node. s∆ = (1/4) √(a+b+c)(a-b+c)(b-a+c)(c-a+b) ; New sensor nodes are deployed in uncovered regions by following two methods: After calculating area of the hole, healing process starts. Each hole is healed by a single sensor node. New sensor nodes are deployed in uncovered regions by following two methods: • If the sensing area of the sensor is greater than area of the hole than circumcircle methods is used as shown in fig 24. The circumcircle is obtained by the perpendicular bisectors of the three sides of the triangle. The point where these perpendicular bisectors intersect is called the circumcircle center of the triangle and this is the point where new nodes is deployed. Figure 24. Calculation for circumcircle center of the triangle.[3] Figure 24. Calculation for circumcircle center of the triangle.[3] • If the sensing area of the sensor is less than the area of the hole than incircle method is used as shown in fig 25.The incircle is obtained by the angle bisectors of the three angles of triangle. The point where these three angle bisectors intersect is called incircle center of the triangle and this is the point where new node is deployed. The incircle is inscribed inside the triangle and it is tangent to each side of the triangle. 15 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 national Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 20 Figure 25. Calculation for incircle center of the triangle.[3] Figure 25. Calculation for incircle center of the triangle.[3] Figure 25. Calculation for incircle center of the triangle.[3] 3.3. Delaunay’s Triangulation Delaunay’s Triangulation[8][18][19] is a geometrical structure which is formed by a given set of points. It is a triangulation method in which triangles are formed in such a way that circumcircle of each triangle will not contain any other point of the given set. This means the vertices of the triangle will lie on the circumference of the circle and the circle will be empty inside. In fig 26(a), the triangulation formed is Delaunay’s triangulation because each circumcicle is empty and 26(b) is not Delaunay’s triangulation because each cicumcircle encloses a point inside it. Also the minimum angle of a triangle formed by Delaunay’s Triangulation is maximum for other triangulations for the same set of points. Figure 26. Triangulation for a given set of four points. The minimum angle α shown in (a) is greater than the minimum angle shown in (b).[8] Figure 26. Triangulation for a given set of four points. The minimum angle α shown in (a) is greater than the minimum angle shown in (b).[8] In[4], an algorithm is proposed to heal coverage holes using Delaunay’s triangulation as shown in fig 27. Holes with irregular shapes and boundaries are analyzed and the point where the irregular curve turns is considered as a vertex. All these vertices together form a closed polygon and then the Delaunay’s triangulation is performed over the polygon vertices. The radius for the circumcircle of the triangles formed is calculated and new node is deployed at the center of the circumcircle whose radius is maximum. Figure 27. Delaunay Triangulation of holes.[4] Figure 27. Delaunay Triangulation of holes.[4] Figure 27. Delaunay Triangulation of holes.[4] 2.4. Equally Divided Path Selection Method 2.4. Equally Divided Path Selection Method This method says that new nodes should be patched along the angle bisector of the hole angle discussed in section 2.5 for healing the hole. This path is the shortest path for data to travel to the sink. To prove this, a hole Hi is considered inside a parallelogram TUVW as shown in fig 28. A and B are two nodes equidistant and symmetrical from the sink. Let q is a point on AB and p is a point on TU such that Aq=x, Bq=y, Ap=a, Bp=b and the distance between line AB and p i.e. qp=h. Now using the property of a right angled triangle[6]: a2 = h2 + x2 ; a2 = h2 + x2 ; b2 = h2 + y2 ; b2 = h2 + y2 ; 16 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 20 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 Let a function f(x) = a+b; and AB=x+y=L; => f(x) = √ h2 + x2 + √ h2 + (L-x)2 ; => f(x) = √ h2 + x2 + √ h2 + (L-x)2 ; First and Second order derivative of f(x) is given as[6]: First and Second order derivative of f(x) is given as[6]: Now by using maximum value theorem, the minimum value of x for the function f(x) comes out to be L/2. This means minimum value for a+b will be obtained when x and y are equal i.e. q lies in the center and passes through the angle bisector. Hence, the shortest path to reach sink through the hole will be along the angle bisector. New nodes will be patched along this path. Figure 28. Calculation for shortest path along the angle bisector.[6] Figure 28. Calculation for shortest path along the angle bisector.[6] 3.5. Comparative Study The experimental results for direction adjustment and sensor movement with direction adjustment is given in fig 29 and fig 30. The results shows that out of these two voronoi diagram based healing techniques, sensor movement with direction adjustment is better. The results are obtained for sensor nodes with angle of view α=120o and sensing radius=50metres and field size=500m*500m. This shows that providing sensor nodes with moving capabilities helps in improving overall coverage of the network. Figure 29. IDA represents Direction Adjustment.[1] Figure 29. IDA represents Direction Adjustment.[1] 17 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, Febr Figure 30. DVSA represents Sensor Movement with Direction Adjustment.[2] Figure 30. DVSA represents Sensor Movement with Direction Adjustment.[2] The graph for Delaunay’s triangulation and equally divided path selection method is discussed earlier in section 2.6. with fig 9 and fig 8, where they are implemented along with travelling salesman method and weighted method. The equally divided path selection method is evaluated in terms of average delivery hops whereas other methods are evaluated based on coverage ratio. This is because equally divided path selection method focus on improving connectivity by providing shorter routes to the active nodes in the network. A comparative study of these methods is given in table 2. Table 2. Comparative study for healing process methods. Healing Process Method Mechanism Utility Type of Sensor Network Voronoi Diagram Based Approach Direction Adjustment Voronoi cell vertex is selected as working direction of the sensor. Can be used for both hole detection and Healing Directional Sensor Network Sensor Movement with Direction Adjustment Voronoi cell vertex is selected as the new location for the sensor. Triangular Diagram Based Approach New node is deployed either on circumcircle center or incircle center of the triangle, based on the size of the hole. Hole Detection and Healing Omni- Directional Delaunay’s Triangulation Method Hole is divided into triangles and new node is deployed on circumcircle center of the largest triangle. Hole Healing Omni- Directional Equally divided path selection Method New nodes are deployed along the angle bisector of the hole angle. Hole Healing Omni- Directional Table 2. Comparative study for healing process methods. REFERENCES [1] T.-W. Sung and C.-S. Yang, (2013) “Voronoi-based coverage improvement approach for wireless directional sensor networks”, Journal of Network and Computer Applications. [2] T.-W. Sung and C.-S. Yang, (2013) “Distributed voronoi-based self-redeployment for coverage enhancement in a mobile directional sensor network,” International Journal of Distributed Sensor Networks, Article ID 165498, 15 pages. [3] S. Babaie and S. S. Pirahesh, (2012) “Hole detection for increasing coverage in wireless sensor network using triangular structure,” IJCSI International Journal of Computer Science Issues, vol. 9, no.1, article 2. [4] P. Si, C. Wu, Y. Zhang, Z. Xia, and L. Cheng, (2011) “A hole detection and redeployment strategy in wireless sensor network,” Journal of Information and Computational Science, vol. 8, no. 13, pp.2577– 2585. [5] C. Zhang, Y. Zhang, and Y. Fang, (2010) “A coverage inference protocol for wireless sensor networks,” IEEE Transactions on Mobile Computing, vol. 9, no. 6, pp. 850–864. [6] Fu-Tian Lin, Tien-Wen Sung, and Chu-Sing Yang, (2013) “A Coverage Hole Healing Strategy with Awareness of Data Delivery Time in Wireless Sensor Networks,” International Journal of Distributed Sensor Networks, Volume 2013, Article ID 790794, 11 pages. [7] Waltenegus Dargie and Christian Poellabauer (2010) Fundamentals of Wireless Sensor Networks, Theory and Practice; A John Wiley and Sons, Ltd., Publication. [8] F. Aurenhammer, (1991) “Voronoi diagrams—a survey of a fundamental geometric data structure,” ACM Computing Surveys, vol. 23, no. 3, pp. 345–405. [9] Junbin LIANG, Ming LIU and Xiaoyan KUI, (2014) “A Survey of Coverage Problems in Wireless Sensor Networks,” Sensors & Transducers, Vol. 163 , Issue 1, pp. 240-246. [10] J. Yick, B. Mukherjee and D.Ghosal, (2008) “Wireless sensor network survey,” Computer Networks, vol. 52, no. 12, pp. 2292–2330. [11] F. Ye, G. Zhong, S. Lu and L. Zhang, (2002) “PEAS, a robust energy conserving protocol for long lived sensor networks,” in Proceedings of 10th IEEE International Conference on Network Protocols (ICNP’02), Paris, France, pp. 200-201. [12] D. Tian and N. D. Georganas, (2003) “A node scheduling scheme for energy conservation in large wireless sensor networks,” Wireless Communications and Mobile Computing, Vol. 3, Issue 2, pp. 271-290. [13] J. Wu and S. Yang, (2004) “Coverage issue in sensor networks with adjustable ranges,” in Proceedings of International Workshop on Mobile and Wireless Networking (MWN’04) in conjunction with International Conference on Parallel Processing Workshops (ICPPW'04), Montreal, Quebec, Canada, pp. 61-68. [14] M. Cardei, M. Thai, Y. Li, and W. 4. CONCLUSION In this study, we discussed about various coverage problems and hole healing strategies in WSN. It can be noticed that with the heterogeneity in sensor nodes we get different type of coverage problems and hence will need different strategies to overcome these problems e.g. in mobile sensor networks, coverage is improved by moving the sensors to appropriate locations, in directional sensor networks, coverage is improved by adjusting the sensing directions of these sensors, and in static sensor networks, we can patch failed nodes or can use redeployment methods. The coverage holes can be healed in a random order or a proper sequence can be followed. This sequence can be obtained by converting the problem into travelling salesman problem, covering each hole once and returning back to the initial hole[4]. Priority healing is another option where holes can be assigned a priority value based on some properties of the hole[6]. All these techniques have their own advantages and disadvantages. Some algorithms are centralized while others are distributed. The centralized algorithms results in optimal routes while poses communication overhead. The distributed algorithms may not result in optimal routes but 18 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 20 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 they are energy efficient and can be used for scalable networks. Random healing result in increasing the coverage ratio only while priority healing not only increases coverage but it can also reduce energy consumption of the network during the healing process. Keeping in mind all these factors we can choose an appropriate healing method suitable for our application. they are energy efficient and can be used for scalable networks. Random healing result in increasing the coverage ratio only while priority healing not only increases coverage but it can also reduce energy consumption of the network during the healing process. Keeping in mind all these factors we can choose an appropriate healing method suitable for our application. REFERENCES Wu, (2005) “Energy efficient target coverage in wireless sensor networks,” in Proceedings of the 24th Conference of the IEEE Communications Society (INFOCOM’05), pp. 1976-1984. 19 International Journal of Ad hoc, Sensor & Ubiquitous Computing (IJASUC) Vol.7, No.1, February 2016 [15] Hang Ding Xing, Zhang Bo Yun and Tang Wen Shen, (2009) “Distributed connected algorithm for wireless sensor networks,” Computer Engineering and Application, Vol. 45, Issue 7, pp. 17-19. [16] I. Vasilescu, K. Kotay, D. Rus, M. Dunbabin and P. Corke, (2005) “Data collection, storage, retrieval with an underwater sensor network,” in: Proceedings of the Third International Conference on Embedded Networked Sensor Systems (Sensys), SanDiego, CA. [17] Smita Karmakar and Alak Roy, (2014) “Holes Detection in Wireless Sensor Networks: A Survey,” I.J. Modern Education and Computer Science, 4, 24-30 [18] D. Du, F. Hwang, S. Fortune, (1992) “Voronoi diagrams and Delaunay triangulations,” Euclidean Geometry and Computers. [19] ChunHsien Wu, KuoChuan Lee, YehChing Chung, (2006) “A Delaunay Triangulation Based Method for Wireless Sensor Network Depoyment,” In Proceedings of the 12th International Conference on Parallel and Distributed Systems, pp. 1521-1530 [20] A. Okabe, B. Boots, and K. Sugihara, (2000) Spatial Tessellations, Concepts and Applications of Voronoi Diagrams, John Wiley & Sons, 2nd edition. AUTHORS Latesh Mehta is pursuing his Master degree in Computer Science Engineering from Chitkara University, Himachal Pradesh, India. He is enrolled in Integrated Bachelor + Master Degree course in Computer Science Engineering from Chitkara University, Himachal Pradesh, India. His main research area includes, Wireless Sensor Network. Manik Gupta is Assistant Professor in the Department of Computer Science at Chitkara University, Himachal Pradesh, India. He has a good number of publications in the field of wireless sensor networks in various international conferences and journals of good repute. He had been awarded for Best Research Paper, in December, 2010. He also worked as Software Developer for one year after completing his Bachelors in Engineering and as Consultant in LSI, Research and Development. He also remained a regular columnist of column “Manik’s Tech Tonics” in the newspaper- “Student Age”. His research area is Security, Energy Efficiency, Fault Tolerance, Fault Revoking, Coverage, Connectivity and Mobility in Wireless Sensor Networks and Body Area Sensor Networks. Latesh Mehta is pursuing his Master degree in Computer Science Engineering from Chitkara University, Himachal Pradesh, India. He is enrolled in Integrated Bachelor + Master Degree course in Computer Science Engineering from Chitkara University, Himachal Pradesh, India. His main research area includes, Wireless Sensor Network. Manik Gupta is Assistant Professor in the Department of Computer Science at Chitkara University, Himachal Pradesh, India. He has a good number of publications in the field of wireless sensor networks in various international conferences and journals of good repute. He had been awarded for Best Research Paper, in December, 2010. He also worked as Software Developer for one year after completing his Bachelors in Engineering and as Consultant in LSI, Research and Development. He also remained a regular columnist of column “Manik’s Tech Tonics” in the newspaper- “Student Age”. His research area is Security, Energy Efficiency, Fault Tolerance, Fault Revoking, Coverage, Connectivity and Mobility in Wireless Sensor Networks and Body Area Sensor Networks. 20 20
https://openalex.org/W3021002523	https://zenodo.org/record/1284588/files/09_IJETMR18_A05_414.pdf	English	null	ANALYSIS OF RETAIL MIX STRATEGIES: A SPECIAL FOCUS ON MODERN RETAIL FORMATS	International journal of engineering technologies and management research	2,020	cc-by	2,441	Abstract: A shopper usually comes across many retailers in the market place, each having its own overall product offering. One retailer may be attracting customers due to its unbeatable location and visibility, other on the basis of discounted prices, someone on the basis of variety of merchandise or specialised product offering. If small retailers promise to offer the convenience of fulfilling the daily needs at your doorstep, the big retailers promise to offer better value in terms of competitive pricing and convenience of making all products available under the same roof. Then online retailers are trying to outsmart conventional retailers by killing time and distance constraints. Thus, it is seen that retail market place has become very competitive and challenging over a period of time. Market has already become tough due to increasing cultural and demographic diversity and changing lifestyles over a period of time. Every market has its own set of needs. Like any marketer, a retailer has to decide which kind of market it should cater to in a satisfactory manner. As a result, the retailer has to explore many options to differentiate themselves in the market place and appeal to its target customers. The options are available in the form of combining different elements of retail mix. Retail mix is mainly deciding about merchandise, price, promotional mix, location, services extended etc. to serve the needs of target market in a satisfactory manner. Retail format is the popular term used for retail mix. It bears close resemblance with marketing mix. In the ensuing study, discussion about various retail formats is carried on. Keywords: Retail Format; Retail Mix Strategies; Customer Retention Strategies; Image Enhancement Strategies; Departmental Stores; Hypermarkets. Cite This Article: Dr. K. Aparna, Dr. P. Murali Krishna, and Dr. V. Satish Kumar. (2018). “ANALYSIS OF RETAIL MIX STRATEGIES: A SPECIAL FOCUS ON MODERN RETAIL FORMATS.” International Journal of Engineering Technologies and Management Research, 5(5), 71-76. DOI: 10.5281/zenodo.1284588. Cite This Article: Dr. K. Aparna, Dr. P. Murali Krishna, and Dr. V. Satish Kumar. (2018). “ANALYSIS OF RETAIL MIX STRATEGIES: A SPECIAL FOCUS ON MODERN RETAIL FORMATS.” International Journal of Engineering Technologies and Management Research, 5(5), 71-76. DOI: 10.5281/zenodo.1284588. ANALYSIS OF RETAIL MIX STRATEGIES: A SPECIAL FOCUS ON MODERN RETAIL FORMATS Dr. K. Aparna 1, Dr. P. Murali Krishna 2, Dr. V. Satish Kumar 3 1 Assistant Professor in Humanities & Basic Science Department, G. Pulla Reddy Engineering College (Autonomous) Kurnool - 518007, A.P., India 2 Professor of Management & Dean, Industry Interface & International Collaboration (IIIC) Sri Krishnadevaraya University, Anantapur, A.P. - 515003, India 3 Professor of Industrial Management, G. Pulla Reddy Engineering College (Autonomous) Kurnool - 518007, A.P., India Dr. K. Aparna 1, Dr. P. Murali Krishna 2, Dr. V. Satish Kumar 3 1 Assistant Professor in Humanities & Basic Science Department, G. Pulla Reddy Engineering College (Autonomous) Kurnool - 518007, A.P., India 2 Professor of Management & Dean, Industry Interface & International Collaboration (IIIC) Sri Krishnadevaraya University, Anantapur, A.P. - 515003, India 3 Professor of Industrial Management, G. Pulla Reddy Engineering College (Autonomous) Kurnool - 518007, A.P., India ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 [Aparna et. al., Vol.5 (Iss.5): May 2018] 1. Introduction The Indian retail market is witnessing a dramatic transformation. Liberalisation in the 90s, increasing incomes and foreign trips, exposure to the internet, growing confidence in one’s [71] Http://www.ijetmr.com©International Journal of Engineering Technologies and Management R ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 [Aparna et. al., Vol.5 (Iss.5): May 2018] DOI: 10.5281/zenodo.1284588 personal success, etc. are making people to splurge on luxury goods. With retail sector developing quickly and becoming more competitive, retailers need to understand the importance of meeting consumer demand. personal success, etc. are making people to splurge on luxury goods. With retail sector developing quickly and becoming more competitive, retailers need to understand the importance of meeting consumer demand. To enjoy their increased spending power, Indians typically prefer to shop in the company of their families by visiting malls, looking at brands, experimenting between categories, etc. Some retailers and brands have launched family stores to cater to men, women and children. Modern trade has made shopping enjoyable, with malls offering multiplexes, restaurants and food courts, a range of shops, arcades for children, etc. and becoming more competitive. Retailers need to develop and offer products which meet consumer demands and also delight and excite them. Different retail formats evolve in response to changes in the competitive environment. The basic components for retail formats are the same but it is the relative degree of focus by retailer on these components which becomes basis for formation of different retail formats. Retail format can be termed as combination of various elements of retail mix, to offer value to the target customers. It is interface between retailer and the customers. It makes customers visit the store to obtain goods and services and desired value. Sometimes, it is defined as Business Model of the retailer. It is important to mention that every store has its own personality to appeal to clientele. The importance of retail formats can be gauged from the fact that most of the retail organisations equate them with strategic business units and can have separate operational infrastructure and organisational set-up for them. The retail format is the store ‘package’ that the retailer presents to the shopper. A format is defined as a type of retail mix, used by a set of retailers. Store Formats are formats based on the physical store where the vendor interacts with the customer. 1.1. Objective of the Study To study major retail mix strategies adopted by retailers of various formats 1. Introduction It is the mix of variables that retailers use to develop their business strategies and constitute the mix as assortment, price, transactional convenience and experience. Therefore each retailer needs to evaluate the enablers and deterrents in the retail marketplace. This primarily involves identifying the key drivers of growth, the shoppers’ profile and shopper expectations .It also means evaluating the nature of competition and challenges in the market place. Then the retailer decides the elements of the retail mix to satisfy the target markets’ needs more effectively than its competitors. The choice of retail mix elements will enable it to decide the type of format or structure of business. Format-wise preference of retail mix strategies as depicted in figure 1 highlight that the retailers of departmental stores prefer customer retention strategies followed by image enhancement and innovative strategies. Retailers of hypermarkets are in favour of customer retention, promotional and store operational strategies whereas supermarkets adopt store operational, growth and improvement strategies followed by promotional strategies. [72] Http://www.ijetmr.com©International Journal of Engineering Technologies and Management Rese [Aparna et. al., Vol.5 (Iss.5): May 2018] ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 [Aparna et. al., Vol.5 (Iss.5): May 2018] 2. Framework for Retail Mix Strategies The framework for retail mix of emerging retail formats has been developed with the help of results and analysis on retailers' perspective. The Figure 2 depicts various formats of emerging retail outlets as and the retail mix strategies adopted by them to enhance sales and customer satisfaction. With the help of factor analysis, retail mix strategies have been divided into seven major strategies as customer retention, image enhancement, store operational, promotional, innovative, growth & improvement and competitive strategies which are depicted in the framework. Figure 2: Frame Work for Retail Mix Strategies Figure 2: Frame Work for Retail Mix Strategies The related hypothesis is Ho: there is no significant difference in the strategies adopted by retailers of various formats. H1: there is significant difference in the strategies adopted by retailers of various retail formats. Figure 1: Format Wise Preference of Retail Mix Strategies Source: Statistical analysis on field data Figure 1: Format Wise Preference of Retail Mix Strategies Source: Statistical analysis on field data Figure 1: Format Wise Preference of Retail Mix Strategies Source: Statistical analysis on field data Figure 1: Format Wise Preference of Retail Mix Strategies While specialty stores like to implement innovative and image enhancement strategies followed by competitive strategies, convenience stores prefer customer retention, followed by store operational and promotional strategies. Discount stores choose to adopt store operational, promotional and growth and improvement strategies. With this brief explanation it can be inferred that store operational and promotional strategies are adopted by most of the formats followed by customer retention strategies. Image enhancement, innovative and growth and improvement strategies take the next precedence and competitive strategy is the least adopted strategy. The mean values of different retail mix strategies adopted by various formats are depicted in the following table. [73] Http://www.ijetmr.com©International Journal of Engineering Technologies and Management Rese [Aparna et. al., Vol.5 (Iss.5): May 2018] ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 Table 1: Format Wise Preference of Retail Mix Strategies Retail Formats Retail Mix Strategies Customer Retention Strategies Promotional Strategies Competitive Strategies Growth and Improvement Strategies Innovative Strategies Image Enhancement Strategies Store Operational Strategies Mean Mean Mean Mean Mean Mean Mean Departmental Stores 4.24 1.63 1.86 2.15 3.07 3.6 1.36 Hypermarkets 4.63 4.24 3.60 2.81 1.66 2.15 4.15 Supermarkets 2.15 3.63 3.64 3.64 2.84 2.55 4.29 Specialty Stores 2.81 3.61 4.15 2.15 4.65 4.26 1.66 Discount Stores 3.10 3.72 2.95 3.70 2.58 2.18 3.95 Convenience Stores 4.35 3.36 3.24 3.29 2.81 2.76 3.83 Source: Statistical analysis on field data Source: Statistical analysis on field data 3. Statistical Analysis 3. Statistical Analysis ANOVA Test for retail Mix Strategies and retail Formats The study uses ANOVA analysis for finding out whether there is a significant difference between retail mix strategies and emerging retail formats. [74] [Aparna et. al., Vol.5 (Iss.5): May 2018] ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 DOI: 10.5281/zenodo.1284588 Table 2: Anova-Retail Formats and Retail Mix Strategies of Retailers ANOVA Sum of Squares df Mean Square F Sig. Customer Retention Strategies Between Groups 53.081 5 10.616 10.473 .000 Within Groups 59.805 59 1.014 Total 112.886 64 Image Enhancement Strategies Between Groups 29.269 5 5.854 4.432 .002 Within Groups 77.921 59 1.321 Total 107.190 64 Promotional Strategies Between Groups 37.323 5 7.465 4.888 .001 Within Groups 90.108 59 1.527 Total 127.431 64 Store Operational Strategies Between Groups 24.191 5 4.838 3.033 .017 Within Groups 94.103 59 1.595 Total 118.294 64 Growth and Improvement Strategies Between Groups 55.167 5 11.033 6.551 .000 Within Groups 99.365 59 1.684 Total 154.532 64 Competitive Strategies Between Groups 27.642 5 5.528 3.963 .004 Within Groups 82.311 59 1.395 Total 109.953 64 Innovative Strategies Between Groups 21.349 5 4.270 3.488 .008 Within Groups 72.217 59 1.224 Total 93.566 64 Source: Statistical analysis on field data Table 2: Anova-Retail Formats and Retail Mix Strategies of Retailers ANOVA ANOVA results in the table show significant differences in all seven retail mix strategies of retailers across different retail formats. Competitive Strategies, Store Operational Strategies, Innovative Strategies, Customer Retention Strategies, Image Enhancement Strategies and Promotional Strategies are significant at 1 per cent level and Growth and Improvement Strategies are significant at .01 level. Thus the ANOVA results also throw light on the fact that there is a significant difference in retail strategies on the basis of retail formats. 4. Conclusion Retailing is a vast and fast growing industry as compared to other sectors of the economy. Therefore managing a retail business has become a complex task. It necessitates planning approach that integrates the efforts and activities of the different departments of the retail organization and results in achieving the companies’ objectives. [75] Http://www.ijetmr.com©International Journal of Engineering Technologies and Management Rese ISSN: 2454-1907 DOI: 10.5281/zenodo.1284588 ISSN: 2454-1907 [Aparna et. al., Vol.5 (Iss.5): May 2018] Further, there is a paradigm shift from traditional forms to emerging forms. The organized retail segment is growing at blistering pace. However, the success of retail industry would depend on the ways and means a retailers is trying to retain the customers, the manner by which a retailer is trying to enhance the image of the store, how efficiently retail stores perform their day-to-day operations and many more tactics. The store operations play very important role in the profitability and long-term survival. The rate at which new and new stores are coming up and are getting out of the competition is a serious issue for Indian retail industry. The approach, a store has towards merchandising placement, the visual aspects, and the customers and team work, affects the store's bottom line. With the increasing competition, they necessitate to be updated when it comes to cost cutting techniques and managing store's day-to-day operations. Corresponding author. E-mail address: nkv.aparna@ yahoo.co.in References [1] PricewaterhouseCoopers. (2011). Winning India's Retail: Factors for success, pp 3-5. [2] Swapna Pradhan (3rd Edition) Retailing Management: Text & cases New Delhi: Tata McGraw Hill. pp-137-147 aan. (2009). Fundamentals of Retailing New Delhi: Tata McGraw Hill pp 75-91 pp [3] KVS Madaan. (2009). Fundamentals of Retailing New Delhi: Tata McGraw Hill p [3] KVS Madaan. (2009). Fundamentals of Retailing New Delhi: Tata McGraw Hill pp 75-91 [4] K tl K ll K h Jh (2007) M k ti M t A S th A i P ti N D lhi 4] Kotler, Keller, Koshy, Jha. (2007). Marketing Management A South Asian Perspective Pearson. pp 145-170 [5] Michael Levy, Barton A Weitz (2003). Retailing Management NewDelhi: Tata McGraw-Hill.pp 151-157 [6] Field, A. (2005). Discovering Statistics Using SPSS (Second Edition ed.). London: Sage. [7] Grewal, D., Levy, M., and Kumar, V. (2009). Customer Experience Management in Retailing: An Organizing Framework. Journal of Retailing, 85(1), 1-14. [8] Gupta, S., Jain, k., and Jain, D. (2009). Retention Strategies for Organized Retailers in Semi-Urban Markets. ICFAI University Journal of Marketing and Management, VIII (2), 24-37. [9] Jain, R., and Bagdare, S. (2009). Determinants of Customer Experience in New Format Retail Stores. Journal of Marketing & Communication, 5 (2), 34-44. KVS Madaan.(2009). Fundamentals of Retailing New Delhi: Tata McGraw Hill pp 192-199 [11] Jasola, M. (2007). Emerging Trends in Retail Sector. Journal of IMS Group, 4 (2), 22-28. [12] Rajagopal. (2007). Leisure Shopping Behaviour and Recreational Retailing: A Symbiotic Analysis of Marketplace Strategy and Consumer Response. Journal of Hospitality & Leisure Marketing, 15(2), 5-31 [13] Tendai, M., and Crispen, C. (2009). In-store shopping environment and impulsive buying. African Journal of Marketing Management, 1(4), 102-108. [14] Anand, K.S., and Sinha, P.K. (2009). Store format choice in an evolving market: role of affect, cognition and involvement. International Review of Retail, Distribution and Consumer Research, 19(5), 505-534. [15] Goldman, A. (2001). The transfer of retail formats into developing economies: The example of China. Journal of Retailing, 77(2), 221-242 [16] Guy, C.M. (1998) Classifications of retail stores and shopping centres: Some methodological issues. Geo Journal, 45, 255-264. Corresponding author. E-mail address: nkv.aparna@ yahoo.co.in [76] Http://www.ijetmr.com©International Journal of Engineering Technologies and Management R
https://openalex.org/W3006927004	https://philpapers.org/archive/KNUSFT-3.pdf	English	null	Synthetic fictions: turning imagined biological systems into concrete ones	Synthese	2,020	cc-by	10,573	Synthese (2021) 198:8233–8250 https://doi.org/10.1007/s11229-020-02567-6 Synthese (2021) 198:8233–8250 https://doi.org/10.1007/s11229-020-02567-6 UNREALISTIC MODELS Abstract The recent discussion of ﬁctional models has focused on imagination, implicitly con- sidering ﬁctions as something nonconcrete. We present two cases from synthetic biology that can be viewed as concrete ﬁctions. Both minimal cells and alternative genetic systems are modal in nature: they, as well as their abstract cousins, can be used to study unactualized possibilia. We approach these synthetic constructs through Vai- hinger’s notion of a semi-ﬁction and Goodman’s notion of semifactuality. Our study highlights the relative existence of such concrete ﬁctions. Before their realizations nei- ther minimal cells nor alternative genetic systems were any well-deﬁned objects, and the subsequent experimental work has given more content to these originally schematic imaginings. But it is as yet unclear whether individual members of these heteroge- neous groups of somewhat functional synthetic constructs will eventually turn out to be fully realizable, remain only partially realizable, or prove outright impossible. Keywords Fiction · Models · Synthetic biology · Modality · Semifactuality · Unrealisticness Keywords Fiction · Models · Synthetic biology · Modality · Semifactuality · Unrealisticness Synthetic ﬁctions: turning imagined biological systems into concrete ones Tarja Knuuttila1 · Rami Koskinen1 Received: 28 February 2019 / Accepted: 3 February 2020 / Published online: 28 February 2020 © The Author(s) 2020 B Tarja Knuuttila tarja.knuuttila@univie.ac.at 1 Department of Philosophy, University of Vienna, Universitätsstraße 7, 1010 Vienna, Austria 1 Introduction Scientiﬁc models are unrealistic in various ways. The epistemic status of these sim- pliﬁed and highly idealized, and thus strictly speaking false depictions of reality, has become a central philosophical question. To make sense of the seeming falsity of models, several philosophers have entertained the idea of likening models to liter- ary ﬁctions. They have typically highlighted the ﬁctional nature of such idealized mathematical models as the harmonic oscillator, the Lotka–Volterra predator and prey system, Carnot’s ideal steam machine, economic theories of the ﬁrm, and artiﬁcial neural network models, to name just a few examples of a potential list that could go on 12 12 3 8234 Synthese (2021) 198:8233–8250 and on. What is puzzling about these models is that the systems that they appear to be describing cannot strictly speaking exist. For instance, the harmonic oscillator model assumes, among other things, that the string is massless, the bob is a point mass, and there is no friction. The Lotka–Volterra model, in turn, assumes that the populations increase or decrease continuously (although actual populations consist of discrete individuals), the environmental conditions do not change, the prey population ﬁnds enough food at all times, and so on. Thus, these models posit systems with properties that any known real systems do not, or even cannot, have. How should such model systems be understood? Invoking ﬁction provides one solution to this problem: these model systems could be imaginary systems, or “imagined-objects” (Godfrey-Smith 2006; Frigg 2010; Frigg and Nguyen 2016; Salis 2019).1 gg gg g y The ﬁctionalist solution to how these models can represent worldly target sys- tems thus casts them as nonconcrete, imagined systems (Godfrey-Smith 2006; Frigg 2010; Frigg and Nguyen 2016). The idea is that, if mathematical models are not to be identiﬁed with their targets or their contingent material instantiations (e.g. partic- ular equations on a blackboard), they might be thought of as some kind of imaginary constructs that resemble works of literary ﬁction. However, such a focus on the onto- logical status of models tends to overshadow another important locus of ﬁctionality and unrealisticness, namely the cases in which the target of the model is a non-existent system. In this article, we wish to rehabilitate this latter notion of ﬁction because of its important function in science. 1 Another way of approaching such systems in terms of ﬁction would be to address them as ﬁctional descriptions of real systems (Toon 2012; Levy 2015). We do not discuss this alternative as it leads to cumbersome analysis of modeling as it does not really recognize such objects as models. We focus on those accounts that take as their starting point the idea that models are surrogate systems (Swoyer 1991) or indirect representations (Weisberg 2007; Godfrey-Smith 2006). For a critical view on various ﬁctionalist epistemologies of modeling, see Magnani (2012). 2 At the time of its writing, neither Fine’s article on Vaihinger nor Vaihinger’s work awakened too much interest among philosophers of science. Fine (1993) is reprinted in Suárez’s collection on ﬁction (2009), in which several authors discuss or refer to Vaihinger. Mäki (1980) discusses Vaihinger, but rather in relation to positivist philosophy of science. 1 Introduction Consequently, establishing a biological or any other possibility is not just a question of imagination—what is ﬁctional may, or may not, prove to be realizable. We proceed as follows: before addressing two at least partially realizable biological ﬁctions, minimal cells and alternative genetic systems (Sect. 3), we will discuss ﬁction in philosophy of science (Sect. 2). In Sect. 4, we analyze the ﬁctional character of these synthetic constructs and argue for why we think that the ﬁctional perspective should be widened to cover also concrete ﬁctions, and what implications this move has for the discussion of ﬁctional modeling. Section 5 concludes the article. 1 Introduction There is a prominent class of scientiﬁc models, whose main purpose is not to provide scientiﬁc explanations of actual phenomena, but rather to explore unactualized possibilia. Interestingly, models studying such possibilia may also be concrete things, and hence they could be considered, we argue, as concrete ﬁctions. We will study concrete ﬁctions through two examples from synthetic biology: minimal cells and alternative genetic systems. Apart from modeling such systems in conceptual and theoretical terms, the scientists studying them also seek to mate- rially realize them. We call these biological constructs synthetic ﬁctions and analyze their various ﬁctional and modal aspects. The research practice of synthetic biology shows that it can be more fruitful to approach ﬁction in terms of modality (i.e. possi- bility) than verisimilitude (i.e. outright falsity). This is because the difference between the ﬁctional and the actual is often not ﬁxed, but dependent also on available tech- nologies. We argue that many synthetic ﬁctions are semi-ﬁctions and may later on ﬁnd partial or alternative realizations through semifactual strategies. That these biologi- cal realizations remain often only partial, or incomplete with respect to the functions sought for, indicates that although they are concrete, their existence is only relative. Another aspect of their relative existence is due to their dependence on the technolog- ical apparatus and laboratory conditions needed for their construction and continued functioning. In other words, although concrete, the synthetic constructs we study still 12 3 Synthese (2021) 198:8233–8250 8235 embody a ﬁctional dimension. Indeed, we claim that one of the advantages of viewing synthetic constructs as concrete ﬁctions is the recognition of the laborious processes through which scientists seek to realize some ﬁctional, though theoretically and empir- ically grounded ideas. Through these processes, scientists are probing whether those imagined systems are biologically possible, giving also more theoretical ﬂesh to the original imaginary speculations. Consequently, establishing a biological or any other possibility is not just a question of imagination—what is ﬁctional may, or may not, prove to be realizable. embody a ﬁctional dimension. Indeed, we claim that one of the advantages of viewing synthetic constructs as concrete ﬁctions is the recognition of the laborious processes through which scientists seek to realize some ﬁctional, though theoretically and empir- ically grounded ideas. Through these processes, scientists are probing whether those imagined systems are biologically possible, giving also more theoretical ﬂesh to the original imaginary speculations. 2.1 Vaihingerian fictions Writing about Vaihinger’s classic The Philosophy of ‘As if’ (1924), Fine (1993) antic- ipated the revival of the notion of ﬁction within the context of modeling, observing that “the industry devoted to modeling natural phenomena, in every area of sci- ence, involves ﬁctions in Vaihinger’s sense” (p. 6).2 Vaihinger understood ﬁction through its deviation from reality. These deviations are due to “thought-processes” and “thought-constructs” that contradict reality, or even themselves, but are neverthe- less “intentionally thus formed in order to overcome difﬁculties of thought by this artiﬁcial deviation” (Vaihinger 1924, p. xlvii). Vaihinger continues: “These artiﬁcial thought-constructs are called Scientiﬁc Fictions and distinguished as conscious cre- ations by their “As if” character” (ibid.). In highlighting the artiﬁcial character of ﬁction, Vaihinger invokes to the etymology of ﬁction in the Latin ﬁctio, and ﬁngere, that is the activity of constructing, forming, conceiving, inventing and so on, referring also to the products of such creative activities. Indeed, ﬁctions are omnipresent in Vaihinger’s view. Art, science and the social world are permeated by ﬁctions. Vaihinger discusses as ﬁctions artiﬁcial classiﬁca- tion,abstractiveﬁctions,andschematic,paradigmatic,symbolic,legal,personiﬁcatory, summational, heuristic and mathematical ﬁctions. He considers also various kinds of concepts or theoretical constructs as ﬁctions such as the atom, inﬁnity, the Absolute, and Kantian things in themselves. Many methods are ﬁctional as well: e.g. the method of abstract generalization, the method of unjustiﬁed transference, and mathematics as a method of thought. 12 3 3 8236 Synthese (2021) 198:8233–8250 Given the inclusive way Vaihinger talks about ﬁction, it is difﬁcult to think of any dimension of human language and thought that would not, ineluctably, involve ﬁction. Yet, Vaihinger warns of taking ﬁctions for real things, of “personiﬁcating” or “hypostatizing” them. Fictions are best used, knowingly and reﬂectively, as provisional scaffolding for thinking, providing useful thought-constructs that eventually should be abandoned in the quest for “real knowledge”.3 Consequently, a ﬁction cannot be veriﬁed, it can only be justiﬁed, for, at best, it presents an expedient, though “circuitous approach” (1924, p. 88) on the way to knowledge. Despite the abundance of ﬁctions that Vaihinger enumerates and analyzes, his gen- eral characterization of ﬁction, i.e. his “logic of ﬁctions”, is straightforward. There are two kinds of ﬁctions: real ﬁctions and semi-ﬁctions. Both kinds of ﬁctions are in con- tradiction with reality, and generally understood to be so. Additionally, real ﬁctions are in contradiction with themselves. 2.1 Vaihingerian fictions While semi-ﬁctions are “historically provisional”, real ﬁctions are “logically provisional” (p. 80). Vaihinger further explains the distinc- tion in the following way: “semi-ﬁctions are in conﬂict with the objective state of affairs, while with real ﬁctions we get essentially formal mistakes in thought, logical mistakes” (p. 81). Semi-ﬁctions may be discarded as the investigation proceeds, but they may also develop into real ﬁctions. We are interested in the opposite movement, that of turning semi-ﬁctions into concrete things that shows, we argue, the scientiﬁc, semifactually anchored, character of some semi-ﬁctions. While they amount to “a deviation from reality”, as Vaihinger puts it, they do not need to be false, but point instead toward unactualized possibilities. But before turning to study this insight, we take a look at the recent discussion of models and ﬁctions where the notion of ﬁc- tion has gravitated from the consideration of non-existent targets towards scientists’ imaginings without too much explicit notice. 3 Vaihingerian ﬁctionalism has been given both anti-realist (e.g. Fine 1993) and realist readings (e.g. Mäki 1980). 2.2 Fictions as imagined-objects In contrast to viewing ﬁctions in terms of their falseness, the contemporary discus- sion has focused on their imaginary nature. Godfrey-Smith’s (2006) seminal article on model-based theoretical strategy has provided a starting point for several articles addressing ﬁction (e.g. Frigg 2010; Knuuttila 2017; Salis 2019). In discussing models as indirect representations, Godfrey-Smith (2006) comes up with two perspectives on ﬁction that do not necessarily align. On the one hand, and in accordance with Vaihinger, he approaches models as tightly constrained systems through the study of which modelers seek to understand complex real-world systems. Godfrey-Smith notes that this strategy of model-based science has characteristic strengths and weaknesses, and possibly a “historical signature”. Importantly, it has one epistemic advantage in particular: the modal reach of modeling. Godfrey-Smith studies the work of Maynard Smith and Szathmáry (1995) on major transitions in evolution that proceeds stage by stage through presenting “idealized causal mechanisms”, i.e. models. If these “how possibly” models, Godfrey-Smith points out, “work at all, [they] would work just as 123 12 Synthese (2021) 198:8233–8250 8237 well in a range of nearby possible worlds that happen to be inhabited by different organisms.” (p. 732). well in a range of nearby possible worlds that happen to be inhabited by different organisms.” (p. 732). On the other hand, Godfrey-Smith views model systems as particular kinds of nonconcrete things, as “‘imagined concrete things’—things that are imaginary or hypothetical but would be concrete if they were real” (2006, pp. 734–735). In other words, “they do not exist, but at least many of them might have existed and if they had, they would have been concrete, physical things, located in space and time […]” (2009, p. 101). In the aforementioned quote Godfrey-Smith comes close to the prospect of realizing unactualized possibilities but falls back on the position that model systems are non-existent nonconcrete things.4 The current discussion has picked the latter notion of ﬁction that has provided a seemingly convenient way of handling the ontological question of what kind of objects nonconcrete model systems are—i.e. imagined-objects—and how they might represent. While in the case of concrete physical models, the material 3-D object is recognized as the model, in the case of nonconcrete models like mathematical models, the model is the imagined-object generated by a model description, e.g. a set of equations (Frigg and Nguyen 2016, pp. 225–242, see also Salis 2019). 4 In Godfrey-Smith’s thinking the two perspectives are two sides of the same coin since he combines the notion of indirect representation by Weisberg (2007) with Giere’s analysis of model-based representation (Giere 1988). Consequently, a model description speciﬁes a model system, understood as an imagined system that then provides a candidate for representation in terms of resemblance (Godfrey-Smith 2009). 2.2 Fictions as imagined-objects Kendall Walton’s pretence theory of ﬁction has been especially inﬂuential (e.g. Barberousse and Ludwig 2009; Frigg 2010; Frigg and Nguyen 2016; Salis 2019; Toon 2012; Levy 2015). A partial explanation for Walton’s popularity among philosophers of science is due to his antirealist answer to the puzzling ontological nature of ﬁctions: there are no such things as model systems. The basic idea of Walton’s pretence theory is that humans can imagine various kinds of things by making use of a diversity of objects as props. Accordingly, scientists can be seen to use models as props to envisage imaginary or ideal systems. Nearly anything can function as a prop, but it becomes such only as a result of rules for its use, i.e. “principles of generation” that are part of a “game of make-believe”. Thus what is ﬁctional becomes synonymous with what is “true in the appropriate game of make-believe” (Walton 1990, p. 34). Frigg (2010) uses Walton’s theory to formulate an account of ﬁction and scien- tiﬁc representation. According to this account, model descriptions serve as props that “p-represent” an imagined system that then “t-represent[s]” a target system (Frigg 2010). Three things are crucial in this construal of representation. First, representation is indirect in that whatever we often empirically treat as models (e.g. equations, dia- grams or scale models) are considered model descriptions that create in the minds of scientists an imagined entity that represents the target system. Second, this imagined entity typically has also features that are not contained in the model description (and, presumably, the other way around). Third, the ﬁctional model system has no existence of its own, it is just something imagined in a scientiﬁc game of make-believe. Obvi- 3 3 8238 Synthese (2021) 198:8233–8250 ously, the question then becomes that of explaining how such imagined entities are supposed to (t-)represent actual targets.5 In a later series of articles, Frigg and Nguyen (e.g. 2016, 2018) have developed the account presented in Frigg (2010) into a fully-ﬂedged account of scientiﬁc rep- resentation. Their DEKI account of scientiﬁc representation is a rather complicated amalgamation of resources drawn from Walton as well as from Goodman (1976) and Elgin(e.g.2004),coveringbothmaterial(i.e.“concrete”)modelsandnon-physical(i.e. “nonconcrete”) models. Exempliﬁcation and denotation are two of the key constituents of the DEKI account; a (representational) vehicle both Denotes a target system, and Exempliﬁes some properties. 5 For a critique, see Knuuttila (2017) and Salis (2019). Both Knuuttila and Salis point out, though approach- ing scientiﬁc modeling from different directions, that for a model to represent the model descriptions should be considered as parts of models. 6 To be more speciﬁc, Frigg and Nguyen consider the nonconcrete model to be composed of both the vehicle X and interpretation, although in many cases the interpretation boils down to a simple identity, see e.g. Frigg and Nguyen (2016, p. 238). 7 Knuuttila and Loettgers (2017b) refer to synthetic biology constructs as synthetic ﬁctions in their study of synthetic genetic circuits. 2.2 Fictions as imagined-objects That the DEKI account becomes unduly convoluted in the case of synthetic ﬁctions is due to the fact that it is ﬁrst and foremost an account of representation, yet the ﬁctionality 12 123 Synthese (2021) 198:8233–8250 8239 of synthetic systems is not primarily related to the problem of how to turn them, by imagining, into representations of yet another kind of (material) things or systems. of synthetic systems is not primarily related to the problem of how to turn them, by imagining, into representations of yet another kind of (material) things or systems. It does seem, then, that for all its sophisticated machinery, the DEKI account does not readily account for the evolving, self-referential, and simultaneously concrete and ﬁctional nature of synthetic systems. What is distinctive about minimal cells and alter- native genetic systems is that they are initially very schematically imagined systems referring to unactualized possibilia before (at least partially) successful attempts to realize them. Once realized, they furnish their own targets, to be worked on in suc- cessive rounds of experimentation and modeling, remaining still partially ﬁctional for reasons that will become clearer in our discussion of these synthetic constructs below. 2.2 Fictions as imagined-objects These properties, or related ones, are Imputed to a tar- get through a Key that translates the exempliﬁed properties to properties that can be ascribed to a target. In the case of 3-D models, such as the Phillips-Newlyn hydraulic model of macroe- conomy, there is a concrete object that exempliﬁes, under interpretation, the features to be imputed (Frigg and Nguyen 2018). However, the case of nonconcrete models, such as mathematical models, is trickier, since there is no concrete model object pro- viding the “base” for representation. Instead, it is the imagined-object that becomes the locus of representation (as in Frigg 2010). Frigg and Nguyen are explicit on this: “By mandating those involved in a certain game to imagine certain things, the model description generates the imagined-object that serves as the vehicle X of a represen- tation as.” (2016, p. 13, emphasis added).6 In other words, models are objects that represent by exemplifying, and the notion of ﬁction is employed to explain how mathe- matical models and other nonconcrete models can achieve this. (DEKI does not address ﬁctional targets in particular, since it aims to be a general account of representation, whose targets can be actual or ﬁctional systems). In contrast, ﬁction does not play any prominent part in Frigg and Nguyen’s treat- ment of concrete models. Imagination only enters in the symbolic rendering of the material object into a Z-representation, for example, turning the physical hydraulic machine into an economy-representation. Frigg and Nguyen explain: “In the concrete case X [i.e. the representational vehicle] is a physical object and claims about X are true or false; the imagination only comes into play when explaining how X becomes a Z-representation.” (Frigg and Nguyen 2016, p. 238). While this seems reasonable with regard to the Phillips-Newlyn machine, the cases of minimal cells and alter- native genetic systems pose speciﬁc challenges. Are we supposed to imagine how particular realizations of minimal cells or alternative genetic systems become mini- mal cell-representations or alternative genetic system-representations, for the purpose of imputing their properties to minimal cells and alternative genetic systems? 3.1 Minimal cells The basic motivation of the bottom-up approach is to construct minimal cells from nonliving material, and so being able to answer, at least partially, the puzzle concerning the origin of life. But this goal has still remained beyond the reach of scientiﬁc research. A more restrained goal than creating a minimal cell would be that of estimating a minimal genome that would contain the smallest number of genetic elements suf- ﬁcient to build and support a free-living cellular organism in an ideal environment (see Mushegian 1999, p. 709; Acevedo-Rocha et al. 2013, p. 273). The ﬁrst functional minimal cell, JCVI-syn3.0, created in the J. Craig Venter Institute (Hutchison et al. 2016), was a result of a top-down approach that begins with an existing cell and tries to reduce its genome to the minimum number of genes required to maintain cellular life. In fact, although the JCVI-syn3.0 cell is a reduced version of the Mycoplasma mycoides bacterium, it is actually a minimized version of an earlier synthetic version of the M. mycoides, called JCVI-syn1.0 (Gibson et al. 2010). JCVI-syn1.0 was hailed as the ﬁrst synthetic cell. The design process of JCVI- syn1.0 went as follows. First, the scientists picked the sequenced genomic data of a cell,whosesequencewasalreadyverysmall(M.mycoides).Thentheyfurtherdesigned the genome by deleting some genes and adding others including the watermarking of the genome by adding sequences that would distinguish it from the natural M. mycoides sequence. The names of the researchers, their URL and even some famous quotes were added. This modiﬁed genome was chopped into 1100 pieces that were sent to a DNA- synthesis company. Yeast cells that are among the workhorses of synthetic biology, were used to stich the overlapping synthesized fragments together into a synthetic genome that was through laborious trial and error procedures transplanted into M. capricolum bacteria. When the synthetic genome took over (some) of the recipient cells, the resulting JCVI-syn1.0 cells provided a proof of a principle that genomes can be designed on a computer, synthetized in laboratory, and booted up to produce a self-replicating cell controlled by a synthetic genome (see American Association for the Advancement of Science 2016). One of the original aims of creating synthetic cells was to eventually succeed in engineering a “minimal cell”. 3.1 Minimal cells The project of constructing artiﬁcial and minimal cells dates back at least to the 1950s. The notion of an artiﬁcial cell does not refer to any speciﬁc physical entity, or “an attempt to reproduce biological cells,” as one of its pioneers has put it (Chang 2007). “It is an idea involving the preparation of artiﬁcial structures of cellular dimensions” performing some tasks of cells, where “different approaches can be used to demon- strate this idea” (Chang 1972). While most artiﬁcial cells can only mimic certain behaviors of cells, the idea of a minimal cell is more ambitious: “a minimal cell is a hypothetical biological system that possess only the necessary and sufﬁcient attributes to be considered alive” (Gil 2011, pp. 1065–1066). Alternatively, minimal cells have been characterized as “the simplest collection of interacting molecules that can show signs of cellular life, under speciﬁc environmental conditions” (Ehmoser-Sinner and Tan 2018, p. 12). Such a cell would need to have, ﬁrst, a metabolism for maintaining life, second, a genetic program, and, third, a boundary that separates the cell from its environment (e.g. Acevedo-Rocha et al. 2013, p. 276; Xavier, Patil and Rocha 2014, p. 487). These three requirements are usually considered a part of a minimal deﬁnition of life, although there exists a considerable latitude as to what is included in such def- initions (see Knuuttila and Loettgers 2017a). What these considerations already hint at, however, is that the minimal cell is not going to be very minimal. There are two principal methods of creating minimal cells, the bottom-up and the top-down method. The bottom-up method is the more demanding of the two. It aims to approximate a minimal cell by assembling one such candidate from biomolecules such as proteins, lipids, carbohydrates, nucleic acids etc. Protocells are examples of this line of research. They are compartmentalized self-organized aggregations of abiotic componentsthathavebeenusedasmodelstostudytheemergenceoflifecharacteristics such as self-organization and replication, and, more generally, the transition towards living cells. One of the foci of this line of research has been on encapsulating genetic and metabolic material within vesicle membranes. A milestone in this line of research 12 3 3 8240 Synthese (2021) 198:8233–8250 was the protocell in which the researchers succeeded to implement RNA replication within a fatty acid vesicle (Adamala and Szostak 2013, pp. 1098–1100). 3.1 Minimal cells This goal was reached in 2016 with the JCVI-syn3.0 cell that only contained what researchers called essential and quasi-essential genes (the latter genes are not essential yet needed for robust growth) (Hutchison et al. 2016). The JCVI-syn3.0 cell is a minimized version of the JCVI-syn1.0. With improved trans- poson methods (involving foreign genetic sequences) the functions of genes were disrupted in order to ﬁnd out which ones were essential for the functioning of the bac- terial cell. The resulting JCVI-syn3.0 cell contained only 473 genes, yet the function of about 30% of the genes remained unknown. The researchers of the Venter team conclude: “That we have no clear idea of the functions performed by 149 of 473 genes in the minimal gene set makes it clear how incomplete our knowledge of the cellular biology really is” (Glass et al. 2017, p. 9). What is important to notice is that researchers do not believe any minimal cell to be exhibited. They rather stress the conceptual or imaginary nature of the synthetic cell. For example, Chang (1972) refers to the notion of artiﬁcial cells as an idea (see above), and other researchers discuss minimal cells and minimal genomes as 8 Glass et al. (2017) write interchangeably about ‘minimal genomes’ and ‘minimal cells’. This suggests that they think of a cell with a minimal genome as an approximation of a minimal cell. 123 123 Synthese (2021) 198:8233–8250 8241 concepts (e.g. Mushegian 1999; Koonin 2000; Delaye and Moya 2010; Acevedo- Rocha et al. 2013; Xavier et al. 2014; Glass et al. 2017). Xu et al. (2016, pp. 516–532) consider minimal and artiﬁcial cells as theoretical cells, while Moya et al. (2009, p. 225) canvass “different minimal hypothetically viable cells”. Ehmoser-Sinner and Tan (2018) explain: “While it is uncertain whether the minimal cell ever existed, or is still in existence, in the natural environment, this idea has spurred researchers into seeking to identify, or even create, the minimal cell” (p. 12). The authors of the JCVI-syn3.0 cell considered their creation as an instance of a real minimal cell, summarizing their achievement in the following way: “No longer are we limited to working with imaginary minimal cells or naturally occurring organisms with small genomes as surrogates. A minimal cell8 has now been constructed” (Glass et al. 2017, p. 10). Moreover, in their original article published in Science, the scientists of the J. Craig Venter Institute refer to JCVI-syn3.0 as “a working approximation to a minimal cell” (Hutchison et al. 2016, aad6253-1). Another important observation concerns the fact that the minimality of an organ- ism’s genome depends on its environment. Glass et al. (2017) discuss mycoplasma bacteria as “near-minimal cells” that were recognized as such even before the genomic era. Already Morowitz (1984) suggested that they could be used as models for understanding basic principles of life. As discussed above, Mycoplasma mycoides functioned as the template organism for JCVI-syn3.0 (via JCVI-syn1.0). What is inter- esting about mycoplasmas is that they were not originally so simple but evolved from other more conventional bacteria through a massive gene loss as a result of adopting parasitic lifestyles in nutrient-rich stable environments (e.g. Woese et al. 1980), such as e.g. human urogenital tract. The environment of their synthetic cousin JCVI-syn3.0 is even more ideal, a stress-free laboratory environment, where the growth media pro- vides all needed nutrients. Finally, there can be different kinds of minimality that do not go hand in hand. Multiple studies have shown that the genome size does not often correlate with the complexity of other cellular features of an organism (see Xavier et al. 2014). The minimality in the components does not imply the simplicity of interactions. 3.2 Alternative genetic systems Alternative genetic systems are synthetically designed chemical structures that are conjectured to have the ability to carry and transmit genetic information—arguably some of the most important and basic functions of any living system. Given that all known life is based on DNA (and RNA), successful alternative realizations of genetic systems would amount to a ground-breaking scientiﬁc and technological achieve- ment. Although such xenobiological alternatives are foreign to Terran biology, the basic ontological toolkit of synthetic biologists constructing them consists of chemi- cal elements that obey known chemical and physical principles. It is only at the level of biologically functional macromolecules and their purported ability to support life that things become (partially) ﬁctional. 3 3 12 8242 Synthese (2021) 198:8233–8250 Many of the alternative genetic systems that are currently being studied are based on permutations and reconﬁgurations of the classical Watson–Crick model of DNA. Changes to this structure can be classiﬁed into two main categories: those targeted at the backbone material (so-called xeno nucleic acids, or XNAs) and those targeted at the nucleobases (artiﬁcial or expanded genetic alphabets). Thus, models for alternative genetic systems consist of molecular building blocks, like hexose sugar in place of the natural deoxyribose backbone of DNA, or as yet unfamiliar, but chemically synthe- sized, alternative genetic alphabets, like P and Z in place of, or as complements to, the natural nucleotides A, T, C and G. Natural evolution has opted for a certain structural design in DNA, but as far as we know, there might be other ways to implement genetic material, too.9 Although no living self-sustaining xeno-organism exists yet, many alternative genetic systems have already been constructed, be they XNA-based systems or unnat- ural genetic alphabets (see, e.g., Anosova et al. 2016; Benner et al. 2016). For example, in the case of TNA, the natural deoxyribose backbone of the DNA molecule has been replaced with the molecule threose, while PNA would stand for peptide nucleic acid (Anosova et al. 2016, p. 1012). Alternative genetic alphabets range from single nucle- obase changes to systems consisting of completely new foreign base pairs (Marlière et al. 2011; Malyshev et al. 2014). Unnatural base pairs can also be merged together with the A-T and C-G pairs, forming artiﬁcially expanded genetic information systems (Zhang et al. 2017). Most recent developments include the so-called hachimoji10 system of eight genetic letters, forming the alphabet GACTPZSB (Hoshika et al. 2019). 9 Already some years before Watson and Crick’s discovery of the exact structure of the DNA molecule in 1953, Schrödinger (1944) had proposed that genes could be made up from a certain kind of as-yet-unknown aperiodic crystal. Although his hypothesis about the actual material medium of the genetic material turned out to be false, the general structural insights associated with it are still appreciated by working synthetic biologists (Karalkar and Benner 2018; Hoshika et al. 2019). 11 Although no self-sustaining life is yet based on the GACTPZSB system, as an alphabet it nevertheless already surpasses in size a famous imaginary case from sci-ﬁ. In the Steven Spielberg movie The Extrater- restrial from 1982, E.T.’s genome is said to consist of a six-letter genetic alphabet, featuring a foreign base pair in addition to the two familiar pairs found in the DNA of Terran life. In this regard, E.T. comes closer to an earlier achievement of the Foundation for Applied Molecular Evolution, the six-letter system GACGPZ (Benner et al. 2016). https://blogs.scientiﬁcamerican.com/news-blog/artiﬁcial-life-was-steven- spielber-2009-02-15/ (Link last accessed 20.11.2019). 3.2 Alternative genetic systems According to a recent report in Science, the system is designed to strike a delicate balance between structural stability and mutability. Like natural DNA and RNA, it is able to maintain its overall structure while its particular building blocks change, making it a “mutable information storage system” (Hoshika et al. 2019, p. 363). A system with these properties is central to Darwinian evolution.11 Of course, in and of themselves, these artiﬁcial genetic systems cannot be taken as deﬁnitive proofs about the nature of the genetic basis of life on Earth, or elsewhere in the universe. Rather, we suggest that they are like synthetic minimal cells in that they are best seen as more or less successfully realized concrete ﬁctional model systems that can be used to explore the range of biologically feasible possibilities. Though some theoretical models have been used to explore the in-principle viability of these kinds of 9 Already some years before Watson and Crick’s discovery of the exact structure of the DNA molecule in 1953, Schrödinger (1944) had proposed that genes could be made up from a certain kind of as-yet-unknown aperiodic crystal. Although his hypothesis about the actual material medium of the genetic material turned out to be false, the general structural insights associated with it are still appreciated by working synthetic biologists (Karalkar and Benner 2018; Hoshika et al. 2019). 11 Although no self-sustaining life is yet based on the GACTPZSB system, as an alphabet it nevertheless already surpasses in size a famous imaginary case from sci-ﬁ. In the Steven Spielberg movie The Extrater- restrial from 1982, E.T.’s genome is said to consist of a six-letter genetic alphabet, featuring a foreign base pair in addition to the two familiar pairs found in the DNA of Terran life. In this regard, E.T. comes closer to an earlier achievement of the Foundation for Applied Molecular Evolution, the six-letter system GACGPZ (Benner et al. 2016). https://blogs.scientiﬁcamerican.com/news-blog/artiﬁcial-life-was-steven- spielber-2009-02-15/ (Link last accessed 20.11.2019). 123 9 Already some years before Watson and Crick’s discovery of the exact structure of the DNA molecule in 1953, Schrödinger (1944) had proposed that genes could be made up from a certain kind of as-yet-unknown aperiodic crystal. Although his hypothesis about the actual material medium of the genetic material turned out to be false, the general structural insights associated with it are still appreciated by working synthetic biologists (Karalkar and Benner 2018; Hoshika et al. 2019). 10 Inspired by the Japanese words for “eight” and “letter”. 11 Although no self-sustaining life is yet based on the GACTPZSB system, as an alphabet it nevertheless already surpasses in size a famous imaginary case from sci-ﬁ. In the Steven Spielberg movie The Extrater- restrial from 1982, E.T.’s genome is said to consist of a six-letter genetic alphabet, featuring a foreign base pair in addition to the two familiar pairs found in the DNA of Terran life. In this regard, E.T. comes closer to an earlier achievement of the Foundation for Applied Molecular Evolution, the six-letter system GACGPZ (Benner et al. 2016). https://blogs.scientiﬁcamerican.com/news-blog/artiﬁcial-life-was-steven- spielber-2009-02-15/ (Link last accessed 20.11.2019). 10 Inspired by the Japanese words for “eight” and “letter”. 123 12 Synthese (2021) 198:8233–8250 8243 molecules (e.g., Wagner 2005; Henderson et al. 2019), this work is now complemented and seriously reﬁned by concrete wet-lab probing of these constructions by synthetic biologists and chemists. molecules (e.g., Wagner 2005; Henderson et al. 2019), this work is now complemented and seriously reﬁned by concrete wet-lab probing of these constructions by synthetic biologists and chemists. At the current stage, these systems have a distinctly partial existence. On the one hand, they are chemically synthesized material entities exhibiting many important desiderata of a functional genetic molecule. On the other hand, it is not yet clear whether they can support life to its fullest, especially when it comes to developmental and multi-generational evolutionary processes. Successful incorporations of foreign bases have been reported in some constrained cases, like the replacement of thymine by 5-choloruracil in a laboratory strain of E. coli (Marlière et al. 2011). However, to achieve completely self-sustaining life that fully incorporates alternative genetic molecules is a big challenge. Recently, researchers managed to have E. coli cell read and sustain the pair d5SICS–dNaM as an extension of its natural genetic system, providing a proof of principle that “increased” genetic information can be both stored and retrieved by the resulting “semi-synthetic organism” (Zhang et al. 2017). 13 Semi-ﬁctions are related to “anchored possibilities” in the empirical literature on human counterfactual thought. There are certain cognitive and social factors that affect what people prefer to keep immutable when considering a particular ﬁctional situation, although these boundaries are not categorically immovable (Byrne 2005, pp. 171–173). 12 Though semi-ﬁctions can of course turn out to be false. The phlogiston element is a case in point. From our current epistemic situation, it seems to provide a borderline case between a semi-ﬁction and a real ﬁction. 4 Synthetic semi-ﬁctions and the semifactual strategy Of the different philosophical notions of ﬁction discussed above, Vaihingerian semi- ﬁctions seem most useful in capturing the ﬁctional, yet concrete and partially realizable character of synthetic models. Although being at variance with what is actually the case, semi-ﬁctions are not in principle contradictory with what we know about possible reality.12 At the very least, there are no nomological reasons to deem them impossible. In other words, their nonexistence in the (known part of the) actual world may be a contingent fact. One way to look at synthetic biology is through its distinctively modal research practice that materially explores biological possibilities (Elowitz and Lim 2010; Knuuttila and Loettgers 2017b; Koskinen 2017). What may begin as a more general idea, as a scientiﬁc ﬁction, provides a concept and impetus for various attempts at concretely realizing it. However, such scientiﬁc semi-ﬁctions need to be restricted in scope and keep many factual elements unchanged in order to point towards biologically realizable possibilities. In the case of modeling, such factual parts can amount to explicitly stated parameters and highly situational factors, but also the background knowledge, e.g., general scientiﬁc theories and empirical regularities play a role.13 Minimal cells and alternative genetic systems can be viewed as particular kinds of scientiﬁcally constrained semi-ﬁctions: synthetic ﬁctions. The scientiﬁcally constrained nature of many synthetic ﬁctions can be further ana- lyzed, we suggest, by the notion of semifactuality. Possibilities are often linked to the idea of counterfactuals. Whereas counterfactuals as standardly conceived address 13 Semi-ﬁctions are related to “anchored possibilities” in the empirical literature on human counterfactual thought. There are certain cognitive and social factors that affect what people prefer to keep immutable when considering a particular ﬁctional situation, although these boundaries are not categorically immovable (Byrne 2005, pp. 171–173). 12 3 3 8244 Synthese (2021) 198:8233–8250 “what-if-things-were-different” questions, semifactuals highlight what might stay the same in the face of certain changes, taking rather an “even-if-things-were-different” line of approach to inquiry. “what-if-things-were-different” questions, semifactuals highlight what might stay the same in the face of certain changes, taking rather an “even-if-things-were-different” line of approach to inquiry. The term “semifactual” goes back to Goodman (1947, 1954), who used it to denote a conditional with a false antecedent, but a true consequent. He viewed semifactuals in relation to counterfactuals, stating that “in practice full counterfactuals afﬁrm, while semifactuals can deny what is afﬁrmed by the counterfactuals.” (Goodman 1947, p. 115). 4 Synthetic semi-ﬁctions and the semifactual strategy The examples Goodman (1947) gives are “Had the match been scratched, it would have lighted” for the counterfactual, and “Even if the match had been scratched, it still would not have lighted” for the semifactual. On closer inspection, one can distinguish between two types of semifactuals (McCall 1983). First, there is the case in whichnoconnectionbetweentheantecedentandconsequentexists,andtheconsequent is true regardless of the truth of the antecedent. But in the second case, a connection exists, and the antecedent can be viewed as an alternative way of generating the consequent. The earlier literature on semifactuals is rather thin. We propose a new reading of semifactuals that seeks to give more substance to the notion in a pragmatic and nat- uralistic context. Previous authors like Goodman treated semifactuals as linguistic constructions. We acknowledge that history, and draw some inspiration from it, but analogously to how counterfactuals are treated in contemporary philosophy of science, we do not want to limit our understanding of science to sentences (or propositions). Indeed, in the more recent literature, semifactuals have been given, besides their met- alinguistic interpretation, a possible worlds reading (Barker 2006). According to this reading, semifactuals highlight how an important factual aspect of the actual world would hold even in nearby possible, but slightly different worlds. Semifactuals thus point to a converging picture of some aspect of reality; to different historical trajecto- ries or causal processes leading to the same outcome (McCall 1983). Drawing inspiration from the possible worlds reading, we suggest a third inter- pretation of semifactuality as the exploration of the potential multiple realizability of biological (and also other) systems. Under this interpretation, some higher-level func- tion or organizational feature in the actual world is kept invariant while its underlying material makeup is being varied as in the case of our examples of minimal cells and alternative genetic systems. A familiar example comes from a philosophy of mind context where multiple realizability is used to argue that it is possible to hold mental properties ﬁxed while their physical or neural realizers are manipulated (Pernu 2014, p. 529). The semifactual formulation of this idea would be: Even if the realizer was (at least partially) different, the property of interest would still hold. Thus, while the semifactual strategy highlights the invariance of some factual components of many semi-ﬁctional models, it also employs in model construction counterfactual changes or partially ﬁctional realizers. 4 Synthetic semi-ﬁctions and the semifactual strategy While the philosophical tradition has related ﬁction largely to thought processes and imagination, technoscience has provided a new material scaffolding for ﬁctional thinking and theoretical reﬂection. Novel technologies prompt researchers to adopt a modal mode as they acquire new ways to effectively manipulate nature. From a cog- nitive standpoint, directing one’s attention to a possibility in the world—or, indeed, recognizing it as a possibility to begin with—is correlated with how controllable the 8245 Synthese (2021) 198:8233–8250 target is assumed to be (Byrne 2005, p. 100). The rapidly advancing toolkit of biolog- ical engineering, and the consequent capabilities on intervening in the living world makes it easier to see possibilities where they earlier remained hidden. In contrast to previous armchair/work desk/dry lab approaches, synthetic biology can experimen- tally study alternative biological designs that are not found in nature, but which could nevertheless realize familiar biological functions, when concretized (see Koskinen 2019b). Both minimal cells and alternative genetic systems keep some fundamental biological functions ﬁxed, while their naturally evolved material realizers, or at least some important parts of them, are changed. We suggest that such synthetic systems allow us to draw semifactual inferences in that they employ modal “even if” reasoning in constructing novel biological systems or parts to implement familiar functions. Thus, instead of ontologizing the concept, or viewing semifactuals solely from a linguistic perspective, we suggest that they are best understood in terms of particular types of modelling/research strategies. We want to know whether something that is true of the actual world also holds in nearby, but different possible worlds. XNA systems, should they prove as functional as RNA/DNA, provide a relatively straightforward example of a semifactual strategy. The actual known world has genes made out of DNA. A possible alternative world has genes made out of XNA. Because in the actual world we only have one kind of genetic molecule, the only realistic way of testing out alternative possibilities is to construct them in a lab. Because the function of these XNA molecules is purported to be the same as that of DNA-based genes, successful synthesis and incorporation of XNA molecules would amount to showing the multiple realizability of genes. 4 Synthetic semi-ﬁctions and the semifactual strategy Accordingly, in the study of minimal cells the goal of the various lines of investigation is to develop a minimal artiﬁcial cell that would nevertheless be able to fulﬁll at least some basic requirements for qualifying as a living thing (in addition to possibly also modifying these requirements). The semifactual research strategy has a number of epistemic beneﬁts. Firstly, it restricts the search space for biological possibilities. Just compare the semifactual strategy to a purely blind-shot exploration where no clear actual world function is kept ﬁxed as a goal. Semifactuals thus provide a ready-made standard for evaluat- ing success. Secondly, and as mentioned above, since semifactuals are also attached to the actual world, modal inferences drawn from them tend to be more connected and robust, both biologically and conceptually. For example, a successful realization of an XNA-lifeform would show that it is not necessary to have DNA to support life; in other words, we would know that DNA is contingent as a material medium for genes. Thus, by achieving knowledge about a possibility, we also learned something new about the actual world. The semifactual strategy can generate understanding of the robustness and gener- ality of various biological functions—to the extent that they can or cannot be multiply materially realized. The (supposedly) multiply realizable character of biological sys- tems is also importantly related to their modularity, a cornerstone of the synthetic biology practice. Alternative genetic systems like XNA (pick your favourite in place of the “X”) or the eight-letter hachimoji DNA provide a nice example of how synthetic biologists proceed in a modular manner, in addressing one central part of biological organization at a time. The research on minimal cells, in turn, takes it for granted 12 3 3 8246 Synthese (2021) 198:8233–8250 that there are genes that are responsible for the development of cell structures and the regulation of their behavior—and can leave it open how exactly they are constituted. g p y y In view of considering minimal cells as ﬁctions, Vaihinger’s discussion of Goethe’s original plant and original animal proves intriguing.14 Vaihinger approaches Goethe’s discussion as a ﬁctive judgement that all plant and animal species should be regarded ‘“as if they had been formed according to the standard of an animal or plant archetype” (1926, p. 267). In the same vein he notes that Lotze’s “hypothetical animal” should rather be considered as ﬁctional. 14 In the present-day scientiﬁc discussion of minimal cells, one line of research concerns the Last Universal Common Ancestor (LUCA), the supposed ancestor of all the life forms on Earth. 4 Synthetic semi-ﬁctions and the semifactual strategy While the complete minimal cell can best be consid- ered as a theoretical ﬁction, it is important to note that this ﬁction can have different kinds of realizations. Indeed, one central motivation for discussing minimal cells has precisely been the attempt by bioengineers, biochemists and biophysicists to create such cells in laboratories. As these usually rudimentary ideas can be realized very dif- ferently, the minimality of an actual biological system is always of a relative nature. For example, for biological reasons, one cannot strive to construct the minimal genome, but just a minimal genome of a particular organism. The estimates of the shared min- imal sets of genes have dropped eventually to even zero as more genomes have been added to comparison. In their study of 1000 genomes, Lagesen et al. (2010) found out that there “is an incredible diversity within these genomes with essentially no genes being conserved across all prokaryotes” (p. 607). Moreover, minimal sets of genes vary considerably in terms of gene number and even identity not only between different organisms but also in the same organism in different conditions. Consequently, the notion of a minimal gene set is relativized not only to the organism in question but also to its environment and so the condition of ideal (laboratory) environment is typically added to the deﬁnition of a minimal cell (e.g. Mushegian 1999; Koonin 2000; Glass et al. 2017). The same kinds of consid- erations apply also to alternative genetic systems whose foreign metabolic needs are only satisﬁed in a highly artiﬁcial laboratory environment. One intriguing theoretical question, then, that these semi-ﬁctional constructs raise, is when should we count a synthetic system as a living entity, or an essential part of such an entity. 5 Conclusions Synthetic biology investigates the realizability of biological possibilities: various orga- nizational principles and mathematical and other models are turned into synthetic constructs that are not only scaffolds towards a better understanding of already exist- ing actual biological entities (Elowitz and Lim 2010; Knuuttila and Loettgers 2017b; Koskinen 2017). Rather, these synthetic constructs extend the boundaries of biological knowledge also beyond natural evolution on Earth. For example, minimal cells provide insight into how life could have evolved from non-living materials, and how life could be deﬁned. Artiﬁcial genetic alphabets address, among other things, the question of whether the genetic code would need to be RNA/DNA-based, or if that is simply a contingent fact concerning all known life on Earth. We have argued that minimal cells 12 3 Synthese (2021) 198:8233–8250 8247 and alternative genetic systems are both concrete and ﬁctional in that they are (par- tial) realizations of unactualized but possible biological systems (see also Koskinen 2019a). and alternative genetic systems are both concrete and ﬁctional in that they are (par- tial) realizations of unactualized but possible biological systems (see also Koskinen 2019a). In order to analyze the concrete, yet ﬁctional, character, of synthetic constructs we reviewed some, in our view, promising discussions of ﬁctions in science. Two different approaches were discerned: the more contemporary theorizing focusing on imagina- tion as mental activity (e.g. Godfrey-Smith 2006; Frigg 2010; Frigg and Nguyen 2016), and the earlier Vaihingerian account of ﬁction as a deviation from reality (Vaihinger 1924; Mäki 1980; Fine 1993). Of the former accounts, the DEKI account (e.g. Frigg and Nguyen 2016, 2018) has the advantage of analyzing both the cases of noncon- crete and concrete models, imagination playing partially different role in them. That the DEKI account does not, in our view, accommodate the most pertinent features of synthetic ﬁctions is due to, ﬁrst, the fact that it is not dynamic,15 and so not designed to address the changing ﬁctional-cum-concrete status of synthetic models. Second, the DEKI account is an account of representation and as such unduly convoluted to study synthetic ﬁctions, since they do not primarily function as representations. Syn- thetic constructs can rather be viewed as biological research objects in their own right, whose epistemic functioning can be better accommodated from the artefactual than representational perspective. 15 We thank the anonymous reviewer for urging us to make this point explicit. 5 Conclusions The artefactual account of models (Knuuttila 2011, 2017) attributes the epistemic value of models, ﬁrst, to the scientiﬁc questions that they are designed to probe, and, second, to the representational modes and material media used in model construction. Both of these aspects of the artefactual account are crucial for the practice of synthetic modeling. Through turning ﬁctional ideas into concrete entities, scientists study modal questions concerning whether speciﬁc biological materials, principles and organiza- tional features are necessary for some biological function, or might they be contingent, and if so, how else could the function in question be realized. That synthetic systems are constructed from biological material, according to particular design choices, is crucial for answering these questions, underlining the epistemic importance of the representational modes and media used. Apart from synthetic systems, the practice of synthetic biology combines many other kinds of models (i.e. mathematical, dia- grammatic and digital). Such models tend to remain merely hypothetical, however, as they do not allow scientists to judge whether the mechanisms studied could in fact be realized, or realized by different biochemical materials. The artefactual understanding of models can be aligned with the Vaihingerian account of ﬁctions as “artiﬁcial deviations” from reality intended “to overcome dif- ﬁculties of thought” (see above Sect. 2.1.). Synthetic constructs can be conceived as Vaihingerian semi-ﬁctions since they do not contradict, at the outset, the exist- ing scientiﬁc knowledge, and so amount to the exploration of unactualized biological possibilia. We proposed that one central strategy that synthetic biologists employ in studying the realizability of biological semi-ﬁctions can be characterized as semifac- tual. We then interpreted such semifactual strategy through its utilization of multiple realizability, that is, keeping some higher level biological function ﬁxed and studying experimentally its possible realizations. 3 12 8248 Synthese (2021) 198:8233–8250 The notion of ﬁction, we suggest, is valuable for even such a material practice as synthetic biology, if only because not all prima facie possibilities will turn out to be genuine biological possibilities. Viewing synthetic constructs as concrete ﬁctions addresses also the important question of how to understand those synthetic systems which wind up being only partially realizable (in view of their intended functions). These systems might prove to be eventually impossible, or, even more likely, doomed to occupy the “modal in-between”. 5 Conclusions What synthetic biology teaches us, is that ﬁctions do not wear their justiﬁcation on their sleeves and so we should not take unaided human imagination for a very good guide on what is genuinely possible. Indeed, before their realizations neither minimal cells nor alternative genetic systems were any well-deﬁned imagined-objects, they were rather preliminary sketches largely based on general criteria derived from known features of actual cells and genetic systems. Attempts at realizing them gave more content and coherence to these imaginings. It is no wonder, then, that such ﬁctional systems start multiplying when technological possibilities for their realization are emerging. Acknowledgements Open access funding provided by University of Vienna. This paper has been presented in “What to make of highly unrealistic models” symposium (TINT, University of Helsinki, 13.10.2017), the 7th SPSP conference (University of Ghent, 30.6.2018) and “Varieties of experiment and measurement in technoscience” workshop (Technische Universität Darmstadt, 4.9.2018). We thank the audiences for their valuable comments. We are also grateful to the anonymous reviewer, who went to great lengths in commenting our paper and providing constructive suggestions. Funding European Research Council (ERC) under the European Union’s Horizon 2020 research and inno- vation programme (Grant Agreement No 818772) and the Academy of Finland (Grant No 290079). 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W4200478249.txt	https://www.frontiersin.org/articles/10.3389/fmed.2021.796009/pdf	en		Data_Sheet_1.DOCX	null	2,021	cc-by	7,894	SYSTEMATIC REVIEW published: 09 December 2021 doi: 10.3389/fmed.2021.796009 Physical Exercise vs. Metformin to Improve Delivery- and Newborn-Related Outcomes Among Pregnant Women With Overweight: A Network Meta-Analysis Carlos Pascual-Morena 1 , Iván Cavero-Redondo 1,2 , Celia Álvarez-Bueno 1,3 , José Alberto Martínez-Hortelano 1,4 , Sara Reina-Gutiérrez 1 , Alicia Saz-Lara 1 , Sergio Núñez de Arenas-Arroyo 1* and Vicente Martínez-Vizcaíno 1,5 Health and Social Research Center, Universidad de Castilla—La Mancha, Cuenca, Spain, 2 Rehabilitation in Health Research Center (CIRES), Universidad de las Américas, Santiago, Chile, 3 Universidad Politécnica y Artística del Paraguay, Asunción, Paraguay, 4 Guadalajara University Hospital, Health Service of Castilla-La Mancha (SESCAM), Guadalajara, Spain, 5 Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Talca, Chile 1 Edited by: Alessandra Familiari, Università Cattolica del Sacro Cuore, Italy Reviewed by: Daniele Di Mascio, Sapienza University of Rome, Italy Caterina Neri, Fondazione Policlinico Gemelli, Italy *Correspondence: Sergio Núñez de Arenas-Arroyo sergio.nunezdearenas@uclm.es Specialty section: This article was submitted to Obstetrics and Gynecology, a section of the journal Frontiers in Medicine Received: 15 October 2021 Accepted: 22 November 2021 Published: 09 December 2021 Citation: Pascual-Morena C, Cavero-Redondo I, Álvarez-Bueno C, Martínez-Hortelano JA, Reina-Gutiérrez S, Saz-Lara A, Núñez de Arenas-Arroyo S and Martínez-Vizcaíno V (2021) Physical Exercise vs. Metformin to Improve Delivery- and Newborn-Related Outcomes Among Pregnant Women With Overweight: A Network Meta-Analysis. Front. Med. 8:796009. doi: 10.3389/fmed.2021.796009 Frontiers in Medicine \| www.frontiersin.org Background: Overweight/obesity is associated with the risk of delivery- and newborn-related complications in pregnancy. Interventions such as exercise or metformin could reduce the risk of these complications. Objective: To estimate and compare the effects of different types of exercise interventions (i.e., aerobic, resistance, combined exercise) and metformin on delivery- and newborn-related outcomes among pregnant women with overweight/obesity. Methods: MEDLINE, Scopus, Web of Science, Cochrane Library databases and the gray literature were searched from inception to September 2021. This systematic review was registered in PROSPERO (CDR: 42019121715). Randomized controlled trials (RCTs) of metformin or an exercise intervention aimed at preventing cesarean section, preterm birth, macrosomia, or birth weight among pregnant women with overweight/obesity were included. Random effects meta-analyses and frequentist network meta-analyses (NMA) were conducted for each outcome. Results: Fifteen RCTs were included. In the NMA, metformin reduced the risk of cesarean section (RR = 0.66, 95% CI: 0.46, 0.95), combined exercise reduced the risk of macrosomia (RR = 0.37, 95% CI: 0.14, 0.95), and aerobic exercise reduced birth weight (mean difference = −96.66 g, 95% CI: −192.45, −0.88). In the subgroup among pregnant women with obesity, metformin reduced the risk of cesarean section (RR = 0.66, 95% CI: 0.45, 0.97). Conclusions: Combined exercise could reduce the risk of macrosomia in pregnant women with overweight, whereas metformin could reduce the risk of cesarean section in pregnant women with obesity. However, previous evidence suggests a larger effect of physical exercise in other outcomes for this population group. Therefore, 1 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy the medicalization of healthy pregnant women with obesity is not justified by the current evidence. Systematic Review Registration: PROSPERO: CRD42019121715; https://www.crd. york.ac.uk/prospero/display_record.php?ID=CRD42019121715 Keywords: pregnancy, exercise, metformin, overweight, obesity, systematic review, network meta-analysis INTRODUCTION METHODS Pre-pregnancy overweight and obesity are global public health problems that affect ∼40% of women (1). The risk of numerous adverse deliveries and newborn events are increased by overweight and obesity, such as the risk of gestational diabetes mellitus, hypertensive disorders of pregnancy, macrosomia, preterm birth, or cesarean sections delivery (2–4). Furthermore, overweight and obesity also cause long-term health problems in offspring through epigenetic and microRNA interaction mechanisms, such as obesity or type II diabetes (5–7). The rate of total cesarean sections is too high according to WHO recommendations, which estimate that only 10% of cesarean sections are actually necessary (8), but in the United States, 18.5% of deliveries in 2010 were by cesarean section (9). Preterm birth is defined as birth occurring before 37 weeks of gestation, and its incidence globally and in the United States is estimated to be approximately 10% (10). Additionally, macrosomia is usually defined as a birth weight greater than 4,000–4,500 g (depending on the guideline or author) (11), with an incidence of 9% in the United States (considering macrosomia as birth weight > 4,000 g) (12). International guidelines recommend at least 30 mins of moderate-vigorous physical activity per day, including aerobic or combined (resistance and aerobic exercise) activity (13). It is not ruled out that exercise could have a protective effect on the development of fetal macrosomia and cesarean sections (14, 15). For preterm birth, in the past, exercise in pregnancy was discouraged because of theoretical risks. However, currently, some authors have even proposed that it is beneficial, but that is not without controversy (16). Metformin has also been proposed for pregnant women with gestational diabetes mellitus, polycystic ovary syndrome, and obesity. Despite being a safe drug and having some benefits in women with gestational diabetes mellitus or polycystic ovary syndrome, the benefits of prescribing metformin in non-diabetic women with obesity are unclear (17). In a previous network meta-analysis (18), metformin reduced the risk of cesarean section. Moreover, a previous network metaanalysis by our group (19) showed that the type of exercise (i.e., aerobic, resistance, or combined) could determine the effect obtained, as observed with aerobic exercise and the risk of gestational diabetes mellitus. Therefore, the aim of this systematic review and network meta-analysis is to estimate the effect of metformin and different types of exercise on the development of delivery and newborn complications, including the risk of cesarean section, preterm birth, macrosomia, and birth weight among pregnant women with overweight/obesity. This systematic review and network meta-analysis was conducted according to the Cochrane Collaboration Handbook and the Preferred Reporting Items for Systematic Review incorporating Network Meta-analysis (PRISMA-NMA) (20, 21). The study protocol was registered in PROSPERO (registration number: CDR: 42019121715) and published elsewhere (22). Frontiers in Medicine \| www.frontiersin.org Search Strategy MEDLINE, Scopus, Web of Science, and Cochrane Library databases were searched from their inception to September 2021. We also reviewed clinicaltrials.gov, EudraCT, the gray literature, and the reference list of previous systematic reviews and articles included in this review. The databases searched, keywords, and additional information are detailed in Supplementary Appendix S1. The search and selection of studies was conducted independently by two reviewers (CP-M and CA-B), and disagreements were resolved by consensus or by a third reviewer (VM-V). Eligibility The inclusion criteria were as follows: (1) type of study: randomized controlled trials; (2) type of participants: pregnant women with overweight or obesity; (3) type of interventions: structured exercise program (aerobic, resistance or combined exercises) or metformin treatment as the intervention, and (4) type of outcome assessment: delivery-related outcomes (i.e., risk ratio (RR) of cesarean section, RR of preterm birth) or newbornrelated outcomes (i.e., RR of macrosomia, difference in mean birth weight). There was no language restriction. The exclusion criteria were as follows: (1) type of studies: single-arm studies or non-randomized controlled trials; (2) type of participants: studies whose target population was exclusively women with pregestational insulin resistance, polycystic ovary syndrome, or other diseases that could affect the main outcomes; (3) type of intervention: dietary intervention as the primary cointervention, nutraceutical or dietary supplement interventions, or unstructured exercise intervention. Data Extraction CP-M and IC-R extracted the data from the included studies according to the following predetermined information for each study: (1) reference, (2) country, (3) design), (4) participants (sample size, age, weight status), (5) intervention (type of intervention, frequency, length, intensity), (6) outcomes: risk of cesarean section, preterm birth, macrosomia, and/or birth weight. 2 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy Categorization of Available Evidence Consistency was assessed by checking whether intervention effects estimated from indirect comparisons were consistent with those estimated from direct comparisons. We conducted the Wald test, and due to the low statistical power, the side-splitting assessment was also used (33). For statistically significant effects, the number needed to treat was estimated using the risk ratio obtained in the network meta-analysis and the basal risk. We conducted a standard meta-analysis and frequentist network meta-analysis for direct and indirect comparisons between interventions and control groups (34, 35). Statistical heterogeneity was examined by the I2 statistic, was classified as not important (<40%), moderate (30–60%), substantial (50– 90%), or considerable (>75%) (20). The p-values were also considered. The τ 2 statistic was calculated to determine the size and clinical relevance of the heterogeneity. τ 2 = 0.04 was considered a low, 0.14 was considered moderate, and 0.40 was considered a substantial degree of clinical relevance of the heterogeneity (36, 37). We displayed these results by creating both forest plots and a league table. The transitivity requirement was assessed, checking that the synthesis of direct comparisons of two treatments had been conducted in similar studies on the most important clinical and methodological characteristics, including basal age and basal body mass index (38). We conducted a relative ranking of treatments to identify superiority (31), and we estimated the surface under the cumulative ranking for each intervention (32). We conducted a sensitivity analysis through a subgroup analysis with pregnant women with obesity using a random effects meta-analysis and a frequentist network meta-analysis for each outcome. Additionally, we conducted a sensitivity reanalysis from a Bayesian perspective. To rule out a dependent effect of maternal body mass index or maternal weight gain, meta-regressions were performed using the risk ratios as the dependent variable. Finally, we also performed random effects meta-regression models, using as independent variables age, body mass index, intensity of intervention (or dose of metformin), weekly exercise frequency, duration of exercise session, gestational age at baseline, length of intervention, and total number of exercise sessions. Finally, we used a funnel plot to visually examine the symmetry criterion to determine the presence of bias due to the small study effect (39). We conducted all analyses in Stata 15.0 (Stata, College Station, Texas, United States). We used the original studies’ classification to categorize body mass index as overweight or obese. When they did not report this categorization, the baseline body mass index was considered overweight (body mass index: 25–30 kg/m2 ) and obese (body mass index ≥ 30 kg/m2 ) (23). Exercise was defined as a subset of structured and repetitive physical activity with the objective of improving or maintaining physical fitness (24). We classified exercise interventions into three categories: (1) aerobic exercise, (2) resistance training, and (3) combined exercises. Aerobic exercises are aimed at increasing energy expenditure and include walking, running, cycling, jogging, swimming, or interval exercise. Strength training was aimed at increasing muscle strength and included exercises with elastic bands or dumbbells, among others. Combined exercise includes, alternately or in combination, aerobic and strength exercises. The intensity of the exercise intervention was reported by the authors and was classified as vigorous, moderate-vigorous, moderate, light-moderate, or light. When the authors did not report intensity, we used the criteria from the American College of Sports Medicine guidelines to estimate it (25–27) based on the percentage of maximum heart rate, percentage of heart rate, percentage of maximum oxygen uptake, or rating of perceived exertion reported by the studies. Risk of Bias Assessment The risk of bias assessment of the included randomized controlled trials was conducted by two researchers (CP-M and IC-R) using the Cochrane Collaboration’s tool for assessing risk of bias (28). This tool assesses the risk of bias of six domains: (1) randomization process, (2) assignment to intervention, (3) adherence to intervention, (4) missing outcome data, (5) measurement of the outcome, (6) selection of the reported result. Finally, the overall bias is scored as high/low/moderate (some concerns) risk of bias. Any disagreements were resolved by consensus or by a third reviewer (VM-V). Grading the Quality of Evidence We used the Grading of Recommendations, Assessment, Development and Evaluation tool to assess the quality of evidence and make recommendations (29, 30). Each outcome obtained a high, moderate, low, or very low level of evidence, depending on several domains pre-established by the tool. Data Synthesis Modifications to the Initial Protocol We summarized the clinical trials in an ad hoc table describing the types of direct and indirect comparisons. Our network meta-analyses were conducted following the PRISMA-NMA statement (21). We used a network geometry graph to assess the robustness of the evidence. The size of the nodes was proportional to the sample size of the trials, the thickness of the continuous line connecting the nodes was proportional to the sample size in trials directly comparing the two treatments, and the dashed lines represented indirect comparisons (31, 32). Frontiers in Medicine \| www.frontiersin.org In the protocol, the target population included all pregnant women, and the inclusion of all types of trials. It was decided to limit it to pregnant women with overweight/obesity in randomized clinical trials to improve the transitivity principle and the quality of the final analyses. Finally, the protocol established the performance of Bayesian network metaanalysis. Subsequently, it was decided to conduct frequentist network meta-analyses and a sensitivity reanalysis using a Bayesian perspective. 3 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy RESULTS Preterm Birth Figure 2B, Supplementary Table S4B, and Supplementary Figure S1B show that no intervention had a statistically significant effect on either the pairwise comparisons or the network meta-analysis. Fifteen randomized controlled trials (40–54) were included in the analyses (Table 1, Figure 1, Supplementary Table S1), and 48 studies were excluded for various reasons (Supplementary Table S2). Of the included trials, 6 included pregnant women with overweight, and 13 pregnant women with obesity. The trials were conducted in 10 countries: 8 in Europe, including two in the Netherlands (48, 51), two in Spain (43, 49), two in the United Kingdom (52, 54) and one each in Ireland (45) and Norway (46), 5 in America, including three in Brazil (47, 50, 53), one in Canada (44) and one in the United States (40), one in Asia (China) (42), and one in Oceania (New Zealand) (41). A total of 2,759 pregnant women were included in the trials (412 in aerobic exercise, 1,021 in combined exercise, and 1,326 metformin interventions). Exercise frequency was two to five times per week, lasting 12–30 weeks. The dose of metformin was between 1,000 and 3,000 mg per day, lasting ∼25 weeks. The details of the interventions are described in Supplementary Table S3. Macrosomia Figure 3A, Supplementary Table S4C, and Supplementary Figure S1C show that combined exercise reduced the risk of macrosomia in the network meta-analysis estimates (RR = 0.37, 95% CI: 0.14, 0.95). The number needed to treat of the combined exercise was 12 women to prevent one case. Birth Weight Figure 3B, Supplementary Table S4D, and Supplementary Figure S1D show that aerobic exercise reduces birth weight in the network meta-analysis (Mean Difference = −96.66 g, 95% CI: −192.45, −0.88). Cesarean Section Risk of Bias Figure 2A, Supplementary Table S4A, and Supplementary Figure S1A show the standard pairwise comparisons (upper diagonal) and the network meta-analysis (under diagonal). Metformin reduced the risk of cesarean section in pairwise comparisons and in the network meta-analysis (RR = 0.79, 95% CI: 0.63, 0.99, and RR = 0.66, 95% CI: 0.46, 0.95, respectively). The number needed to treat of metformin was 8 women to prevent one case. According to the Cochrane Collaboration’s tool for assessing risk of bias, 10 out 15 (66.7%) showed a high risk of bias for overall bias, and five (33.3%) showed some concerns. By domain, 13.3% of the studies showed high risk for assignment to intervention, 66.7% showed some concerns, 46.7% showed high risk, 20.0% showed some concerns for adhering to intervention, 20.0% showed high risk, 26.7% showed some concerns for missing outcome data, and 66.7% showed some concerns for TABLE 1 \| Characteristics of included trials. References Country Participants NT NI NC AgeI AgeC Intervention Weight status Outcomes RR CS RR PR RR MA BW ✓ Kong et al. (40) United States 19 9 9 26.2 ± 2.6 27.3 ± 3.6 Overweight Aerobic exercise ✓ ✓ ✓ Kong et al. (40) United States 18 9 10 28.6 ± 5.3 25.7 ± 4.0 Obesity Aerobic exercise ✓ ✓ ✓ ✓ Seneviratne et al. (41) New Zealand 75 38 37 NA NA Obesity Aerobic exercise ✓ ✓ ✓ ✓ Wang et al. (42) China 300 150 150 32.1 ± 4.6 32.5 ± 4.9 Overweight Aerobic exercise ✓ ✓ ✓ ✓ Barakat et al. (43) Spain 168 90 78 – – Overweight Combined exercise – ✓ ✓ – Barakat et al. (43) Spain 54 25 29 – – Obesity Combined exercise – ✓ ✓ – Bisson et al. (44) Canada 50 25 25 30.5 ± 3.7 31.0 ± 4.0 Obesity Combined exercise ✓ – – ✓ ✓ Daly et al. (45) Ireland 88 44 44 30.0 ± 5.1 29.4 ± 4.8 Obesity Combined exercise ✓ ✓ ✓ Garnæs et al. (46) Norway 91 46 45 31.3 ± 3.8 31.4 ± 4.7 Obesity Combined exercise ✓ ✓ ✓ ✓ Nascimento et al. (47) Brazil 82 40 42 29.7 ± 6.8 30.9 ± 5.9 Overweight/Obesity Combined exercise ✓ – – ✓ Oostdam et al. (48) Netherlands 121 62 59 30.8 ± 5.2 30.1 ± 4.5 Obesity Combined exercise ✓ – – ✓ Ruiz et al. (49) Spain 275 146 129 – – Overweight/Obesity Combined exercise ✓ ✓ ✓ ✓ Santos et al. (50) Brazil 92 46 46 26.0 ± 3.4 28.6 ± 5.9 Overweight Combined exercise – ✓ – ✓ Brink et al. (51) Netherlands 49 24 25 29.3 ± 5.2 30.7 ± 5.2 Obesity Metformin ✓ ✓ – ✓ Chiswick et al. (52) United Kingdom 449 226 223 28.7 ± 5.8 28.9 ± 5.1 Obesity Metformin ✓ ✓ – ✓ Nascimento et al. (53) Brazil 378 189 189 28.6 ± 6.2 29.6 ± 6.1 Obesity Metformin ✓ ✓ – – Syngelaki et al. (54) United Kingdom 450 225 225 32.9 30.8 Obesity Metformin ✓ ✓ ✓ – NT , sample size; NI , sample size in intervention group; NC , sample size in control group; AgeI , mean age in intervention group; AgeC , mean age in control group; RR CS, risk ratio of cesarean section; RR PR, risk ratio of preterm birth; RR MA, risk ratio of macrosomia; BW, birthweight (mean differences). Frontiers in Medicine \| www.frontiersin.org 4 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy FIGURE 1 \| PRISMA flowchart of study selection. Grades of Recommendation, Assessment, Development, and Evaluation measurement of the outcome. No significant risk of bias was detected for the randomization process or for selection of the reported results. The total risk of bias is shown in the Supplementary Figure S2. Frontiers in Medicine \| www.frontiersin.org According to the Grading of Recommendations, Assessment, Development and Evaluation tool, all interventions showed very 5 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy FIGURE 2 \| Network meta-analyses for delivery-related outcomes. It includes cesarean section (A) and preterm birth (B). The network mapping is shown on the left, and the network meta-analysis estimates on the right, measured as risk ratio (RR) and 95% confidence interval (95% CI). Probabilities low certainty for all outcomes. Only the metformin intervention showed low certainty for the risk of cesarean section. The most affected domains were risk of bias, inconsistency, indirectness and imprecision. The complete assessment is detailed in Supplementary Table S5. The Metformin intervention showed the highest probability of being the best intervention for preventing cesarean section (Probability of best intervention = 83.3%, surface under the cumulative ranking = 0.936), combined exercise was best for preventing macrosomia (Probability of best intervention = 78.6%, surface under the cumulative ranking = 0.912), and aerobic exercise was best for reducing birth weight (Probability of best intervention = 78.7%, surface under the cumulative ranking = 0.910). No intervention showed a high probability of preventing preterm birth (Figure 4, Supplementary Figure S3). Transitivity There were no statistically significant differences in baseline age or body mass index between the two interventions for cesarean section and birth weight. There were statistically significant differences between the baseline body mass index of the intervention groups for preterm birth, with the baseline body mass index of the metformin intervention being higher than the exercise interventions. There were insufficient data to conduct a transitivity analysis for the risk of macrosomia. The complete assessment is detailed in Supplementary Table S6. Frontiers in Medicine \| www.frontiersin.org Subgroup Analysis Subgroup analysis examining only pregnant women with obesity showed no effect of any intervention to prevent preterm birth or macrosomia or to modify birth weight, but metformin significantly reduced cesarean sections (RR 6 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy FIGURE 3 \| Network meta-analyses for newborn-related outcomes. It includes macrosomia (A) and birth weight (B). The network mapping is shown on the left, and the network meta-analysis estimates on the right, measured as risk ratio (RR) and 95% confidence interval (95% CI). association was observed for the variables studied for each intervention (Supplementary Table S9). = 0.79, 95% CI: 0.63, 0.99, and RR = 0.66, 95% CI: 0.45, 0.97, in the pairwise comparisons and the network meta-analysis, respectively). The number needed to treat of metformin was 8 women to prevent one case. The complete subgroup analysis is detailed in Supplementary Table S7, Supplementary Figure S4. Heterogeneity and Publication Bias Combined exercise showed moderate heterogeneity for the risk of macrosomia (I2 = 53.48%) and metformin showed moderate heterogeneity for the risk of cesarean sections and birth weight (I2 = 55.24% and I2 = 46.71%, respectively), but the rest of the interventions did not show statistically significant heterogeneity in the studied outcomes. Aerobic and combined exercise showed moderate and substantial degrees of clinical relevance in the heterogeneity for the risk of macrosomia (τ 2 = 0.11 and τ 2 = 0.88, respectively), but the remaining interventions had a low degree of clinical relevance of the heterogeneity (Supplementary Figure S1). There was no evidence of publication bias in funnel plot asymmetry for any outcome (Supplementary Figure S5). Sensitivity Analysis With Bayesian Methods The sensitivity reanalysis using Bayesian methods did not produce statistically different results from the frequentist analyses. Meta-Regressions Models There was no statistically significant association between body mass index or maternal weight gain and the included outcomes (Supplementary Table S8). No statistically significant Frontiers in Medicine \| www.frontiersin.org 7 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy FIGURE 4 \| Relative rankings of treatments for delivery- and newborn-related outcomes. It includes cesarean section (A), preterm birth (B), macrosomia (C), and birth weight (D). Each image shows the rankogram, and a summarized table of the probability of being the best intervention (PrBest) and the surface under the cumulative ranking (SUCRA). DISCUSSION reduced the incidence by 34%. The observed effect is due to the inclusion of new studies with respect to previous metaanalyses, in which there was a non-significant trend to benefit. Interestingly, the meta-regressions did not show the effect of covariates such as dose and length of the intervention. Moreover, considering the dose and length of the individual studies with their risk ratios, there does not appear to be a dose-response association, which suggests that at moderate doses the desired effect could be achieved. The mechanism by which metformin reduces the risk of cesarean section is also unclear. Considering that body mass index is associated with birth weight and risk of cesarean section, the hypothesis that obesity and excessive maternal weight gain increases the deposition of fatty tissue in the maternal pelvis and increases birth weight, causing obstructed labor, is attractive (58). This hypothesis is supported by the reduction of maternal weight gain with metformin (19). However, in the meta-regressions, no association was observed between the maternal weight gain and the risk of cesarean Main Findings Metformin in women with overweight/obesity was found to reduce the risk of cesarean section by 34% with a number needed to treat of 8, and combined exercise reduced the risk of macrosomia by 63%, with a number needed to treat of 12. Additionally, aerobic exercise reduced birthweight by 96.7 g. No effect was found on the risk of preterm birth for any intervention. No statistically significant association was observed in the metaregressions. Finally, the study in subgroups of women with obesity confirmed the effect of metformin in reducing the risk of cesarean section. Interpretation Regarding the cesarean section, our results confirm the findings of the previous network meta-analysis (18), in contrast to previous meta-analyses (55–57). Thus, in our study, metformin Frontiers in Medicine \| www.frontiersin.org 8 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy of the combined exercise on the risk of macrosomia. Based on one study (42), international guidelines (13) suggest that aerobic exercise could reduce the incidence of macrosomia in pregnant women with overweight. Although the scarcity of studies does not allow us to reject this hypothesis, our study shows that the most effective exercise is combined, at light-moderate to moderate intensity, 50–60 mins per session, 3 times per week. Second, it is also suggested (13) that exercise could reduce the risk of preterm birth in women with overweight or obesity. Our study could not replicate these findings, however, it was shown to be safe, this being a relevant aspect due to the traditional fear of recommending exercise during pregnancy. Third, the effect of exercise on the risk of cesarean section pointed out by other authors (13, 68) could not be confirmed either. This is probably due to the inclusion of exclusively women with overweight and the lack of effect it may have on this outcome in this population. Fourth, metformin had no effect on the risk of macrosomia, which was interesting, but it did have a significant effect on the risk of cesarean section. Since metformin could have some effect on maternal weight gain and cesarean section, and it appears to be safe for newborns (19, 56), its use in specific cases and with a thorough assessment of the benefit-risk profile cannot be ruled out. However, the lack of effect on other outcomes, the low quality of the evidence (GRADE) and the caution in the administration of drugs during pregnancy do not allow to recommend a generalized medicalization of healthy pregnant women with obesity. section. In addition to the above, combined exercise, which reduces the incidence of macrosomia, had no effect on the risk of cesarean section. Another hypothesis is the possibility that metformin improves lipid profiles, including cholesterol, lowdensity lipoprotein and very low-density lipoprotein, which may negatively affect the contractility of the myometrium because of alterations in the fluidity and viscosity of cell membranes, which in turn alter the function of the calcium in muscle contraction (58), as well as the increase in glycogen stores in myometrial cells necessary to perform vigorous contractions (59). However, these two hypotheses have not been confirmed (58, 59), requiring future research. Our results showed a reduction in the risk of macrosomia and birth weight with combined and aerobic exercise, respectively, which contrasts with the results of two previous meta-analyses (60, 61) which found no effect. These results pose a challenge, since the same type of intervention that decreased birth weight did not reduce macrosomia, and vice versa. There are several mechanisms that could explain it. The Pederson theory (62) established that maternal hyperglycaemia caused fetal hyperinsulinemia, with increased fetal weight and macrosomia. Although some effect cannot be ruled out, the lack of effect of metformin in these outcomes rules out that it is the main mechanism. In fact, exercise also improves the glycaemic profile (63–66), which does not necessarily translate into an improvement in fetal weight. Some authors (67) have recently proposed that the decrease in body fat with exercise is not directly because of the oxidation of fatty acids during exercise sessions, but to the uptake of fatty acids after exercise to repair tissue damage, which is especially the case with anaerobic or high intensity sessions. Thus, combined exercise, with the inclusion of strength exercises, could deprive the fetus of excess energy from fatty acids and reduce the risk of macrosomia in fetuses predisposed to it. The reason that aerobic exercise reduced birth weight was due to one trial (42) using moderate-vigorous intensity. At this intensity, in addition to having a possible anaerobic component and tissue damage, it could increase catecholamine levels causing lipolysis (67), and decrease uterine blood flow depending on time and intensity (66). As expected, in the study by subgroups among pregnant women with obesity, the effect of metformin was maintained because of the inclusion of the same studies. However, no effect of combined exercise was found on the risk of macrosomia. In addition to the scarcity of studies, the exclusion of two inputs (43, 49) for not exclusively including women with obesity reduced the effect obtained, probably due to the high adherence of the participants in these two trials compared to other included trials. Finally, no effect on birthweight was observed in obese women, which was also expected, because of the exclusion of the trial with the greatest effect (42), suggesting the importance of intensity for this outcome. Although there was variability in the cohorts of women and the interventions (i.e., body mass index, metformin dose, exercise intensity, length of interventions), we attempted to control by assessing the transitivity principle, meta-regressions, and subgroup analyses to provide consistent evidence to aid decision-making. First, the most interesting result is the effect Frontiers in Medicine \| www.frontiersin.org Limitations Some limitations should be acknowledged. First, the main limitation is related to the scarcity of studies, which could affect the effect estimates, especially for the aerobic exercise and metformin intervention, the statistical power of the network meta-analysis, the publication bias analysis, and the assessment of the transitivity requirement. Second, the lack of studies limited additional analysis (i.e., meta-regressions) by covariates or mediators that could determine the possible effect of length, frequency, or intensity of interventions. Third, only four outcomes were considered. This was because most trials only report these outcomes. Therefore, future research is needed to determine, by meta-analysis or NMA, the influence of exercise type and other covariates, on outcomes such as placental weight, gestational age at delivery, other birth weight categories (i.e., low and adequate birthweight) and Apgar score in 1 or 5 min. Fourth, neonatal glycemia is associated with long-term adverse events. However, the studies did not report these data, therefore, it was not possible to estimate the effect of the interventions on this parameter, nor the association between neonatal glycemia and the included outcomes. Fifth, for the diagnosis of macrosomia, the cut-off weight for categorization as macrosomia varied by author between 4,000 and 4,500 g, which could slightly affect the estimate of effect. Sixth, no differentiation was made between emergency and elective cesarean sections, which could affect the estimate of effect, although it is unlikely to have a statistically significant effect since both types of cesarean sections are associated with overweight/obesity. Seventh, no resistance exercise interventions were found, which can be a problem for 9 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy DATA AVAILABILITY STATEMENT understanding what effect adding resistance exercise to aerobic exercises can have. Eighth, we found a moderate to high risk of bias in most studies, with the domains ’adhering to intervention’ and ’missing outcome data’ being the ones that could most affect the effect estimates in our analyses. Nineth, although there were no statistically significant differences between the baseline age and body mass index for exercise and metformin on cesarean section and birth weight, overall there was high heterogeneity across studies. Tenth, we were unable to perform transitivity analysis for macrosomia due to lack of studies on metformin. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. AUTHOR CONTRIBUTIONS CP-M and VM-V conceptualized the study. CP-M, IC-R, and CA-B planned and carried out the study, data curation, and investigation. CP-M, SR-G, JAM-H, AS-L, and SN-A-A carried out the formal analysis. CP-M, IC-R, and VM-V wrote the manuscript. VM-V provided the funding. All authors reviewed the manuscript. CONCLUSIONS Metformin reduces the risk of cesarean section in pregnant women with obesity, and combined exercise reduces the risk of fetal macrosomia in pregnant women with overweight/obesity. Aerobic exercise could also reduce birth weight. Additionally, exercise was safe for the risk of preterm birth, something that has been debated for decades. The meta-regressions were limited by the number of included studies, and therefore, further research is needed to determine the effect of the length, frequency, and intensity of each type of exercise, and the length and dosage of metformin, on the risk of the outcomes studied. Considering the limitations of the study and the quality of the evidence, the systematic medicalization of pregnancy among women with overweight/obesity is not justified. However, it is highly recommended that women without exercise contraindications perform structured exercise, including aerobic and strength exercises, and achieve high adherence. FUNDING This study was funded by the Consejería de Educación, Cultura y Deportes—Junta de Comunidades de CastillaLa Mancha and European Regional Development Fund (SBPLY/17/180501/000533). CP-M was supported by a grant from the Universidad de Castilla-La Mancha (2018-CPUCLM-7939). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2021.796009/full#supplementary-material REFERENCES 9. Ananth C V, Friedman AM, Keyes KM, Lavery JA, Hamilton A, Wright JD. Primary and Repeat cesarean deliveries: a population-based study in the United States, 1979–2010. Epidemiology. (2017) 28:567– 74. doi: 10.1097/EDE.0000000000000658 10. Behrman RE, Butler AS. Preterm Birth: Causes, Consequences, and prevention. Preterm Birth: Causes, Consequences, and Prevention. Washington, DC: National Academies Press (2007), p 1–772. 11. Hoopmann M, Abele H, Wagner N, Wallwiener D, Kagan KO. 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Medicine. (2019) 98:e16038. doi: 10.1097/MD.0000000000016038 23. Body mass index—BMI. (2021). Available online at: https://www.euro.who. int/en/health-topics/disease-prevention/nutrition/a-healthy-lifestyle/bodymass-index-bmi (accessed Apr 9, 2021). 24. Caspersen CJ, Powell KE, Christenson GM. Physical activity, exercise, and physical fitness: definitions and distinctions for health-related research. Public Health Rep. (1902) 100:126–31. 25. Riebe D, Ehrman J, Liguori G, Magal M. Medicine AC. of S. In: ACSM’s guidelines for Exercise Testing and Prescription. 10th ed. Philadelphia: Wolters Kluwer (2018). 26. Mcnair D, Lorr M, Droppleman LF. Manual for the Profile of Mood States. San Diego: Educational and Industrial Testing Service (1971). 27. Borg GA. Psychophysical bases of perceived exertion. Med Sci Sports Exerc. (1982) 14:377–81. doi: 10.1249/00005768-198205000-00012 28. Eldridge S, Campbell MK, Campbell MJ, Drahota-Towns A, Giraudeau B, Higgins JPT, et al. Revised Cochrane risk of bias tool for randomized trials (RoB 2.0): additional considerations for cluster-randomized trials. (2016). Available online at: https://researchportal.port.ac.uk/en/publications/revisedcochrane-risk-of-bias-tool-for-randomized-trials-rob-20-a (accessed February 12, 2021). 29. Neumann I, Pantoja T, Peñaloza B, Cifuentes L, Rada G. El sistema GRADE: Un cambio en la forma de evaluar la calidad de la evidencia y la fuerza de recomendaciones. Rev Med Chil. (2014) 142:630–5. doi: 10.4067/S0034-98872014000500012 30. Salanti G, Del Giovane C, Chaimani A, Caldwell DM, Higgins JPT. Evaluating the quality of evidence from a network meta-analysis. PLoS ONE. (2014) 9:e99682. doi: 10.1371/journal.pone.0099682 31. Chaimani A, Higgins JPT, Mavridis D, Spyridonos P, Salanti G. Graphical tools for network meta-analysis in STATA. PLoS ONE. (2013) 8:e76654. doi: 10.1371/journal.pone.0076654 32. Salanti G, Ades AE, Ioannidis JPA. Graphical methods and numerical summaries for presenting results from multiple-treatment meta-analysis: an overview and tutorial. J Clin Epidemiol. (2011) 64:163–71. doi: 10.1016/j.jclinepi.2010.03.016 33. Veroniki AA, Vasiliadis HS, Higgins JP, Salanti G. Evaluation of inconsistency in networks of interventions. Int J Epidemiol. (2013) 42:332–45. doi: 10.1093/ije/dys222 34. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials. (1986) 7:177–88. doi: 10.1016/0197-2456(86)90046-2 35. White IR. Network meta-analysis. Stata J. (2015) 15:951– 85. doi: 10.1177/1536867X1501500403 36. Spiegelhalter DJ, Abrams KR, Myles JP. Bayesian Approaches to Clinical Trials and Health-Care Evaluation. Bayesian Approaches to Clinical Trials and Health-Care Evaluation. Chichester: John Wiley & Sons, Ltd. (2003). doi: 10.1002/0470092602 37. Stettler C, Allemann S, Wandel S, Kastrati A, Morice MC, Schömig A, et al. Drug eluting and bare metal stents in people with and Frontiers in Medicine \| www.frontiersin.org 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 11 without diabetes: collaborative network meta-analysis. BMJ. (2008). 337:a1331. doi: 10.1136/bmj.a1331 Cipriani A, Higgins JPT, Geddes JR, Salanti G. Conceptual and technical challenges in network meta-analysis. Ann Intern Med. (2013) 159:130– 7. doi: 10.7326/0003-4819-159-2-201307160-00008 Egger M, Smith GD, Schneider M, Minder C. Bias in metaanalysis detected by a simple, graphical test. BMJ. (1997) 315:629–34. doi: 10.1136/bmj.315.7109.629 Kong KL, Campbell CG, Foster RC, Peterson AD, LanninghamFoster L. A pilot walking program promotes moderate-intensity physical activity during pregnancy. Med Sci Sports Exerc. (2014) 46:462–71. doi: 10.1249/MSS.0000000000000141 Seneviratne SN, Jiang Y, Derraik J, McCowan L, Parry GK, Biggs JB, et al. Effects of antenatal exercise in overweight and obese pregnant women on maternal and perinatal outcomes: a randomised controlled trial. BJOG. (2016) 123:588–97. doi: 10.1111/1471-0528.13738 Wang C, Wei Y, Zhang X, Zhang Y, Xu Q, Sun Y, et al. A randomized clinical trial of exercise during pregnancy to prevent gestational diabetes mellitus and improve pregnancy outcome in overweight and obese pregnant women. Am J Obstet Gynecol. (2017) 216:340–51. doi: 10.1016/j.ajog.2017.01.037 Barakat R, Pelaez M, Cordero Y, Perales M, Lopez C, Coteron J, et al. Exercise during pregnancy protects against hypertension and macrosomia: Randomized clinical trial. Am J Obstet Gynecol. (2016) 214:649.e1– e8. doi: 10.1016/j.ajog.2015.11.039 Bisson M, Alméras N, Dufresne SS, Robitaille J, Rhéaume C, Bujold E, et al. A 12-week exercise program for pregnant women with obesity to improve physical activity levels: an open randomised preliminary study. PLoS ONE. (2015) 10:e0137742. doi: 10.1371/journal.pone.0137742 Daly N, Farren M, McKeating A, O’Kelly R, Stapleton M, Turner MJ, et al. A medically supervised pregnancy exercise intervention in obese women: a randomized controlled trial. Obstet Gynecol. (2017) 130:1001– 10. doi: 10.1097/AOG.0000000000002267 Garnæs KK, Mørkved S, Salvesen Ø, Moholdt T. Exercise training and weight gain in obese pregnant women: a randomized controlled trial (ETIP Trial). PLoS Med. (2016) 13:1002079. doi: 10.1371/journal.pmed.1002079 Nascimento SL, Surita FG, Parpinelli MA, Siani S. Pinto e Silva JL. The effect of an antenatal physical exercise programme on maternal/perinatal outcomes and quality of life in overweight and obese pregnant women: a randomised clinical trial. BJOG. (2011) 118:1455–63. doi: 10.1111/j.1471-0528.2011.03084.x Oostdam N, Van Poppel MNM, Wouters MGAJ, Eekhoff EMW, Bekedam DJ, Kuchenbecker WKH, et al. No effect of the FitFor2 exercise programme on blood glucose, insulin sensitivity, and birthweight in pregnant women who were overweight and at risk for gestational diabetes: results of a randomised controlled trial. BJOG. (2012) 119:1098– 1107. doi: 10.1111/j.1471-0528.2012.03366.x Ruiz JR, Perales M, Pelaez M, Lopez C, Lucia A, Barakat R. Supervised exercise-based intervention to prevent excessive gestational weight gain: a randomized controlled trial. Mayo Clin Proc. (2013) 88:1388–97. doi: 10.1016/j.mayocp.2013.07.020 Santos IA, Stein R, Fuchs SC, Duncan BB, Ribeiro JP, Kroeff LR, et al. Aerobic exercise and submaximal functional capacity in overweight pregnant women: a randomized trial. Obstet Gynecol. (2005) 106:243– 9. doi: 10.1097/01.AOG.0000171113.36624.86 Brink HS, Alkemade M, van der Lely AJ, van der Linden J. Metformin in women at high risk of gestational diabetes mellitus. Diabetes Metab. (2018) 44:300–2. doi: 10.1016/j.diabet.2018.01.008 Chiswick C, Reynolds RM, Denison F, Drake AJ, Forbes S, Newby DE, et al. Effect of metformin on maternal and fetal outcomes in obese pregnant women (EMPOWaR): a randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. (2015) 3:778–86. doi: 10.1016/S2213-8587(15)00219-3 Nascimento IB. do, Sales WB, Dienstmann G, Souza MLR de, Fleig R, Silva JC. Metformin for prevention of cesarean delivery and large-for-gestational-age newborns in non-diabetic obese pregnant women: a randomized clinical trial. Arch Endocrinol Metab. (2020) 64:290–7. doi: 10.20945/2359-3997000000251 Syngelaki A, Nicolaides KH, Balani J, Hyer S, Akolekar R, Kotecha R, et al. Metformin versus placebo in obese pregnant women without diabetes mellitus. N Engl J Med. (2016) 374:434–43. doi: 10.1056/NEJMoa1509819 December 2021 \| Volume 8 \| Article 796009 Pascual-Morena et al. Exercise vs. Metformin in Pregnancy 64. Moghetti P, Bacchi E, Brangani C, Donà S, Negri C. Metabolic effects of exercise. Front Horm Res. (2016) 47:44–57. doi: 10.1159/000445156 65. Yaribeygi H, Atkin SL, Simental-Mendía LE, Sahebkar A. Molecular mechanisms by which aerobic exercise induces insulin sensitivity. J Cell Physiol. (2019) 234:12385–92. doi: 10.1002/jcp.28066 66. Pourranjbar M, Arabnejad N, Naderipour K, Rafie F. Effects of aerobic exercises on serum levels of myonectin and insulin resistance in obese and overweight women. J Med Life. (2018) 11:381–6. doi: 10.25122/jml-2018-0033 67. Harris MB, Kuo CH. Scientific challenges on theory of fat burning by exercise. Front Physiol. (2021) 12:685166. doi: 10.3389/fphys.2021.685166 68. Magro-Malosso ER, Saccone G, Di Tommaso M, Roman A, Berghella V. Exercise during pregnancy and risk of gestational hypertensive disorders: a systematic review and meta-analysis. Acta Obstet Gynecol Scand. (2017) 96:921–31. doi: 10.1111/aogs.13151 55. Dodd JM, Grivell RM, Deussen AR, Hague WM. Metformin for women who are overweight or obese during pregnancy for improving maternal and infant outcomes. Cochrane Datab Syst Rev. (2018) 2018:CD010564. doi: 10.1002/14651858.CD010564.pub2 56. D’Ambrosio V, Brunelli R, Vena F, Di Mascio D, Marchetti C, Boccherini C, et al. Metformin reduces maternal weight gain in obese pregnant women: a systematic review and meta-analysis of two randomized controlled trials. Diab Metab Res Rev. (2019) 35:e3164. doi: 10.1002/dmrr.3164 57. Elmaraezy A, Abushouk AI, Emara A, Elshahat O, Ahmed H, Mostafa MI. Effect of metformin on maternal and neonatal outcomes in pregnant obese non-diabetic women: A meta-analysis. Int J Reprod BioMed Res Clin Center Infertitlity. (2017) 15:461–70. doi: 10.29252/ijrm.15.8.461 58. Kiran U, Evans J. Poor uterine contractility in obese women. BJOG. (2007) 114:1304–5. doi: 10.1111/j.1471-0528.2007.01470.x 59. Chiswick CA, Reynolds RM, Denison FC, Drake AJ, Forbes S, Newby DE, et al. Does metformin reduce excess birthweight in offspring of obese pregnant women? A randomised controlled trial of efficacy, exploration of mechanisms and evaluation of other pregnancy complications. Effic Mech Eval. (2016) 3:1–800. doi: 10.3310/eme03070 60. Magro-Malosso ER, Saccone G, Di Mascio D, Di Tommaso M, Berghella V. Exercise during pregnancy and risk of preterm birth in overweight and obese women: a systematic review and metaanalysis of randomized controlled trials. Obstet Gynecol Surv. (2017) 72:457–8. doi: 10.1097/01.ogx.0000521862.17087.d3 61. Du MC, Ouyang YQ, Nie XF, Huang Y, Redding SR. Effects of physical exercise during pregnancy on maternal and infant outcomes in overweight and obese pregnant women: A meta-analysis. Birth. (2019) 46:211– 21. doi: 10.1111/birt.12396 62. Pedersen J. Diabetes and Pregnancy; Blood Sugar of Newborn Infants During Fasting and Glucose Administration. Copenhagen: Danish Science Press (1952). 63. Consitt LA, Dudley C, Saxena G. Impact of endurance and resistance training on skeletal muscle glucose metabolism in older adults. Nutrients. (2019) 11:2636. doi: 10.3390/nu11112636 Frontiers in Medicine \| 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. 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 © 2021 Pascual-Morena, Cavero-Redondo, Álvarez-Bueno, MartínezHortelano, Reina-Gutiérrez, Saz-Lara, Núñez de Arenas-Arroyo and MartínezVizcaíno. 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. 12 December 2021 \| Volume 8 \| Article 796009
https://openalex.org/W2148077930	https://www.scielo.br/j/bor/a/MCPZrqyq4D4ZWZKWc3LWwKp/?lang=en&format=pdf	English	null	Diagnosing non-cavitated lesions in epidemiological studies: practical and scientific considerations	Brazilian Oral Research	2,014	cc-by	4,562	Oral Health Oral Health Joana Christina Carvalho(a) Heliana Dantas Mestrinho(b) Abstract: Over the last decade, there has been growing interest in di- agnosing non-cavitated lesions in epidemiological studies involving large numbers of preschool children, schoolchildren and young adults. In this context, assessment of lesions characteristics indicating whether or not there is ongoing mineral loss is also considered relevant. The reasoning sustained by these studies is that diagnosis of the caries process limited to the cavitated level is no longer in accordance with current state-of-the-art knowledge in cariology. This paper highlights one topic of the lecture en- titled “Caries Process: Evolving Evidence and Understanding,” presented at the 18th Congress of the Brazilian Association for Oral Health Pro- motion (Associação Brasileira de Odontologia de Promoção de Saúde - ABOPREV) in April 2013. In the framework of epidemiological stud- ies, the interest in diagnosing active and inactive non-cavitated lesions was elucidated. However, relevant questions associated with the diagno- sis of non-cavitated lesions that might raise concerns among researchers and health administrators were not addressed. The present paper aims to bring these questions into discussion. The contribution of this discussion in terms of developing the understanding of caries decline is analyzed by using data from a caries trends study of Brazilian preschool children residing in the Federal District of Brazil as an example. The inclusion of active and inactive non-cavitated lesions in the diagnosis of the car- ies process allowed us to demonstrate that, in Brazilian 1- to 5-year-old children, caries prevalence decreased significantly from 1996 to 2006, simultaneously with a reduction in the rate of caries progression. (a) School of Medicine and Dentistry, Catholic University of Louvain, Belgium. (b) Department of Odontology, Faculty of Health Sciences, Universidade de Brasília - UnB, Brasília, DF, Brazil. * Paper presented at the “Equity, Social Inclusion and Oral Health Promotion: Major Challenges” International Symposium, Held at the 18th Congress of the Brazilian Association for Oral Health Promotion (Associação Brasileira de Odontologia de Promoção de Saúde - ABOPREV), April 2013, Bauru, SP, Brazil. * Paper presented at the “Equity, Social Inclusion and Oral Health Promotion: Major Challenges” International Symposium, Held at the 18th Congress of the Brazilian Association for Oral Health Promotion (Associação Brasileira de Odontologia de Promoção de Saúde - ABOPREV), April 2013, Bauru, SP, Brazil. Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. Descriptors: Dental Caries; Diagnosis; Epidemiology. * Paper presented at the “Equity, Social Inclusion and Oral Health Promotion: Major Challenges” International Symposium, Held at the 18th Congress of the Brazilian Association for Oral Health Promotion (Associação Brasileira de Odontologia de Promoção de Saúde - ABOPREV), April 2013, Bauru, SP, Brazil. Diagnosing non-cavitated lesions in epidemiological studies: practical and scientific considerations* Joana Christina Carvalho(a) Heliana Dantas Mestrinho(b) Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Are the field conditions suitable for the diagnosis of non-cavitated lesions? lored to the aim of the epidemiological study, since arguments can be made both ways.8 This is indeed a relevant aspect to consider because objectives may in some cases be achieved with the estimate mea- sured only at a more severe stage of caries develop- ment. The need for operative treatment at the pop- ulation or individual levels may well be measured by the diagnosis of the caries process into dentin or even into dental pulp. While this information is in itself important, when evaluated alone, it perpetu- ates the outdated concept that the caries process should be treated by restorations and extractions only. Thus, the contemporary needs for planning, delivery and monitoring of oral health care servic- es at the population level are not fulfilled.9 This is true for populations of children, adolescents, young adults and, to some extent, adults up to 45 years of age.2,3,10,11 A major challenge in carrying out epidemiologi- cal studies is to develop the conditions required to perform a thorough clinical examination for caries and other oral health conditions as these studies are carried out at nurseries, schools, nursing homes and in-home settings. This includes training and calibration of the examiners and positioning of the participant for clinical examination, in addition to obtaining good lighting, and clean and dry teeth. The field equipment should include at least material and instruments such as toothbrushes, dental floss, portable lights, mouth mirrors, periodontal probes, dental probes and gauze bandages. Additional mate- rial and equipment may be required according to the subject of study and the resources available to the researcher, institution or government responsible for carrying out the epidemiological survey. Currently, the major challenge in the manage- ment of the caries process is to control caries pro- gression, mainly by delivering non-operative treat- ments and by limiting the number of individuals in a population subject to operative treatment.12 In this context, it is important to bear in mind that not all non-cavitated lesions progress to dentinal lesions requiring operative treatment,8 but many of them certainly require non-operative treatment to control further caries progression. Are the field conditions suitable for the diagnosis of non-cavitated lesions? Thorough clinical examination may be per- formed under field conditions, including the diag- nosis of non-cavitated lesions, by having calibrated dentists assess caries activity and severity at the non-cavitated level, by having participants lie down on tables and having their teeth professionally brushed, flossed and dried with gauze bandage and by working with portable lights. Good lighting may be obtained by means of frontal lamps. It is recom- mended that the professional perform the brushing and flossing while the participant lies down and subsequently dry the teeth with a gauze bandage.13 Thus, field conditions may become suitable for the diagnosis of non-cavitated lesions. This paper highlights one topic of the lecture entitled “Caries Process: Evolving Evidence and Understanding” presented at the 18th Congress of the Brazilian Association for Oral Health Promo- tion (Associação Brasileira de Odontologia de Pro- moção de Saúde - ABOPREV) in April 2013. In the framework of epidemiological studies, the interest in diagnosing active and inactive non-cavitated lesions was elucidated. However, relevant questions associ- ated with the diagnosis of non-cavitated lesions that might raise concerns among researchers and health administrators were not addressed. The present pa- per aims to bring these questions into the discus- sion. The contribution of this discussion in terms of developing the available understanding of caries de- cline is analyzed by using data from a caries trends study in Brazilian preschool children residing in the Federal District of Brazil13 as an example. Corresponding Author: Corresponding Author: Joana Christina Carvalho E-mail: joana.carvalho@uclouvain.be Corresponding Author: Joana Christina Carvalho E-mail: joana.carvalho@uclouvain.be Joana Christina Carvalho Over the last decade, there has been growing interest in diagnosing non-cavitated lesions in epidemiological studies involving large numbers of preschool and school-age children.1-7 In this context, the assessment of lesion characteristics indicating whether or not there is ongoing mineral loss is also considered relevant. The reasoning sustained by these studies is that diagnosis of the caries process limited to the cavitated level is no longer in accordance with current state-of-the-art knowledge in cariolo- gy. In particular, it contrasts with the current understanding of the devel- opment and arrest of the caries process at subclinical and clinical levels. http://dx.doi.org/10.1590/S1806-83242013005000036 Epub Jan 14, 2014 http://dx.doi.org/10.1590/S1806-83242013005000036 Epub Jan 14, 2014 http://dx.doi.org/10.1590/S1806-83242013005000036 Epub Jan 14, 2014 Submitted: Jul 22, 2013 Accepted for publication: Sep 01, 2013 Last revision: Oct 18, 2013 One may argue that the threshold for caries diagnosis should be tai- Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Diagnosing non-cavitated lesions in epidemiological studies: practical and scientific considerations Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Is the extra time required to record non- cavitated lesions in the field work a barrier? Under field conditions, the overall intra-examiner agreement showed non-weighted kappa values of 0.88 (95% CI 0.86–0.90) for examiner 1 and 0.83 (95% CI 0.80–0.86) for examiner 2. The inter-ex- aminer value of 0.82 (95% CI 0.81–0.83) demon- strated almost perfect agreement.16 Under field conditions, the overall intra-examiner agreement showed non-weighted kappa values of 0.88 (95% CI 0.86–0.90) for examiner 1 and 0.83 (95% CI 0.80–0.86) for examiner 2. The inter-ex- aminer value of 0.82 (95% CI 0.81–0.83) demon- strated almost perfect agreement.16 Time is an important issue when it comes to epi- demiological surveys at a national level or any oth- er survey including a high number of participants. There is no doubt that more time is required to car- ry out a clinical examination that includes the re- cording of non-cavitated lesions than one limited to cavitated lesions. Depending on the system chosen, the time required to record non-cavitated lesions may be double that of the system recommended by the World Health Organisation.17 We went one step further in our analysis in order to independently verify the inter-examiner reliability in the diagnosis of active and inactive non-cavitated lesions. We also showed the non-weighted Kappa values obtained for active and inactive cavitated le- sions since these values might be of interest. The inter-examiner reliability in the diagnosis of active non-cavitated lesions was 0.75 (95% CI 0.71–0.79), whereas for cavitated lesions the corre- sponding value was 0.81 (95% CI 0.79–0.84). These findings indicated substantial agreement for both non-cavitated and cavitated active lesions. Exam- iner 1 had participated in data collection in previous epidemiological surveys and scored higher numbers of active non-cavitated lesions than Examiner 2 who was dealing for the first time with an epidemiologi- cal survey. However, the difference between them was not significant (p > 0.06). Practitioners who are dealing with the record- ing of non-cavitated lesions for the first time spend more time than those who became familiar with it during their university training. In today’s scenario, these aspects may be seen in the light of the increas- ing number of general practitioners whose university training in diagnosis of the caries process has been updated. In Brazil, this training seems to be spread across the country. In the near future, the recording of non-cavitated lesions will become very natural as a continuation of university training and this issue will lose its importance in due time. Is the diagnosis of non-cavitated lesions reliable under field conditions? Several epidemiological studies recording non- cavitated lesions have shown good reliability of the clinical assessments.3-7,10-12 Moreover, lesion activity assessment indicating whether or not there is ongo- ing mineral loss14,15 provides information of major importance for treatment decisions and implementa- tion of strategies to control caries progression. In our study,13 two calibrated examiners exam- ined 225 preschool children to verify intra- and inter-examiner reliability in the diagnosis of caries. Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 2 2 Carvalho JC, Mestrinho HD Is the extra time required to record non- cavitated lesions in the field work a barrier? The extra time required to record non-cavitated lesions should not be considered a barrier, but should be taken into ac- count when planning the field work. Additionally, the inter-examiner reliability in the diagnosis of inactive lesions was also measured at the non-cavitated and cavitated levels. The inter-ex- aminer reliability for inactive non-cavitated lesions was 0.47 (95% CI 0.40–0.55), whereas that for cavi- tated lesions was 0.88 (95% CI 0.81–0.94). These results showed that the reliability of the diagnosis scores for inactive non-cavitated lesions was moder- ate, in contrast to that of cavitated lesions, which was 0.88 (95% CI 0.82–0.94), almost substantial agreement.16 Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 What is the impact of non-cavitated lesions on caries prevalence and severity? Our data showed significant differences in car- ies prevalence and severity when non-cavitated le- sions were considered in the individual’s caries ex- perience.13 In this context, it is worth mentioning that in our studies very few children presented in- active non-cavitated lesions only. These cases were included in the group of participants classified as caries-free. Figures 1 and 2 illustrate the percentage distribution of caries-free Brazilian 1- to 5-year-old children in Cohort 1996 (n = 1,465) and in Cohort 2006 (n = 2,511) according to two levels of exami- nation. The children were classified as caries-free at two levels of examination. Free from any form of treated or untreated active as well as inactive car- ies lesions, i.e. d1efs = 0; and free from cavitated A more detailed analysis of our data looking at the intra-examiner reliability showed that Examiner 1 had a kappa value of 0.64 (95% CI 0.44–0.85) and the corresponding value for Examiner 2 was 0.57 (95% CI 0.32–082). Whereas Examiner 1 pre- sented substantial agreement, Examiner 2 had mod- erate agreement that was probably reflected in the inter-examiner reliability. These results showed that the diagnosis of active non-cavitated lesions in the field work was reliable. However, the diagnosis of inactive non-cavitated lesions should be improved. 3 Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Diagnosing non-cavitated lesions in epidemiological studies: practical and scientific considerations Diagnosing non cavitated lesions in epidemiological studies: practical and scientific considerations 0 10 20 30 40 50 60 70 80 90 100 1yr 2yr 3yr 4yr 5yr Caries-free (d1efs = 0) Caries-free (d3efs = 0) N=110 N=230 N=360 N=385 N=380 % Cohort 1996 0 10 20 30 40 50 60 70 80 90 100 1yr 2yr 3yr 4yr 5yr Caries-free (d1efs = 0) Caries-free (d3efs = 0) % N=170 N=391 N=678 N=670 N=602 Cohort 2006 Figure 2 - Percentage distribution of caries-free Brazilian 1- to 5-year-old children in Cohort 2006 (n = 2,511) with their d1efs scores equal to “0” and their d3efs scores equal to “0”. Figure 1 - Percentage distribution of caries-free Brazilian 1- to 5-year-old children in Cohort 1996 (n = 1,465) with their d1efs scores equal to “0” and their d3efs scores equal to “0”. The number of children in each age group is denoted by “N”. What is the impact of non-cavitated lesions on caries prevalence and severity? 0 10 20 30 40 50 60 70 80 90 100 1yr 2yr 3yr 4yr 5yr N=110 N=230 N=360 N=385 N=380 % Cohort 1996 Figure 1 - Percentage distribution of caries-free Brazilian 1- to 5-year-old children in Cohort 1996 (n = 1,465) with their d1efs scores equal to “0” and their d3efs scores equal to “0”. The number of children in each age group is denoted by “N”. 5yr Caries-free (d1efs = 0) Caries-free (d3efs = 0) 0 10 20 30 40 50 60 70 80 90 100 1yr 2yr 3yr % N=170 N=391 N=678 N=670 N=602 Cohort 2006 Figure 2 - Percentage distribution of caries-free Brazilian 1- to 5-year-old children in Cohort 2006 (n = 2,511) with their d1efs scores equal to “0” and their d3efs scores equal to “0”. Caries-free (d1efs = 0) Caries-free (d3efs = 0) Caries-free (d1efs = 0) Caries-free (d3efs = 0) 0 10 20 30 40 50 60 70 80 90 100 1yr 2yr 3yr 4yr 5yr % N=170 N=391 N=678 N=670 N=602 Cohort 2006 ibution of -year-old = 2,511) ual to “0” al to “0”. 5yr Caries-free (d1efs = 0) Caries-free (d3efs = 0) lesions, fi llings and extracted teeth, i.e. d3efs = 0 (d1 = decayed at non-cavitated level for both active and inactive lesions, d3 = decayed at cavitated level for both active and inactive lesions, e = extracted, f = fi lled, t = tooth, s = surface). When comparing the percentages of caries-free children according to age and level of examination, signifi cant differences, from the age of 2 years, were observed in Cohort 1996 (p < 0.001). In Cohort 2006, differences were still present, but they were signifi cant at the fi rst lev- el of examination only (p < 0.05). Concerning caries severity, signifi cant differences were observed be- tween the mean number of d1eft/s and d3eft/s scores in Cohort 1996 (p < 0.001), while in Cohort 2006 this difference was important, but no longer signifi - cant (p = 0.10; for details see Carvalho et al.13). The inclusion of active and inactive non-cavitated lesions gives a more realistic estimate of caries prevalence; at the same time, it gives further information about possible changes in caries activity and severity. Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Discussion It is important to bear in mind that, tradition- ally, the caries process has been diagnosed at cavi- tated level for both prevalence and time-trend stud- ies.18-26 Since the 1980s, a leveling out of the decline of caries in the primary dentition has been reported in the literature.18,20,23,24 In some countries, however, recent studies still documented a decline in mean defs scores.2,3,19,21 In agreement with our results, studies in the literature showed significant differ- ences in caries prevalence and severity in children, adolescents and young adults when non-cavitated lesions were considered in the individual’s caries ex- perience.1-4,7,10,11,17 The recording of active and inac- tive non-cavitated lesions had a major impact on the results of our caries trends study, as it clearly dem- onstrated that caries decline was still significant in the population studied.13 The traditional view that the diagnosis of the caries process is equivalent to that of cavitated le- sions and that its treatment should be based on fill- ings and extractions is questioned in the present pa- per. The main findings of the present paper were that the diagnosis of non-cavitated lesions was suitable and reliable when performed under field conditions and that the diagnosis of active and inactive non- cavitated lesions significantly influenced caries out- comes. There is general agreement that obtaining good conditions to perform a thorough clinical exami- nation in the context of an epidemiological study is a major challenge. The present study described a methodology that circumvents this challenge, since it allows data collection with sufficient flexibility to be used in a variety of field settings and situations. It is therefore recommended for field work in countries with limited economic resources. This methodology was first implemented in the mid 1990s1 and since then it has been used by our group for large epide- miological surveys.2,3,13 Concerning the reliability of the diagnosis of non-cavitated lesions in field work, most of the values obtained were compatible, either with almost perfect agreement or with substantial agreement.16 However, more efforts should be made to improve the diagnosis of inactive non-cavitated lesions, since it showed moderate agreement. In ad- dition, experienced examiners seemed to score bet- Finally, one may speculate about the reasons for the observed decline during the study period. The authors have no clear explanation, but they con- sider the following to be important. What is the impact of non-cavitated lesions on caries decline? In our study, caries prevalence in preschool chil- dren decreased almost by half from 1996 to 2006. The number of children free of any form of treated and untreated caries increased signifi cantly in all age groups, as shown in Figures 1 and 2 (p < 0.05). Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Carvalho JC, Mestrinho HD ter than those who were dealing for the first time with an epidemiological survey. A marked reduction in terms of severity was reg- istered for d1eft/s scores from the age of 2 years (p < 0.0005). However, a significant reduction at the d3eft/s level was recorded at the tooth level only and limited to the 5-year-old age group (p < 0.0005). The reduction in the rate of caries progression dur- ing early childhood manifested itself as a significant reduction in the number of cavitated lesions at 5 years of age. The recording of non-cavitated lesions had a major impact on the results of the caries trends study,13 as it clearly showed that caries decline was still significant in the population studied. The ob- served caries decline was related to both active and inactive non-cavitated lesions. Any system that records non-cavitated lesions re- quires more time for the clinical examination than the system recommended by the World Health Or- ganization. This statement is in accordance with the literature, since the time required to record non-cav- itated lesions may be doubled depending on the sys- tem chosen.17 However, it should be appreciated that practitioners who are dealing with the recording of non-cavitated lesions for the first time will spend more time than those who are familiar with it. The information obtained by recording active and inac- tive non-cavitated lesions should, in one way or an- other, compensate for the extra time employed dur- ing field work. Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 References 10. Fontana M, Jackson R, Eckert G, Zandona AF, Ando M, Stookey GK, et al. Identification of caries risk factors in tod- dlers. J Dent Res. 2011 Feb;90(2):209-14. 1. Carvalho JC, Mestrinho HD, Bezerra AC, Maltz M. Onset, development and arrest of dental caries in Brazilian pre-school children. Clin Oral Investig. 1998 Jun;2(2):96-100. 1. Carvalho JC, Mestrinho HD, Bezerra AC, Maltz M. Onset, development and arrest of dental caries in Brazilian pre-school children. Clin Oral Investig. 1998 Jun;2(2):96-100. 11. Ismail AI, Sohn W, Tellez M, Willem JM, Betz J, Lepkowski J. Risk indicators for dental caries using the International Caries Detection and Assessment System (ICDAS). Community Dent Oral Epidemiol. 2008 Feb;36(1):55-68. 2. Carvalho JC, Van Nieuwenhuysen JP, D’Hoore W. The decline in dental caries among Belgian children between 1983 and 1998. Community Dent Oral Epidemiol. 2001 Feb;29(1):55- 61. 12. Carvalho JC, Thylstrup A, Ekstrand KR. Results after 3 years of non-operative occlusal caries treatment of erupting perma- nent first molars. Community Dent Oral Epidemiol. 1992 Aug;20(4):187-92. 3. Carvalho JC, D’Hoore W, Van Nieuwenhuysen JP. Caries decline in the primary dentition of Belgian children over 15 years. Community Dent Oral Epidemiol. 2004 Aug;32(4):277- 82. 4. Machiulskiene V, Nyvad B, Baelum V. Prevalence and severity of dental caries in 12-year-old children in Kaunas, Lithuania 1995. Caries Res. 1998 May-Jun;32(3):175-180. 13. Carvalho JC, Figueiredo MJ, Vieira EO, Mestrinho HD. Car- ies trends in Brazilian non privileged preschool children in 1996 and 2006. Caries Res. 2009 Mar;43(1):2-9. 5. Silva BB, Maltz M. Prevalence of dental caries, gingivitis and fluorosis in 12-year-old students from Porto Alegre-RS, Brazil, 1998/1999. Pesqui Odontol Bras. 2001 Jul-Sep;15(3):208-214. Portuguese. 14. Thylstrup A, Bruun C, Holmen L. In vivo caries models- mechanisms for caries initiation and arrestment. Adv Dent Res. 1994 Jul;8(2):144-57. Review. 15. Nyvad B. Diagnosis versus detection of caries. Caries Res. 2004 May-Jun;38(3):192-8. Review. 6. Alm A, Wendt LK, Koch G, Birkhed D. Prevalence of approxi- mal caries in posterior teeth in 15-year-old Swedish teenagers in relation to their caries experience at 3 years of age. Caries Res. 2007 Aug;41(5):392-8. 16. Landis JR, Koch GG. The measurement of observer agree- ment for categorical data. Biometrics. 1977 Mar;33(1):159-74. 17. Braga MM, Oliveira LB, Bonini GA, Bönecker M, Mendes FM. Feasibility of the International Caries Detection and Assessment System (ICDAS-II) in epidemiological surveys and comparability with standard World Health Organization criteria. Caries Res. 2009 Jul;43(4):245-249. 7. Discussion The children who participated in both cohort studies were from the same geographical area of the Federal District of Brazil, and were from low socio-economic back- grounds based on the family’s monthly income and mother’s educational level. The children had daily access to fluoride as shown by the percentages of water samples, which fell within the standard range of 72%–84% (Cohort 1996) and of 78%–96% (Cohort 2006).13 In addi- 5 Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 Diagnosing non-cavitated lesions in epidemiological studies: practical and scientific considerations tion in the rate of caries progression. The reduction in the rate of caries progression manifested itself first by a significant increase in the percentage of children who were free from any form of clinically treated or untreated caries, including active and inactive non-cavitated lesions, and, sec- ond, by a significant reduction in the mean d1eft/s scores in which non-cavitated lesions were included. tion in the rate of caries progression. tion in the rate of caries progression. tion, daily access to fluoride toothpaste was provid- ed by the nursery schools for children from 3 years of age in Cohort 1996 and from 1 year of age in Co- hort 2006. Fluoride reduces the rate of caries pro- gression and this may partially explain our results. Other factors may have contributed to the caries de- cline, but they were not investigated in our study. The reduction in the rate of caries progression manifested itself first by a significant increase in the percentage of children who were free from any form of clinically treated or untreated caries, including active and inactive non-cavitated lesions, and, sec- ond, by a significant reduction in the mean d1eft/s scores in which non-cavitated lesions were included. Acknowledgment The interest in carrying out clinical examina- tions for caries diagnosis in epidemiological stud- ies, which include diagnosis of active and inactive non-cavitated lesions, is highlighted in the present paper. By doing so, it was possible to demonstrate that caries prevalence and caries activity decreased significantly from 1996 to 2006 among Brazilian 1- to 5-year-old children, simultaneously with a reduc- The author thanks the ABOPREV for the op- portunity to lecture at the “Equity, Social Inclusion and Oral Health Promotion: Major Challenges” In- ternational Symposium, held at the 18th Congress of the Brazilian Association for Oral Health Promo- tion (Associação Brasileira de Odontologia de Pro- moção de Saúde - ABOPREV). Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 7 Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 References Ismail AI, Lim S, Sohn W, Willem JM. Predictors of den- tal caries progression in primary teeth. J Dent Res. 2009 Mar;88(3):270-5. 8. Fejerskov O, Kidd E. Dental Caries. The Disease and its Clini- cal Management. 2nd ed. Oxford: Blackwell Munksgaard; 2008. 126 p. 18. Marthaler TM. Changes in dental caries 1953-2003. Caries Res. 2004 May-Jun;38(3):173-81. Review. 9. Pitts NB. Modern concepts of caries measurement. J Dent Res. 2004 Jul;83 Spec No C:C43-7. Review. 19. Ferro R, Besostri A, Olivieri A, Stellini E, Mazzoleni S. Pre- schooler’s dental caries experience and its trend over 20 years 6 Braz Oral Res., (São Paulo) 2014;28(Spec Iss 1):1-7 6 Carvalho JC, Mestrinho HD (2005/6). Surveys co-ordinated by the British Association for the study of community dentistry. Community Dent Health. 2007 Mar;24(1):59-63. in a North-East Italian Health district. Eur J Paediatr Dent. 2007 Dec;8(4):199-204. 20. Truin GJ, van Rijkom HM, Mulder J, van’t Hof MA. Caries trends 1966-2002 among 6- and 12-year-old children and erosive wear prevalence among 12-year-old children in The Hague. Caries Res. 2005 Jan-Feb;39(1):2-8. 24. Bönecker M, Ardenghi TM, Oliveira LB, Sheiham A, Marce- nes W. Trends in dental caries in 1- to 4-year-old children in a Brazilian city between 1997 and 2008. Int J Paediatr Dent. 2010 Mar;20(2):125-31. 21. Martins RJ, Garben CA, Garbin AJ, Moimaz SA, Saliba O. Declining caries rate in a municipality in northwestern São Paulo State, Brazil, 1998-2004. Cad Saude Publica. 2006 May;22(5):1035-41. Portuguese. 25. Davies GM, Jones CM, Monaghan N, Morgan MZ, Neville JS, Pitts NB. The caries experience of 11 to 12 year-old chil- dren in Scotland and Wales and 12 year-olds in England in 2008-2009: reports of co-ordinated surveys using BASCD methodology. Community Dent Health. 2012 Mar;29(1):8-13. 22. Ferreira SH, Béria JU, Kramer PF, Feldens EG, Feldens CA. Dental caries in 0- to 5-year-old Brazilian children: preva- lence, severity, and associated factors. Int J Paed Dent. 2007 Jul;17(4):289-96. 26. World Health Organization. Oral Health survey – Basic meth- ods. 4th ed. Geneva: World Health Organization (WHO); 1997. 66 p. 23. Pitts NB, Boyles J, Nugent ZJ, Thomas N, Pine CM. The dental caries experience of 5-year-old children in Great Britain
https://openalex.org/W2942668641	https://t-s.today/PDF/14SATS418.pdf	Russian	null	The study of tensile deformations at the lower boundary of the package of asphalt concrete layers based on tests of the installation of shock loading	Transportnye sooruženiâ	2,018	cc-by	3,516	For citation: Tiraturyan A.N., Akulov V.V., Bodrov I.V., Simakova A.A., Farnieva M.V. (2018). The study of tensile deformations at the lower boundary of the package of asphalt concrete layers based on tests of the installation of shock loading. Russian journal of transport engineering, [online] 4(5). Available at: https://t-s.today/PDF/14SATS418.pdf (in Russian). DOI: 10.15862/14SATS418 Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering ISSN 2413-9807 https://t-s.today Аннотация. В настоящее время в дорожной отрасли Российской Федерации наметилась модернизация системы эксплуатации автодорожного комплекса. В связи с этим наблюдается тенденция внедрения новых технологий по контролю за состоянием дорожных одежд. Одной из таких является высокопроизводительная ударная установка динамического нагружения FWD, которая позволяет измерять чаши прогиба дорожной одежды. В данной работе предоставлены примеры и модели, позволяющие оценить достоверность результатов с установки FWD, а также зависимость экспериментальных и программных данных чаши прогиба дорожной конструкции. Актуальность исследования обусловлена тем, что в настоящее время осуществляется значительная переработка методов расчета нежестких дорожных одежд и создание нового метода прогнозирования усталостных разрушений. Тем не менее применение данной эмпирической зависимости требует оценки ее адекватности и возможности применения для конструкций и материалов слоев, используемых в практике РФ. Решить эту задачу возможно путем сопоставления фактических значений растягивающих деформаций с расчетными значениями, определяемыми на основе механико-математической модели напряженно-деформированного состояния дорожной конструкции. В качестве экспериментальных конструкций, на которых производилась отработка методик расчета растягивающих деформаций, были выбраны участки федеральных автомагистралей М-4 «Дон» и М-3 «Украина». Измерения на данных участках осуществлялись установкой ударного нагружения FWD через каждые 100 м. При этом при каждом ударном воздействии осуществлялась регистрация чаш прогиба покрытия. На основе полученных чаш прогибов были рассчитаны растягивающие деформации на нижней границе пакета асфальтобетонных слоев. Далее была произведена статистическая обработка результатов замеров чаши прогибов. Для оценки адекватности полученных результатов была применена американская математическая модель MnLayer. В процессе расчета были определены основные параметры напряженно-деформированного состояния, в том числе растягивающие деформации. Анализируя данные, полученные по результатам расчета растягивающих напряжений в математических моделях заданных дорожных конструкций, был построен сопоставительный график, иллюстрирующий зависимость фактических показателей чаши прогиба с показателями, полученными в программе MnLayer. Ключевые слова: дорожная одежда; чаша прогиба; анализ; растягивающие деформации; напряженно-деформированное состояние; FWD; MnLayer; деформация Ключевые слова: дорожная одежда; чаша прогиба; анализ; растягивающие деформации; напряженно-деформированное состояние; FWD; MnLayer; деформация В настоящее время в дорожной отрасли РФ наметилась модернизация системы эксплуатации автодорожного комплекса. В связи с этим наблюдается тенденция внедрения новых технологий по контролю за состоянием дорожных одежд [1]. Одной из таких технологий является применение высокопроизводительной установки ударного нагружения FWD, позволяющей осуществлять регистрацию чаш прогибов нежестких дорожных одежд. Ключевой особенностью данной установки является возможность регистрации чаши прогиба дорожной одежды на расстоянии до 2,5 м от точки приложения нагрузки с помощью датчиков геофонов, установленных на рейке. ГРНТИ 67.01.77, 67.01.81 Тиратурян Артем Николаевич ФГБОУ ВО «Донской государственный технический университет», Ростов-на-Дону, Россия Доцент кафедры «Автомобильные дороги» Кандидат технических наук E-mail: tiraturjan@list.ru Страница 1 из 10 Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering Механизм ударного нагружения, смонтированный на данной установке, позволяет имитировать нагрузку на покрытие нежёсткой дорожной одежды в пределах от 2 до 150 кН, время контактного взаимодействия на покрытие дорожной одежды составляет 30 мс (рисунок 1). Страница 2 из 10 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2018, №4, Том 5 2018, No 4, Vol 5 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering Рисунок 1. Установка динамического нагружения FWD (источник: http://avtodor-eng.ru/cntnt/novosti-1/n341.html) Рисунок 1. Установка динамического нагружения FWD (источник: http://avtodor-eng.ru/cntnt/novosti-1/n341.html) В странах Европы и Северной Америки установка FWD является незаменимым устройством в современных дорожных лабораториях. Как показывает зарубежный опыт, применение данной установки позволяет получать достоверные сведения о состоянии дорожной конструкции непосредственно по значениям чаши прогиба [2]. В результате могут быть определены критические значения деформаций в каждой точке дорожной конструкции. Одними из таких показателей являются значения растягивающих деформаций на нижней границе пакета асфальтобетонных слоев, которые определяются по эмпирической формуле [3]: log 1.06755 0.56178 log 0.003233 log 0.47462 log 1.15612 log 0.68266 lo 1 1800 300 h d SCI BDI r  = − + + + + − (1) где: SCI300 = d0-d300, µm; BDI = d300-d600, µm; BCI = d600-d900, µm; di – вертикальное перемещение на расстоянии I от точки приложения нагрузк Для РФ данная зависимость является актуальной, так как в настоящее время осуществляется значительная переработка методов расчета нежестких дорожных одежд и создание нового метода прогнозирования усталостных разрушений. Тем не менее применение данной эмпирической зависимости требует оценки ее адекватности и возможности применения для конструкций и материалов слоев, используемых в практике РФ [4]. Решить эту задачу возможно путем сопоставления фактических значений растягивающих деформаций с расчетными значениями, определяемыми на основе механико-математической модели напряженно-деформированного состояния дорожной конструкции. Одним из примеров такой модели является программный комплекс MnLayer, разработанный и активно применяющийся в штате Миннесота, при проведении оценке секций дорожных одежд на полигоне MnDOT [5]. В качестве экспериментальных конструкций, на которых производилась отработка методик расчета растягивающих деформаций были выбраны участки федеральных автомагистралей М-4 «Дон» и М-3 «Украина» (таблица 1). Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 1.06755 0.56178 log 0.003233 log 0.47462 log 1.15612 log 0.68266 log 1 1800 300 h d SCI BDI BCI r  = − + + + + − ISSN 2413-9807 https://t-s.today ISSN 2413-9807 https://t-s.today Таблица 1 Экспериментальные участки и конструкции дорожных одежд № п/п Конструкция дорожной одежды Толщина слоя, см Модуль упругости, Мпа М-4 «Дон» км 296+500-321+000 1 Плотная горячая мелкозернистая а/б смесь, Тип А, I марка 5 4500 2 Горячая крупнозернистая пористая а/б смесь, I марка 7 2800 3 Горячая крупнозернистая пористая а/б смесь, II марка 8 2800 4 ЩПС, укрепленное цементом М60 F25 20 800 5 Технологический слой, уложенный по способу заклинки 15 600 6 Песок мелкий 50 200 7 Грунтовое основание - 120 М-4 «Дон» км 377+000-414+000 1 Плотная горячая мелкозернистая а/б смесь, Тип А, I марка 5 4500 2 Горячая крупнозернистая пористая а/б смесь, I марка 7 2800 3 Горячая крупнозернистая пористая а/б смесь, II марка 8 2800 4 ЩПС, укрепленное цементом М60 F25 20 800 5 Технологический слой, уложенный по способу заклинки 15 600 6 Песок мелкий 50 200 7 Грунтовое основание - 120 М-4 «Дон» км 414+000-440+00 1. ЩМА-15 на ПБВ 60 4 5300 2. Плотный а/б из горячей крупнозернистой смеси, Тип А, I марка 8 4500 3. Пористый, горячий крупнозернистый а/б I марки на БНД 60/90 10 2800 4. Фракционированный щебень М1400, по способу заклинки 36 900 5. Технологический слой втапливания щебня М400 10 300 6. Песчаный подстилающий слой 50 200 7. Подстилающий грунт - 120 М-4 «Дон» км 877+000-877+326 1 ЩМА-20 на БНДУ 60 5 5300 2 Плотный а/б из горячей крупнозернистой щебеночной смеси I марки, Тип А, дисперсно-армированный 7 4500 3 Пористый а/б из горячей крупнозернистой щебеночной смеси I марки, дисперсно-армированный 7 2800 4 Органоминеральная смесь, обработанная жидким органическим вяжущем с минеральным вяжущем 20 950 5 Щебеночная смесь с непрерывной грануломитрией (С4) 26 275 6 Мелкий песок 20 200 7 Грунт зем. Страница 3 из 10 Страница 3 из 10 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com ISSN 2413-9807 https://t-s.today ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today № п/п Конструкция дорожной одежды Толщина слоя, см Модуль упругости, Мпа 5 Щебеночная смесь с непрерывной грануломитрией (С4) 26 275 6 Мелкий песок 20 200 7 Грунт зем. полотна глина 50 М-4 «Дон» км 907+000-913+000 1 ЩМА-15 на вяжущем БИТРЭК 60/90 5 5300 2 Пористый горячий крупнозернистый а/б I марки на БНД 60/90 7 2800 3 Пористый горячий крупнозернистый а/б I марки на БНД 60/90 8 2800 4 ЩПС, обработанное цементом М60 F25 20 800 5 Щебень М-800, уложенный по способу заклинки 15 450 6 Щебень М-800 27 275 7 Подстилающий грунт - 120 М-4 «Дон» км 1038+000-1063+000 1 ЩМА-15 на ПБВ приготовленное без индустриального масла 4 5300 2 А/б плотный из горячей крупнозернистой смеси, Тип А, I марка, на БНД 60/90, полимерно-дисперсно-армированный 6 4500 3 А/б пористый крупнозернистой смеси, Тип А, I марка, на БНД 60/90, полимерно-дисперсно-армированный 6 2800 4 Смесь из а/б гранулята с добавлением 30 % щебня, обработанного комплексным вяжущим 30 3200 5 ГПС С4 25 230 6 Подстилающий грунт - 120 М-4 «Дон» км 1442+000-1458+000 1 ЩМА-15 на БНДУ-60, модифицирован полимерными добавками 5 5300 2 А/б плотный из горячей крупнозернистой смеси, Тип А, I марка, на БНД 60/90, полимерно-дисперсно-армированный 6 4500 3 А/б плотный из горячей крупнозернистой смеси, Тип А, I марка, на БНД 60/90, полимерно-дисперсно-армированный 7 4500 4 Фракционированный щебень 40-70 мм, М1000 20 300 5 ЩГПС С4 20 275 6 ГПС С4 25 275 7 ГПС С4 20 275 М-3 «Украина» км 194+000-264+000 1 ЩМА-15 на ПБВ 4 5300 2 Горячий плотный мелкозернисты а/б, Тип А, I марка, полимерно-дисперсно- армированный 8 4500 3 Горячий пористый крупнозернистый а/б, I марки, полимерно-дисперсно- армированный 9 3050 4 Смесь щебеночно-песчаная из металлургический шлаков 17 275 5 Деструктурированный ц/б существующего основания 21 1000 6 Подстилающий грунт - 120 Разработано автором Измерения на данных участках осуществлялись установкой ударного нагружения FWD через каждые 100 м. При этом при каждом ударном воздействии осуществлялась регистрация чаш прогиба покрытия. На основе полученных чаш прогибов были рассчитаны растягивающие деформации на нижней границе пакета асфальтобетонных слоев [6]. Далее была произведена статистическая обработка результатов замеров чаши прогибов, которые представлены на рисунке 2. ISSN 2413-9807 https://t-s.today полотна глина 50 М-4 «Дон» км 877+326-877+476 (Эталонный участок, запроектированный по нормам и правилам ФРГ) 1 ЩМА на БНДУ60, модифицированный полимерными добавками на щебне (SMA-11S) 4 5300 2 Горячий высокоплотный дисперсно-армированный а/б на БНД 60/90 (AC- 22BS) 8 6000 3 Горячий плотный дисперсно-армированный а/б на БНД 60/90 (AC-32ТS) 22 4500 4 Щебеночная смесь непрерывной грануломитрией С5 56 260 5 Песок 20 200 6 Подстилающий грунт - 120 М-4 «Дон» км 892+000-907+000 1 ЩМА-20 на БНДУ 60, модифицированный полимерной добавкой 5 5300 2 А/б из плотной горячей крупнозернистой смеси I марки, Тип А, дисперсно- армированный 7 4500 3 А/б из пористой горячей крупнозернистой смеси I марки, дисперсно- армированный 7 2800 4 Органоминеральная смесь, обработанная жидким органическим вяжущем с минеральным вяжущем 20 950 Экспериментальные участки и конструкции дорожных одежд Страница 4 из 10 2018, №4, Том 5 2018, No 4, Vol 5 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Страница 5 из 10 Корреляция составила 66 %. y = 0,6617x + 8E-06 R² = 0,8821 0 0,00001 0,00002 0,00003 0,00004 0,00005 0,00006 0,00007 0,00008 0,00009 0 0,00001 0,00002 0,00003 0,00004 0,00005 0,00006 0,00007 0,00008 0,00009 0,0001 и программными данными (рисунок 4). Корреляция составила 66 %. y = 0,6617x + 8E-06 R² = 0,8821 0 0,00001 0,00002 0,00003 0,00004 0,00005 0,00006 0,00007 0,00008 0,00009 0 0,00001 0,00002 0,00003 0,00004 0,00005 0,00006 0,00007 0,00008 0,00009 0,0001 Рисунок 4. Корреляционная зависимость между осредненными фактическими растягивающими деформациями, определенными в натурных условиях с использованием установки FWD, и рассчитанными в программном комплексе MnLayer (разработано автором) Рисунок 4. Корреляционная зависимость между осредненными фактическими растягивающими деформациями, определенными в натурных условиях с использованием установки FWD, и рассчитанными в программном комплексе MnLayer (разработано автором) Как видно из представленного графика фактические значения растягивающих деформаций хорошо коррелируют с расчетными значениями, установленными в программном комплексе MnLayer, что свидетельствует о корректности и возможности применения зависимости (1) при проведении натурных исследований. Обобщая вышесказанное, можно сделать вывод: данные замеров чаши прогиба дорожной одежды, полученные с установки динамического нагружения FWD с использованием математической модели MnLayer, могут быть использованы в качестве экспериментального базиса для прогнозирования накопления усталостных напряжений на нижней границе пакета асфальтобетонных слоев [9, 10]. Таким образом с большой долей вероятности возможно определить превышение максимально допустимых напряжений в дорожной конструкции и, как следствие, предотвратить ее разрушение. Страница 5 из 10 Страница 5 из 10 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today 1.98E-05 7.64E-05 6.50E-05 3.35E-05 1.10E-05 2.65E-05 0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 5.00E-05 6.00E-05 7.00E-05 8.00E-05 9.00E-05 м-4 км 296+500-321+000 (330+000) м-4 км 377+000-414+000 м-4 км 474+000-486+000 м-4 км 877+000-950+000 м-4 км 1038+000- 1063+000 м-3 км 194+000-264+000 Растягивающие деформации Километр Рисунок 2. Результатов расчета фактических растягивающих деформаций, на основе чаш прогибов, зарегистрированных в натурных условиях (разработано автором) Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Рисунок 2. Результатов расчета фактических растягивающих деформаций, на основе чаш прогибов, зарегистрированных в натурных условиях (разработано автором) Для оценки адекватности полученных результатов была применена американская математическая модель MnLayer, используемая в департаменте транспорта штата Миннесота, США [7]. В данном программном комплексе были просчитаны рассмотренные выше дорожные конструкции. В процессе расчета были определены основные параметры напряженно- деформированного состояния, в том числе растягивающие деформации [8]. Анализируя данные, полученные по результатам расчета растягивающих напряжений в математических моделях заданных дорожных конструкций, был построен сопоставительный график, иллюстрирующий зависимость фактических показателей чаши прогиба с показателями, полученными в программе MnLayer (рисунок 3). Рисунок 3. Сопоставление фактических растягивающих деформаций, определенных в натурных условиях с использованием установки FWD и рассчитанных в программном комплексе MnLayer (разработано автором) 0 0,00005 0,0001 0,00015 0,0002 0,00025 0,0003 0 1 2 3 4 5 6 7 FWD MNL Рисунок 3. Сопоставление фактических растягивающих деформаций, определенных в натурных условиях с использованием установки FWD и рассчитанных в программном комплексе MnLayer (разработано автором) Страница 6 из 10 Страница 6 из 10 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today ISSN 2413-9807 https://t-s.today ISSN 2413-9807 https://t-s.today Таким образом, была установлена статическая зависимость между экспериментальными и программными данными (рисунок 4). Корреляция составила 66 %. Таким образом, была установлена статическая зависимость между экспериментальными и программными данными (рисунок 4). Корреляция составила 66 %. и программными данными (рисунок 4). Страница 7 из 10 Страница 7 из 10 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2018, №4, Том 5 2018, No 4, Vol 5 2018, №4, Том 5 2018, No 4, Vol 5 Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering ISSN 2413-9807 https://t-s.today ЛИТЕРАТУРА 1. Snaith, M.S., Robinson, R., Danielson, U. Road Maintenance Management: Concepts and Systems. М.: 1998. 312 p. 2. Углова Е.В., Саенко С.С. Обзор инструментов управления состоянием дорожных конструкций // Интернет-журнал «Транспортные сооружения», Том 3, №1 (2016) URL: t-s.today/PDF/02TS116.pdf (доступ свободный). 3. Fernihough, William J., 1995 "BC Hydro’s Experience with Substation RCM", Proceedings of Substation Reliability Centered Maintenance Conference, Newport Beach, CA, 5 p. 4. Углова Е.В., Тиратурян А.Н. Оценка прочности нежестких дорожных одежд. Опыт применения установки динамического нагружения FWD PRIMAX на участках автомобильной дороги м-4 «дон». Дорожная держава. 2014. № 57. С. 55. 5. Мизонов В.В., Тиратурян А.Н. Использование метода «обратного» расчёта при эксплуатации автомобильных дорог. Наука и техника в дорожной отрасли. 2011. № 1. С. 25-27. 6. Углова Е.В., Тиратурян А.Н., Ляпин А.А. / Комплексный подход к исследованию характеристик динамического деформирования на поверхности нежестких дорожных одежд с использованием методов неразрушающего контроля // Вестник Пермского национального исследовательского политехнического университета. Механика. 2016. № 2. С. 111-130. 7. Horak E. Evaluation of Airport Pavements with FWD Deflection Bowl Parameter Bencmarking Methodology // 2nd European Airport Pavement Workshop. 2007. 8. Тиратурян А.Н. Механико-статистический метод оценки остаточного ресурса нежестких дорожных одежд // Транспортные сооружения, 2018 №4, https://t- s.today/PDF/01SATS418.pdf (доступ свободный). Загл. с экрана. Яз. рус., англ. DOI: 10.15862/01SATS418. 9. Веренько В.А. Надежность дорожных одежд. – 2002. 10. Веренько В.А. Дорожные композитные материалы. Структура и механические свойства / Под ред. И.И. Леоновича. – Минск: Наука и техника, 1993. – 246 с. 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering Tiraturyan Artem Nikolaevich Don state technical university, Rostov-on-Don, Russia E-mail: tiraturjan@list.ru Akulov Vladimir Vladimirovich Don state technical university, Rostov-on-Don, Russia Research institute of road-transport complex problems E-mail: marine_25@inbox.ru Bodrov Ilya Vladimirovich Don state technical university, Rostov-on-Don, Russia E-mail: bodrov_ilya120196@mail.ru Simakova Anastasia Anatolievna Don state technical university, Rostov-on-Don, Russia E-mail: simakova000@gmail.com Farnieva Marina Vyacheslavovna Don state technical university, Rostov-on-Don, Russia E-mail: Marina95farnieva@mail.ru Farnieva Marina Vyacheslavovna Don state technical university, Rostov-on-Don, Russia E-mail: Marina95farnieva@mail.ru The study of tensile deformations at the lower boundary of the package of asphalt concrete layers based on tests of the installation of shock loading Abstract. Currently, in the road sector of the Russian Federation, there has been a modernization of the system of operation of the road complex. In this regard, there is a tendency to introduce new technologies for monitoring the condition of road pavements. One of these is the high- performance FWD dynamic loading drum set, which allows you to measure pavement trough bowls. In this paper, examples and models are given to evaluate the reliability of the results from the FWD installation, as well as the dependence of the experimental and software data on the deflection bowl of the road structure. The relevance of the study is due to the fact that a significant reworking of methods for calculating non-rigid pavements and the creation of a new method for predicting fatigue damage is being carried out. Nevertheless, the application of this empirical dependence requires an assessment of its adequacy and the possibility of using layers for structures and materials used in the practice of the Russian Federation. It is possible to solve this problem by comparing the actual values of tensile deformations with the calculated values determined on the basis of the mechanical-mathematical model of the stress-strain state of the road structure. The sections of the M-4 “Don” and M-3 “Ukraine” federal highways were chosen as experimental constructions that were used to work out methods for calculating tensile deformations. Measurements at these sites were carried out by installing shock loading FWD every 100 m. At the same time, at each impact impact, the plates of the coating deflection were recorded. Based on the resulting bowls of the deflections, tensile deformations were calculated at the lower boundary of the package of asphalt concrete layers. Next, a statistical processing of the measurements of the deflection bowl was made. To assess the adequacy of the results obtained, the American mathematical model MnLayer was applied. During the calculation, the main parameters of the stress-strain state, including tensile deformations, were determined. Analyzing the data obtained from the calculation of tensile stresses in mathematical models of given road structures, a comparative graph was constructed illustrating the dependence of the actual deflection cup indicators with those obtained in the MnLayer program. 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2018, №4, Том 5 2018, No 4, Vol 5 ISSN 2413-9807 https://t-s.today Интернет-журнал «Транспортные сооружения» Russian journal of transport engineering REFERENCES 1. Snaith M.S., Robinson R., Danielson U. (1998). Road Maintenance Management: Concepts and Systems. Мoscow, p. 312. 2. Uglova E.V., Saenko S.S. (2018). Overview of road condition management tools. Russian journal of transport engineering, [online] 1(3). Available at: https://t- s.today/PDF/02TS116.pdf (in Russian). 3. Fernihough, William J. (1995). BC Hydro’s Experience with Substation RCM". Newport Beach, California, p. 5. 4. Uglova E.V., Tiraturyan A.N. (2014). Evaluation of the strength of non-rigid pavement. Experience with the use of the FWD PRIMAX dynamic loading unit on m-4 “don” road sections. Road Power, 57, р. 55 (in Russian). 5. Mizonov V.V., Tiraturyan A.N. (2011). Using the method of "reverse" calculation during the operation of roads. Science and technology in the road sector, 1, рр. 25-27 (in Russian). 6. Uglova E.V., Tiraturyan A.N., Lyapin A.A. (2016). An integrated approach to the study of the characteristics of dynamic deformation on the surface of non-rigid pavements using non-destructive testing methods. Bulletin of the Perm National Research Polytechnic University. Mechanics, 2, рр. 111-130 (in Russian). 7. Horak E. (2007). Evaluation of Airport Pavements with FWD Deflection Bowl Parameter Bencmarking Methodology. 2nd European Airport Pavement Workshop. 8. Tiraturyan A.N. (2018). Mechanical-statistical method of estimation of the residual life of flexible pavements. Russian journal of transport engineering, [online] 4(5). Available at: https://t-s.today/PDF/01SATS418.pdf (in Russian). DOI: 10.15862/01SATS418. 9. Verenʹko V.A. (2002). Nadezhnostʹ dorozhnykh odezhd. [Reliability of pavement.] 10. Verenʹko V.A. (1993). Dorozhnye kompozitnye materialy. Struktura i mekhanicheskie svoystva. [Road composite materials. Structure and mechanical properties.] Minsk: Science and Technology, p. 246. Страница 10 из 10 Страница 10 из 10 The study of tensile deformations at the lower boundary of the package of asphalt concrete layers based on tests of the installation of shock loading Keywords: road clothes; deflection bowl; analysis; tensile deformation; stress-strain state; FWD; MnLayer; deformation Страница 9 из 10 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 2018, №4, Том 5 2018, No 4, Vol 5 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com 14SATS418 Издательство «Мир науки» \ Publishing company «World of science» http://izd-mn.com
https://openalex.org/W2744334288	http://www.scielo.br/pdf/csp/v33n7/1678-4464-csp-33-07-e00206416.pdf	Portuguese	null	O legado de Cabanis: hipótese sobre raízes da educação médica no Brasil	Cadernos de Saúde Pública	2,017	cc-by	9,236	ENSAIO ESSAY ENSAIO ESSAY Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodu- ção em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. The legacy of Cabanis: a hypothesis on the roots of medical education in Brazil El legado de Cabanis: hipótesis sobre las raíces de la educación médica en Brasil Naomar Almeida-Filho 1 doi: 10.1590/0102-311X00206416 Correspondência N. Almeida-Filho Rua Waldemar Falcão 641, apto. 501, Salvador, BA 40295-010, Brasil. naomaralmeida@gmail.com 1 Universidade Federal da Bahia, Salvador, Brasil. Correspondência N. Almeida-Filho Rua Waldemar Falcão 641, apto. 501, Salvador, BA 40295-010, Brasil. naomaralmeida@gmail.com Cad. Saúde Pública 2017; 33(7):e00206416 O legado de Cabanis: hipótese sobre raízes da educação médica no Brasil The legacy of Cabanis: a hypothesis on the roots of medical education in Brazil Educação em Saúde; Ensino; Universidades; História da Medicina Resumo Correspondência N. Almeida-Filho Rua Waldemar Falcão 641, apto. 501, Salvador, BA 40295-010, Brasil. naomaralmeida@gmail.com Cad. Saúde Pública 2017; 33(7):e00206416 Introdução Este é o terceiro artigo de uma série sobre a educação médica brasileira 1,2. Para melhor entender a crise de formação de profissionais e de intelectuais que demarca a atual conjuntura nacional no cam- po da saúde, propõe-se apreciar raízes históricas e fundamentos conceituais dos modelos de ensino médico vigentes no mundo e no Brasil. Para isso, é recomendável reler os clássicos, com cuidado e profundidade. Segundo Foucault 3, no contexto da formação médica na França no final do século XVIII, predo- minam uma perspectiva doutrinária e uma pedagogia retórica, distanciadas dos avanços científicos e filosóficos do Iluminismo. No plano institucional, há excesso de escolas médicas de baixa qualidade, nas quais cátedras são negociadas pela nobreza decadente como fonte de renda para professores autorizados a ministrar cursos pagos. Fora das universidades, estudantes compram a preparação para seus exames, “o que torna os estudos médicos muito custosos, tanto mais que, para ter formação prática, o novo doutor deve seguir, em suas visitas, um médico renomado a quem é preciso indenizar” 3 (p. 48). Tal cenário de elitismo, pouca eficácia técnica, alto custo e baixa qualidade pedagógica do ensino médico na França setecentista parece corresponder, mais de um século depois, ao contexto norte-americano do fim do século XIX sintetizado no Relatório Flexner 4 que, em certa medida, persiste em várias partes do mun- do, incluindo o Brasil neste início do século XXI 5. Para Foucault 3, a pauta política da Revolução Francesa inicialmente incluía medidas radicais como extinção de confrarias e corporações e fechamento de universidades e academias, complemen- tadas por três estratégias de longo prazo: primeiro, regulamentação rigorosa, condicionando o exercí- cio da profissão médica a uma instrução superior credenciada pelo Estado; segundo, reorganização da estrutura de serviços assistenciais; terceiro, revisão profunda do próprio sistema de ensino. Em 1792, o médico e farmacêutico Antoine-François de Fourcroy (1755-1809) propôs a recriação do sistema francês de educação mediante o “ensino livre das ciências e das artes” e de reestruturação da formação em saúde pautada em uma “medicina em liberdade”. O marco fundamental dessa proposta compreen- dia a unificação entre medicina, cirurgia e farmacoquímica, retificando clivagem histórica vigente desde a Idade Média. Rejeitando a formação médica de base retórica, Fourcroy cria uma école de santé em Paris, com cursos integrados de medicina, farmácia, higiene e administração pública para forma- ção de um perfil profissional mais aberto e polivalente 3,6. Correspondência Georges Cabanis foi um reformador da prática clínica e do ensino médico que, na Revolução Francesa, lançou as bases conceituais do modelo de educação implantado na França no decorrer do século XIX. Tal modelo, por sua vez, marcou a organização dos sistemas educacionais de muitos países latino-a- mericanos. Este artigo tem como objetivo apresentar e justificar uma hipótese: o modelo de educação médica ainda hoje hegemônico no Brasil ainda hoje se baseia na reforma cabasiana, mantendo uma perspectiva conceitual linear e cartesiana, com matriz curricular disciplinar, formatos tradicionais de prá- tica pedagógica e submissão à lógica profissional corporativa. Para melhor compreensão do contexto social e político dos processos históricos geradores desse anacronismo, em primeiro lugar, apresento uma súmula da biografia e do conjunto pensamento-obra de Georges Cabanis, introduzindo-o como personagem central em seu contexto histórico. Em segundo lugar, destaco os principais elementos estruturais do modelo cabanisiano, com foco especial no ensino médico, tomando alguns dos seus textos autorais como fonte documen- tal. Finalmente, em breves traços, apresento o projeto de reforma do ensino médico elaborado por Cabanis e seus colaboradores como subsídio para ava- liação preliminar da hipótese proposta. 1 Universidade Federal da Bahia, Salvador, Brasil. 1 Universidade Federal da Bahia, Salvador, Brasil. Educação em Saúde; Ensino; Universidades; História da Medicina 2 Almeida-Filho N Cad. Saúde Pública 2017; 33(7):e00206416 Introdução Aprovada com entusiasmo pelos próceres da Revolução Francesa, a proposta logo se ampliaria para duas outras escolas em Montpellier e Estras- burgo, tradicionais centros de educação médica. Relata Foucault: “O povo será amplamente admitido na nova escola de saúde; (...) serão escolhidos em cada distrito alunos que tenham boa conduta, costumes puros, amor à República e ódio aos tiranos, cultura bem cuidada e, sobretudo, conhecimento de algumas ciências que servem de preliminar para a arte de curar. (...) Em vez de ser, como a antiga faculdade, o lugar de um saber esotérico e livresco, a nova escola de saúde [deve ensinar] em vez das vãs fisiologias, a verdadeira arte de curar” 3 (p. 76). Essa proposta compreende acesso amplo de estudantes oriundos de classes populares à educação universitária anteriormente reservada às elites, visando à formação de profissionais de saúde em escolas próprias de saúde em vez de treinar especialistas em escolas de doença chamadas faculdades de medicina. Impressiona a pertinência e atualidade dessa pauta de reforma social em que, nos momen- tos iniciais da Revolução Francesa, despontavam movimentos de reestruturação do campo da saúde. Infelizmente, ressalta Foucault 3, a ordem política tinha outros planos. Os vetores da restauração iniciada pelo Diretório e posteriormente consolidada no Consulado de Bonaparte neutralizaram pro- postas revolucionárias orientadas à emancipação dos sujeitos. Em vez de uma prática clínica não só como “momento essencial da coerência científica, mas também da utilidade social e da pureza política da nova organização da medicina” 3 (p. 75), fomentava-se a recuperação institucional da medicina para “por fim à perigosa experiência da liberdade, um modo, no entanto, de lhe conceder sentido positivo, como também uma via para restaurar, conforme o desejo de alguns, as estruturas do Antigo Regime” 3 (p. 75). A reforma educacional encaminhada pelos jacobinos em 1792 terminou superada pela restauração das faculdades de medi- cina e direito, que ressurgiram ainda mais poderosas na era napoleônica, operando modelos de edu- cação superior orgânicos à nova ideologia burguesa – materialista, individualista, produtivista e laica. Um dos principais líderes intelectuais nesse contexto de restauração da tradição médica, ajustada à matriz cultural da nascente burguesia, é Pierre-Jean-Georges Cabanis. O modelo educacional con- cebido por ele e seus pares, adotado na reforma napoleônica da educação de 1806-1808, estabeleceu Cad. Introdução Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 3 as bases do sistema de ensino superior e profissional implantado na França oitocentista, extrapolando o âmbito restrito da educação superior em saúde 7 e influenciando outros sistemas educacionais, especialmente na Europa meridional e nos países latino-americanos, como o Brasil do século XIX 8. O papel de Georges Cabanis como líder reformador da educação médica, sob bases científicas e diretrizes tecnológicas oriundas do pensamento cartesiano, tem sido em geral pouco reconhecido entre nós. As raras referências na literatura nacional à obra de Cabanis – suposto como defensor da inferioridade da mulher 9, precursor do conceito de instinto sexual 10, por sua teoria fisiológica das ideias 11 e, curiosamente, valorizador da mestiçagem como fator de igualdade entre os homens, con- siderado nesse caso como um antagonista ao racismo de Gobineau 12 – não incluem sua contribuição ao ensino médico e à educação superior. Este artigo tem por objetivo apresentar e justificar a seguinte hipótese: o modelo de educação médica hoje hegemônico no Brasil se organiza numa perspectiva ainda cabanisiana, definida por uma matriz disciplinar, com formatos tradicionais de estrutura curricular e prática pedagógica, submetida à lógica profissional corporativa. A apreciação dessa hipótese requer, sem dúvida, melhor com- preensão do contexto social e político dos processos históricos geradores desse anacronismo. Nesse sentido, em primeiro lugar, apresenta-se uma súmula da biografia e do pensamento-obra de Georges Cabanis, introduzindo-o como personagem central num dado contexto histórico. Em segundo lugar, destacam-se os principais elementos estruturais do modelo cabanisiano, com foco especial no ensino médico, tomando alguns dos seus textos autorais como fonte documental. Finalmente, em breves tra- ços, apresenta-se o projeto de reforma do ensino médico elaborado por Cabanis e seus colaboradores. Biografia breve de Cabanis Esta seção resume informações constantes em textos selecionados sobre vida e obra de Cabanis 13,14,15,16,17,18. Pierre-Jean-Georges Cabanis (1757-1808) foi um médico francês que, na virada do século XIX, alcançou grande reputação como filósofo e líder político. A Figura 1 mostra sua efígie. Cad. Saúde Pública 2017; 33(7):e00206416 Figura 1 Pierre-Jean-Georges Cabanis (litogravura de Ambroise Tardieu – circa 1790). Pierre-Jean-Georges Cabanis (litogravura de Ambroise Tardieu – circa 1790). Cad. Saúde Pública 2017; 33(7):e00206416 4 Almeida-Filho N Cabanis nasceu em 5 de junho de 1757, em Cosnac. Órfão aos sete anos, o pequeno Cabanis é inicialmente educado na paróquia local. Aos dez anos, admitido no internato de Brive, logo alcança os limites da educação provincial. Com a proteção de Turgot, ministro do Rei Luís XVI, o jovem Cabanis é enviado a Paris em 1771 para completar seus estudos, revelando-se dedicado e brilhante estudioso dos clássicos, com talento inato para aprendizado de línguas. Em 1773, contratado como secretário particular do Príncipe Massalski, bispo polonês em visita à França, muda-se para Varsóvia (Polônia). De volta a Paris em 1775, dedica-se a estudos filosóficos e amplia seu horizonte de interesses para o campo das ciências naturais. Participa com entusiasmo dos famosos saraus do Círculo de Auteil, pro- movidos por Madame Helvétius, que o convida a residir na sua mansão, onde convive com grandes nomes do Iluminismo francês. Nessa época, Cabanis torna-se amigo de personalidades que influenciarão toda sua vida, como Dominique-Joseph Garat, advogado que se tornaria um dos principais líderes políticos da Revolução Francesa, e o Marquês de Condorcet, filósofo e matemático interessado em educação (autor de Cinq Mémoires sur l’Instruction Publique, texto base para várias propostas de reforma da educação pública na era revolucionária). A conselho de Léon Dubreuil, médico clínico e fisiologista, começa estudos pre- paratórios em 1778, acompanhando seu mentor nas rondas no Hospital Charité. Em 1780, Cabanis é finalmente aceito na rigorosa Faculdade de Medicina de Paris. Em 1783, interrompe seus estudos para se tornar, por breve tempo, seguidor do famoso hipnotizador Franz Anton Mesmer. Com a morte de Dubreuil, Cabanis retoma seus estudos na Faculdade de Medicina de Reims, gra- duando-se com louvor em 1784. No ano seguinte, abre uma clínica gratuita para os pobres de Auteuil e se dedica a pesquisas científicas sobretudo em fisiologia humana. Em 1788, escreve sua primeira obra sobre semiologia clínica, um opúsculo intitulado Du Degré de Certitude de la Médecine (publicado em 1797), em que explora as interfaces entre medicina e filosofia, criticando o uso indevido da mate- mática e sua incipiente teoria probabilística no processo de diagnóstico diferencial. Revolucionário de primeira hora, Cabanis participa das conspirações e embates políticos que derrubaram o antigo regime. Cad. Saúde Pública 2017; 33(7):e00206416 Figura 1 Após a tomada da Bastilha, conhece Honoré-Gabriel de Mirabeau, com quem colabora em diferentes ocasiões; durante 1790, redige vários discursos por ele proferidos na Assembleia Nacional, ajudando-o a se tornar um dos maiores oradores e líderes políticos da Revo- lução Francesa. Após a morte precoce de Mirabeau em 1791, Cabanis revisa e mesmo reescreve suas anotações para discursos sobre educação e os reúne numa publicação intitulada Quatre Discours sur l’Éducation Publique, concedendo créditos exclusivos ao grande tribuno. Entre 1790 e 1792, Cabanis torna-se administrador geral dos hospitais de Paris. Como programa de trabalho, publica o ensaio Observations sur les Hôpitaux, apoiando a reforma conduzida por Philippe Pinel. Em 1791, elege-se vice-prefeito da comunidade de Auteuil, confirmando-a como sua terra de adoção. No início da Convenção, apresenta relatórios sobre instrução pública e assistência social que muito influenciam as medidas de reestruturação institucional implantadas pela Revolução Francesa. Nesses textos, considera a distribuição injusta de riqueza do antigo regime como o maior problema político enfrentado pela Revolução e propõe estratégias de redução das desigualdades sociais median- te radical reorganização do Estado. Nessa fase, inicia forte amizade e colaboração com o pedagogo e filósofo Destutt de Tracy, organizando o movimento intelectual que a posteridade chamaria de Les Idéologues 13,18. Em 1793, no auge do terror, ajuda a esconder o Marquês de Condorcet nos arredores de Paris, acolhendo toda a família do amigo perseguido (três anos depois, Cabanis se casaria com Charlotte Grouchy, cunhada de Condorcet). Nesse período turbulento, Cabanis sofre muito com a prisão de seus amigos mais próximos, culminando com a execução de Lavoisier e do poeta Roucher, seu men- tor da juventude, e o suicídio de Condorcet. Deprimido, doente e considerado suspeito pelo regime, Cabanis recolhe-se a Auteuil onde retoma sua clínica de pobres e desvalidos (que enfim o protegem da perseguição das milícias populares). Em meados de 1794, quando o terror finalmente começa a refluir, Cabanis retoma intensa ativi- dade política. Reabre os salões de Madame Helvétius, agora acolhendo o núcleo aglutinador do que Picavet 13 chamou de “segunda geração dos ideólogos”: Pierre-Simon Laplace, célebre matemático, astrônomo e físico; Jean-Baptiste Say, pioneiro das ciências econômicas; Constantin-François de Vol- ney, historiador que introduz Napoleão Bonaparte no grupo; François Daunou, educador e precursor da ciência política; liderados por de Tracy e pelo próprio Cabanis. Cad. Figura 1 Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 5 Colaborando com Pinel, Cabanis renova seu interesse pelos temas de reforma dos hospitais, regulamentação do exercício da profissão médica, teoria das práticas clínicas, modelos assistenciais e ensino médico. Um plano moderado de reforma da medicina e do ensino em saúde, elaborado por uma comissão presidida por Vic D’Azyr, tinha sido apresentado à Assembleia Nacional no primeiro ano da Revolução Francesa, logo rejeitado por sua origem numa instituição oficial do regime anterior. Em 1794, o projeto de reforma do ensino médico apresentado por Fourcroy à Convenção, menciona- do na introdução, preconiza uma formação de base anatomoclínica e introduz o conceito de estágio obrigatório nos serviços de saúde (origem do internat des hôpitaux). Cabanis apoia esses pontos da proposta, porém discorda da farmacoquímica como ciência de base do novo currículo e da criação das écoles de santé. Apesar disso, é nomeado professor adjunto de hygiene da primeira escola de saúde implantada em Paris, junto com Pinel, Fourcroy e Thouret. Após a reabertura das faculdades, Cabanis é nomeado professor adjunto da Faculdade de Medi- cina de Paris, onde implanta um novo modelo de ensino clínico. Seguidor da “semiologia natural” de Corvisart, defende examinar o paciente em profundidade, numa abordagem sistemática e metódica, observando a correlação nosológica diretamente na cabeceira do paciente. Cabanis escreve em 1796 um texto sobre a escola de medicina ideal, intitulado Considérations Genérales sur les Révolutions de l’Art de Guérir, discutido no Institut de France. Em 1798, graças ao seu trabalho filantrópico em Auteuil, elege-se representante do Departamento de Seine no Conselho dos Quinhentos. Aproveitando sua tribuna, propõe ajustar o Plano Vic d’Azyr à proposta de Fourcroy, apresentando sucessivos rapports ao Conselho durante seu mandato. O mais importante desses documentos é sem dúvida Sur un Mode Provisoire de Police Médicale 19, proposta de políticas para reinstitucionalização do exercício da medicina (esse é o sentido do termo police médicale) na era pós-revolucionária, em sintonia com diretrizes curriculares e regras de graduação em nível superior para a profissão médica. Político habilidoso e bem relacionado, Cabanis mostra-se atuante na condição de parlamentar e consegue, não sem dificuldades, aprovar a maior parte de seus projetos. Em 1799, de volta do Egito, Napoleão Bonaparte dá sinais de que pretende adotar as ideias defen- didas pela elite intelectual parisiense, com maior simpatia pelas propostas do grupo liderado por Cabanis e Destutt de Tracy. Figura 1 Entusiasmado bonapartista, Cabanis participa do golpe de Estado do 18 Brumário e é convocado a compor o Senado Conservador durante sua criação. Nesse ano, despontan- do no cenário acadêmico parisiense, é nomeado para a cátedra de História da Medicina da Faculdade de Medicina de Paris. Na virada do século, Madame Helvétius falece e deixa Cabanis como herdeiro. Como muitos dos seus colegas ideólogos, Cabanis esperava que os princípios republicanos defendidos pela Revolução fossem aplicados na reconstrução da sociedade francesa, porém se decepciona com o autoritarismo de Napoleão, já Cônsul Vitalício da República; em protesto, não mais comparece às sessões do Senado. O estado de guerra permanente e o patriotismo exacerbado que dominam a sociedade francesa na era Bonaparte acentuam seu isolamento e depressão. Cabanis recolhe-se à biblioteca de Auteuil e empe- nha-se em registrar de modo sistemático seu legado filosófico, científico e pedagógico. Em 1802, publica Rapports du Physique et du Moral de l’Homme, obra que gerou grande polêmica ao propor uma teoria fisiológica sobre a origem das ideias que, de modo explícito, negava a existência da alma. Em 1803, com a reconstituição das academias com base nas seções do Institut de France, Cabanis é eleito para a entidade que posteriormente se tornaria a Academia Francesa. Nesse mesmo ano, organiza seus escritos sobre história dos saberes em saúde, reforma da medicina e educação médica, no entanto, por razões de saúde, não conclui a revisão e ampliação planejadas; apesar disso, atendendo à pressão dos amigos, autoriza sua publicação como livro-testamento. A partir de 1805, sua delicada saúde piora bastante; impossibilitado de sair do refúgio em Auteuil, desgostoso com a tirania do governo bonapartista, encerra sua carreira política. Georges Cabanis falece no dia 5 de maio de 1808, devido a acidentes vasculares encefálicos 18. Cad. Saúde Pública 2017; 33(7):e00206416 6 Almeida-Filho N Revoluções e reforma da medicina Anotações escritas durante o inverno de 1795, aulas sobre história da medicina ministradas na Facul- dade de Medicina de Paris entre 1797-1798 e propostas sobre reforma do ensino médico submetidas ao Conselho dos Quinhentos entre 1798-1801, foram integradas num volume intitulado Coup d’Oeil sur les Révolutions et la Réforme de la Médecine (Coup d’Oeil) 20, somente publicado em 1804, como se viu. Propõe-se, aqui, tomar esse fascinante documento, representativo do pensamento político-filosófico cabanisiano, cujo frontispício encontra-se na Figura 2, como peça-chave para a presente análise. O título dessa obra justamente indica um balanço do esforço coletivo de reforma da medicina, vitorioso na criação de um modelo de prática médica que, já em 1803, mostrava-se funcional e pro- missor. Entretanto, sua tábua de conteúdos (Figura 3) revela que se trata de detalhado projeto de res- gate da história da prática médica e de reforma da educação médica, ambicioso em termos conceituais, curriculares, pedagógicos e pragmáticos. No Coup d’Oeil, encontra-se uma advertência prévia do autor que narra circunstâncias, motiva- ção e dificuldades (inclusive de saúde) para a elaboração do livro. Como introito, Cabanis confessa que sempre buscou contribuir para o grande projeto político de seu amigo Garat: a reorganização do sistema francês de ensino em todos os setores e níveis. De fato, Cabanis colaborou com o Comité d’Instruction Publique, presidido por Dominique Garat, que conseguiu inserir na Constituição, apro- vada pela Assembleia Constituinte em 1791, a garantia da educação como direito de todos. Figura 2 Frontispício da obra Coup d’Oeil sur les Révolutions et la Réforme de la Médecine 20. Frontispício da obra Coup d’Oeil sur les Révolutions et la Réforme de la Médecine 20. Cad. Saúde Pública 2017; 33(7):e00206416 Cad. Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 7 Figura 3 Tábua de conteúdos do livro Coup d’Oeil sur les Révolutions et la Réforme de la Médecine 20. Tábua de conteúdos do livro Coup d’Oeil sur les Révolutions et la Réforme de la Médecine 20. No prólogo, intitulado simplesmente Objet de Cet Écrit 20, Cabanis apresenta com precisão os objetivos da obra, contrastando com o estilo rebuscado predominante na literatura acadêmica daquela época: • traçar, de maneira rápida e sumária, a história das revoluções da medicina; • caracterizar cada revolução pelas circunstâncias que a fizeram surgir e pelas mudanças que ela produziu no desenvolvimento da ciência; • e, enfim, tentar ver se os diferentes quadros, comparados aos métodos filosóficos modernos, podem fornecer algumas visões úteis para a reforma da medicina e do seu ensino. Ao dar conta do primeiro objetivo do Coup d’Oeil, Cabanis demonstra uma perspectiva inusitada de respeito aos saberes tradicionais, antecipando as bases relativistas da ciência antropológica. Men- ciona que mesmo entre os povos mais primitivos (destaca Nova Zelândia, Lapônia, América do Norte e interior da África) de sua época encontram-se “vestígios de uma medicina e de uma cirurgia verdadeiras”. Cad. Saúde Pública 2017; 33(7):e00206416 8 Almeida-Filho N De imediato, transporta tal analogia para a era pré-histórica, postulando que saberes pertinentes à saúde eram coletivamente apropriados e práticas de saúde “podiam ser exercidas por todas as pessoas dotadas de alguma inteligência” que, ao assumir o papel de cuidador da saúde numa tribo ou aldeia, eram capazes de reconhecer diferentes doenças e aplicar tratamento mais ou menos eficientes. Cabanis propõe peculiar periodização para a pré-história da medicina, correspondente ao papel social dos protomédicos: primeiro, poetas, registrando sentimentos e sofrimentos; depois sacerdotes, com “a linguagem vaga e o sotaque misterioso da superstição”; finalmente, filósofos, que “adotaram prin- cípios de várias ciências que ainda não estavam prontas”. As menções pouco honrosas à competência dos sacerdotes para cuidados em saúde resultam do anticlericalismo dominante entre os ideólogos repre- sentativos do materialismo revolucionário. De todo modo, para Cabanis, os filósofos teriam sido res- ponsáveis pela primeira revolução referida no título de sua obra: “A revolução que os primeiros filósofos fizeram na arte de curar (...) todas as ciências que se encontravam ainda no berço, tornaram-se simultaneamente objeto de suas meditações. (...) Quando estes sábios voltaram seus olhares para a medicina, puderam começar a clareá-la com uma luz mais pura. Acostumados a organizar seus diversos conhecimentos em uma certa ordem, a procurar relações entre eles e a encadeá-los, viram como era indispensável a classificação das observações médicas, para que pudessem ser submetidas ao exame do raciocínio. (...) Estes filósofos (...) transformaram uma doutrina oculta e sacerdotal em ciência comum, em arte usual. Esta revolução foi infinitamente útil, [tanto...] para a medicina como para a filosofia” 20 (p. 65-6). Os fundamentos da medicina hipocrática são transversalmente apresentados por Cabanis em 24 páginas do Coup d’Oeil, com ênfase na teoria da saúde como equilíbrio dinâmico entre ambiente e sujeito enfermo, na doutrina dos humores e na doutrina médica das crises. Em todo o texto, Cabanis se esmera em contextualizar suas relações com escolas filosóficas e outras tradições terapêuticas, narrando episódios e casos, sempre num tom apologético, sem esconder sua imensa admiração pelo “pai da arte de curar”. Além da contribuição à constituição de uma “medicina em sua via natural”, com base numa racionalidade empírica, Cabanis afirma Hipócrates como autor da crucial separação entre medicina e filosofia que dialeticamente permitiu recompor “essas duas ciências uma para dentro da outra”, recriando-as por meio de relações absolutamente novas. Galeno comparece no Coup d’Oeil como o mais ilustre dos médicos romanos de origem grega, que reviu fundamentos da doutrina hipocrática, fez observações próprias e buscou integrar as diver- sas teorias vigentes em sua época. Galeno teria recriado à sua maneira a medicina grega, ampliando sua influência entre as dezenas de seitas que cuidavam da saúde na Roma Antiga. Comenta Cabanis que ao adquirir, nas aplicações galênicas, caráter mais sistemático e até empresarial, a arte/ciência criada por Hipócrates teria perdido “muito de sua pureza”, constituindo “mais um adereço do que uma verdadeira riqueza”. Na seção pertinente à medicina árabe, Cabanis revela-se um crítico ácido, injusto e até mesmo incompreensível. Sem citar fontes, valoriza a tradição ptolomaica da Escola de Alexandria, em que a arte de curar teria sido “ensinada brilhantemente” a estudantes “de todos os cantos, que vinham ouvir os mestres mais célebres do universo”. Imputa a destruição da mais famosa biblioteca da história ao “ignorante furor dos muçulmanos”, assinalando que a destruição foi menor para os livros de medicina, “por causa do interesse que inspira aos homens mais ignorantes – a ciência que promete o alívio dos males” 20 (p. 124). Para Cabanis, a medicina árabe pouco teria contribuído para a história da arte de curar. “As escolas árabes estavam na moda, como as escolas gregas já o tinham sido antigamente. O árabe se tornou a língua dos sábios. Cad. Saúde Pública 2017; 33(7):e00206416 Cabanis destaca a evolução da química como a revolução científica de maior impacto – “a chave geral de todas as ciências naturais, a verdadeira luz das artes industriais” – para a emergência de uma medi- cina de base científica na transição do Renascimento ao Iluminismo. Num tempo em que a população sofria com “preconceitos científicos”, os alquimistas desenvolviam “princípios do sistema natural”. Paracel- so, o mais famoso dos alquimistas, é definido por Cabanis como “um verdadeiro modelo de arrogância, audácia e demência” 20 (p. 135). Nessa altura, Cabanis nos apresenta a Georg Ernst Stahl (1659-1734), médico-alquimista que, em sua opinião, seria “o maior médico desde Hipócrates”, um dos “primeiros a transformar a filosofia em ciência”. Sem indicar referências, considera-o grande conhecedor da matéria médica e comenta que ninguém melhor que ele seria capaz de integrar “as observações e os pensamentos filosóficos dos modernos” à secular arte da cura. Cabanis demonstra grande admiração por essa personagem quase esquecida na história das ciências, a ponto de a ele dedicar dez páginas do Coup d’Oeil. Considerados por muitos autores como fundadores da moderna clínica médica, dois neo-hipo- cráticos históricos – Thomas Sydenham (1624-1689), na Inglaterra; Herman Boerhaave (1668-1738), na Holanda – são destacados por Cabanis, num misto de admiração respeitosa e crítica invejosa, cada um merecendo uma seção inteira no Coup d’Oeil. Para Cabanis, a medicina praticada e ensinada na Inglaterra na virada do século XVIII “era total- mente escolástica” e o “verdadeiro espírito de observação era quase totalmente desconhecido” 20 (p. 160-1). Oficial do exército de Cromwell, Sydenham harmonizou uma carreira militar paralela à prática clíni- ca, exercida inicialmente sob licença precária do College of Physicians. Segundo Cabanis, Sydenham “não conhecia muito bem as teorias que reinavam naquele momento” e, sem acesso a livros caros naquela época, sequer teria estudado Hipócrates. Talvez Cabanis tivesse razão, pois Sydenham completou seus estudos de doutorado na Universidade de Cambridge (Reino Unido) somente aos 56 anos de idade 22. Contudo, Cabanis admite que os novos conceitos de prática clínica que revolucionaram a medicina inglesa e depois influenciaram todo o Norte da Europa são devidos a Sydenham; por isso declara por ele sincera admiração. Não obstante, as referências no Coup d’Oeil revelam profunda ambivalência em relação a Sydenham. Foi através desta língua que a Europa conheceu, primeiramente, as obras de Hipócrates, Galeno, Aristóteles, Euclides e Ptolomeu. Entretanto, no meio deste movimento dos conhecimentos, a medicina não tinha feito nenhum progresso real” 20 (p. 124). No Coup d’Oeil, Cabanis praticamente ignora a contribuição dos médicos-filósofos árabes, sequer nomina Averróis e Avicena, a despeito do Cânon de Medicina de Avicena ter feito parte da bibliogra- fia obrigatória das escolas médicas europeias até o século XVII 21. Pior ainda: aos médicos-filósofos da Pérsia e de Córdoba (Espanha), Cabanis deixa implícita a acusação de plagiadores, saqueadores e usurpadores das raízes gregas da medicina moderna. Em contraste, Cabanis valoriza sobremaneira o papel dos médicos judeus durante a Idade Média. Em tom quase mítico, atribui aos judeus o estabe- lecimento da rede de relações comerciais e financeiras, pela “íntima fraternidade que os unia em todas as partes do mundo”, que lhes permitia operar meios seguros para o deslocamento dos valores monetá- rios. Segundo Cabanis, apesar da “perseguição cruel e constante da qual eram as infelizes vítimas em todas Cad. Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 9 9 as partes do mundo conhecido, (...) eles eram nossos carteiros e nossos banqueiros, antes que soubéssemos ler” e “foram também nossos primeiros médicos” 20 (p. 125). Em um tempo em que Hipócrates e os outros pais da medicina eram conhecidos no Ocidente somente pelas traduções árabes, “os judeus eram quase os únicos que sabiam tratar as doenças com algum método” 20 (p. 126). Comenta Cabanis que tal domínio da arte de curar resultava de uma rede de escolas em Toledo, Córdoba e Granada (Espanha) e no Oriente Médio, onde “a medicina era ensinada com um cuidado todo especial”, omitindo que todas essas escolas se situavam em territórios dominados pelo Islã e desprezan- do registros históricos da estreita colaboração dos médicos judeus na prática da medicina em diferen- tes califados. Sua narrativa revela grande respeito e admiração em relação aos judeus, atribuindo-lhes uma personalidade modal que, para além do estereótipo negativo de agiotas usurários, vigente em sua época, implicava predisposição e mesmo talento para a prática eficiente da medicina. Uma nova pedagogia (para uma nova) clínica Considerando os subsídios arrolados, Cabanis se sente enfim capacitado a elaborar sua concepção de “ensino analítico da medicina” com base em dois pontos. Primeiro, a necessária convergência ideológica entre as revoluções da medicina e do ensino médico. Segundo, a pauta dessa revolução, que combina reengenharia institucional (un bon plan d’écoles) e um novo sistema de ensino (systême de leçons). Como a recriação do sistema de instrução pública foi alcançada mediante luta política específica, trata-se aí de implantar um projeto pedagógico revolucionário, que implica uma didática e uma arquitetura curricular consistentes com a base epistemológica integradora da física e da moral, por uma nova metodologia com tripla amplitude de aplicação: na pesquisa, na clínica, na educação. A pedagogia revolucionária compreende uma didática naturalista, em que a “verdadeira instrução dos jovens médicos é aquela que eles recebem no leito dos doentes e não nos livros, nem em uma fria escola, e sim na presença da própria natureza, (...) que fornecia o texto das lições” 20 (p. 308). Pedagogia revolu- cionária, mas não necessariamente inovadora, pois “os gregos ensinavam a medicina prática no próprio leito dos doentes, (...) daí o nome de clínica”, e em Roma, “os médicos mais reconhecidos levavam seus alunos para a casa dos doentes”. Por outro lado, essa pedagogia, pretendendo ser analítica, traduz-se numa arquitetura curricular fragmentada em matérias, numa trajetória gradual e linear, congruente com a análise de decomposição-recomposição justificada por uma epistemologia cartesiana. Nas palavras de Cabanis 20 (p. 307): “[Trata-se de] apresentar aos alunos os objetos sempre na ordem mais natural, quer dizer, começar pelos objetos mais conhecidos ou pelos mais fáceis de serem conhecidos. Sucessivamente e gra- dualmente, passar para aqueles que demandam uma observação mais profunda, os sentidos mais aguçados ou mesmo novos instrumentos”. Cabanis apresenta uma proposta de organização do currículo médico que em termos gerais de estrutura permanece vigente por pelo menos dois séculos. Enumera como matérias obrigatórias: Anatomia, Fisiologia, Patologia, Ciências da Moral, Semiótica, Nosologia, Química, Farmácia, Botâ- nica, Higiene, Terapêutica, Matéria Médica, Cirurgia, Medicina Veterinária. E como matérias com- plementares, que denomina de “objetos acessórios”: História Natural, Física, Ciências Matemáticas, Métodos Filosóficos, Filosofia Moral, Letras e Artes, Línguas Antigas e Modernas. Na seção dedicada a Herman Boerhaave, Cabanis traz poucas informações sobre vida e obra desse médico e professor que Foucault considera como o mais ilustre e influente dos precursores da clínica moderna. Em tom opinativo e quase sarcástico, prefere apresentá-lo como um estudioso sistemático e metódico, mas não muito criativo, que teve a medicina como última escolha, depois de breve incursão na teologia e que, durante algum tempo, sobreviveu como professor de matemática e física. Reconhe- ce, quase com relutância, os “conhecimentos profundos e vastos” de Boerhaave quase como obstáculo ao sucesso numa carreira médica temporã, na medida em que sua inegável inteligência teria “adquirido muito em força, em desenvolvimento, em hábito de discussões, em tenacidade de atenção, mas sua percepção nunca chegou a um grau de perfeição” 20 (p. 170). Cabanis conclui esse capítulo sobre a história da medicina com uma reflexão, quase confessional, sobre a relação entre a história da prática clínica e a evolução do ensino médico. De início, sombrio, avalia que, nos séculos XVI e XVII, “o progresso da ciência foi grande e rápido, entretanto, em compensação, o progresso do ensino foi quase nulo” 20 (p. 181). Atribui essa defasagem ciência vs. ensino às “concepções servis da maior parte dos professores” e ao “discurso confuso da escolástica” que geravam grande distância entre a doutrina e a escola, “entre a sábia ousadia e a rotina cega”. Em contraste, celebra os “verdadeiros progressos” do Iluminismo no campo do ensino durante a segunda metade do século XVIII. Cad. Saúde Pública 2017; 33(7):e00206416 10 Almeida-Filho N Para a reforma da medicina, tornando-a moderna do ponto de vista científico, Cabanis prescreve teoria rigorosa e metodologia empírica sistemática, aplicada por profissionais capazes de operar novos conceitos, métodos e técnicas. E para isso, é necessário “que o ensino da medicina (...) forme para a prática pensadores profundos, desenvolvidos, firmes e flexíveis, que encontrem nas luzes de uma razão trans- cendente, esse conhecimento da vida e essa sabedoria de aplicação” 20 (p. 7). Como estratégia orientadora desse modelo de ensino médico, propõe adotar uma nova ordem de exposição dos fatos, com métodos seguros de observação, experiência e raciocínio, componentes de uma agenda pedagógica renovada. Cad. Saúde Pública 2017; 33(7):e00206416 Uma nova pedagogia (para uma nova) clínica Com elegante ironia, fala dos arrogantes catedráticos, porta-vozes da pomposa pedagogia retórica da universidade clássica, aquela que teria sido enterrada com o Antigo Regime, prestes a serem supera- dos por um novo perfil de professor, mais comprometido com o ensino dos estudantes do que com a afirmação do seu saber magistral. “...Mais do que apresentar ideias inesperadas e surpreendentes por sua grandeza ou por sua novidade, o pro- fessor deve excitar o interesse e despertar a curiosidade dos alunos. Conforme o caráter dos objetos, o professor deve passar da análise à síntese e da síntese à análise, corrigindo sempre as indicações mais audaciosas da síntese com a ajuda das formas mais regulares e seguras da análise” 20 (p. 310-1). Enriquecido pela disciplinada condução de observações e registros, em ciclos de permanente rea- valiação, aplicando conceitos e pensamentos pertinentes, tal método prático deve retificar, insisten- temente, o que outros métodos, “presos na teoria, apresentam como absoluto e rigoroso” 20 (p. 216-7). Para Cabanis, o “instinto, mais do que o saber”, permite ensaiar as regras provisórias desse método de ensino. No entanto, para “que se possam traçar regras verdadeiras, o saber as esclarece e as amplia, o espírito filosófico as encadeia e as coordena”, e o instinto “aperfeiçoa, sobretudo, sua aplicação”, quando nesse momento “o método se confunde com o talento” 20 (p. 217). Essa dupla valorização do instinto, como fonte do talento e condição da resolutividade, introduz elementos não analíticos numa proposta de pedagogia analítica, tornando recomendável e mesmo justificando a ênfase no fazer (devidamente supervisionado por mestres compreensivos e experientes) como projeto de ensino-aprendizagem. Para cumprir etapas cruciais na transição de uma revolução científica para revoluções na medi- cina e no ensino médico, determinadas pelo progresso das ciências do físico e do moral 23, Cabanis 20 (p. 216) propôs recorrer às “operações de nossa inteligência” a fim de criar “um método experimental e prático, fruto da observação contínua dos objetos e do emprego repetitivo dos instrumentos” (de pesquisa e de ensino). Não obstante a refinada construção teórica, restava uma tarefa hercúlea, em nada trivial: um cuidadoso trabalho de costura política em muitas frentes, especialmente em sua função legislativa como parlamentar, num rico momento de reconstrução de uma nova sociedade civil e de um Estado republicano, após a queda do Ancien Régime. Uma nova pedagogia (para uma nova) clínica Três inserções requerem contextualização: Ciências da Moral, que inclui temas psicológicos e sociológicos (ideias, sentimentos, paixões, virtudes e vícios) de interesse médico; Matéria Médica, que se refere ao con- junto de tecnologias aplicadas ao tratamento e cura; e Medicina Veterinária, área de grande potencial para a nascente cirurgia experimental. A rápida evolução do conhecimento científico nesse período determinou ajustes em conteúdos e na estrutura curricular, com desmembramento de matérias e emergência de novas disciplinas. Veja-se a questão da pedagogia como método prático análogo ao método clínico, finalmente compreendido como aplicação do método científico aos eventos singulares ou casos. Evidentemente, Cabanis pretende construir uma equivalência secundária entre métodos práticos – da terapêutica em medicina à prática pedagógica no ensino médico – em que as regras da clínica “só podem ser desenvolvidas na cabeceira do doente” do mesmo modo que as regras da didática se aplicam na interação mestre-aprendiz. A aplicação dessas regras (outra fórmula para o conhecimento em ato) “só poderá ser bem compreendida depois de longa sequência de exemplos”, esgotando, pela repetição, todas as combinações possíveis. Consistente com sua teoria das ideias, para Cabanis, aprender é memorizar um dado conhe- cimento para aplicá-lo com precisão na prática clínica: “[Nesse processo] é preciso, sobretudo, que esses exemplos deixem na memória [dos estudantes] imagens indeléveis que servirão para reconhecer, no primeiro olhar, o caráter distinto de cada doença, no meio de todas as complicações que podem mascará-lo” 20 (p. 217). Cad. Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 11 Tal como as doenças assumem ampla diversidade de formas, o ensino médico se compõe de matérias de diferentes tipos, distintas também na maneira pela qual podem ser aprendidas. Algumas se desenvolvem bem por meio de lições escritas “ou nas sábias palestras de um bom professor”, podem ser reproduzidas em livros, por várias vezes e sob novas formas. Porém, matérias desse gênero são muito poucas. “Em todas as outras, o professor só poderá ser bem entendido se estiver na presença dos objetos. Querer pintar um músculo, uma doença, uma operação química para aquele que nunca viu esta operação, esta doença e nem este músculo, é querer fazê-lo experimentar o sabor de um fruto que ele não conhece ou o odor de um perfume que ele nunca sentiu” 20 (p. 355). Cabanis nos faz uma advertência: “não é preciso levar a prática deste método à pedantérie” 20 (p. 309). Uma nova pedagogia (para uma nova) clínica Nesse contexto, a narrativa autoral e panorâmica dessa intrigante obra explicita os fundamentos do que se propõe, aqui, designar como Reforma Cabanis. Reforma Cabanis: o império das faculdades Em O Nascimento da Clínica 3, Foucault concede pouca atenção ao Coup d’Oeil. Mais focado numa história crítica da medicina como dispositivo organizador de práticas sociais, toma como fio condu- tor de sua análise operações discursivas normalizadoras de condutas propedêuticas e semiológicas. Interessa-se pouco pelos conhecimentos e métodos advindos da recriação do ensino médico como processo político e institucional que repercutiria na reorganização da educação superior francesa como um todo. Dessa forma, enfatiza a oficialidade e legalidade do que Clavreuil chamaria de “ordem médica”, nova configuração das práticas de intervenção em saúde 24,25,26. Interessado em Cabanis, sobretudo como reformador do sistema francês de assistência à saúde, Foucault concentra-se na análise da proposta da police médicale submetida ao Conselho dos Quinhen- tos. Nesse texto 19, Cabanis aborda a natureza (social, cultural, institucional) da medicina e do cuidado em saúde e propõe, em linhas gerais, uma teoria da economia política da profissão médica capaz de justificar intervenções profundas na incipiente rede social de assistência em saúde, estratégica para o projeto político da Revolução Francesa. Cabanis distingue duas categorias de bens ou objetos eco- Cad. Saúde Pública 2017; 33(7):e00206416 Almeida-Filho N 12 12 Almeida-Filho N 2 nômicos. Para alguns objetos ou mercadorias, os próprios consumidores podem avaliar sua utilidade pelo uso, o que seria suficiente para determinar seu valor. Porém, o valor de outros objetos não pode depender dessa avaliação direta, ou por serem necessários para fixar o valor de outros (como a moeda, incluindo metais preciosos) ou por envolverem riscos à pessoa humana, situação em que qualquer erro ou fraude se torna fatal. Em ambos os casos, como observa Foucault, tanto num mercado real de troca de ativos monetários quanto num quase-mercado de riscos, danos virtuais e bens voláteis (como a saú- de), objetos ou serviços têm valor intrínseco e potencial não imediatamente visível, inevitavelmente vulnerável a erros. Para regular a produção e utilização desses objetos, bens ou serviços, mais do que controle de mercado (diretamente pela oferta e demanda num regime liberal de compra e venda) e controle social pela aceitação tácita de seu valor de uso, a nascente República precisa organizar estru- tura e funcionamento de dispositivos de regulação e controle do Estado sobre a economia, o que inclui sem dúvida o campo da saúde e a prática médica. Reforma Cabanis: o império das faculdades A questão central dessa conjuntura então se apresenta com clareza: como atribuir à corporação médica proteção institucional e reserva de mercado, sem retroceder às estruturas corrompidas e per- versas do Antigo Regime, nem recair em formas arbitrárias de controle do Estado que poderiam trair os valores republicanos? Uma questão preliminar e fundamental seria a delimitação do conjunto de sujeitos que, pelo controle institucional dos órgãos de governo, poderia formar essa nova corporação de profissionais. Para solucionar tal questão num momento de grave carência de pessoal, Cabanis prioriza dois problemas práticos: o credenciamento do exercício profissional dos médicos práticos e o reconhecimento dos oficiais de saúde formados. Engenhosamente, propõe adotar uma única estra- tégia para resolver os dois problemas: um exame de Estado que, para os oficiais de saúde, será limitado ao “conhecimento fundamental da arte, e (...) à sua prática”, mas que, para a validação de estudos médicos regulares, devem incluir provas escrita e oral, bem como “exercícios de anatomia, cirurgia e medicina clí- nica”. O Estado se encarregará de fiscalizar com rigor o exercício profissional: “Todo aquele que praticar medicina sem exames ou escola sem ser colocado perante os júris especiais serão multados e condenados à prisão em caso de reincidência” 3 (p. 91). Foucault relata que a proposta de Cabanis não foi aceita nessa oportunidade, mas, “nas suas linhas fundamentais, sinalizou uma solução que dotaria a medicina do estatuto de profissão liberal e protegida que ela manteria até o século XX” 3 (p. 90). De fato, a legislação sobre a prática da medicina em França, final- mente aprovada pelo Consulado em 1802, mostra-se consistente com as propostas de Cabanis em todas as suas dimensões. Por um lado, estabelece uma hierarquia de dois níveis na profissão médica: doutores em medicina e cirurgia graduados em uma das seis faculdades autorizadas; e agentes de saúde, remanescentes das écoles de santé (substituídas pelos novos estabelecimentos oficiais de ensino médico). Qualquer cidadão fora dessas categorias que se arrisque a praticar a medicina incorrerá em penalidades, de multas à prisão. Por outro lado, conforme preconizado por Cabanis, introduz-se o licenciamento para o exercício profissional mediante aprovação em quatro exames (anatomia e fisio- logia, patologia e nosologia, materia medica, higiene e medicina legal), além de uma prova prática de medicina interna ou externa, respectivamente para os que querem ser médicos ou cirurgiões. Cad. Saúde Pública 2017; 33(7):e00206416 Comentários finais Influenciado pelos iluministas Diderot, Condillac e Helvétius, Cabanis mostrou-se um fiel apologista da analítica cartesiana, conforme demonstram Staum 14 e Williams 23. Com esse embasamento filosó- fico, fundado numa perspectiva disciplinar da ciência, defendeu uma revolução da prática médica, em paralelo a um ambicioso programa de reforma do ensino em saúde. No plano político-institucional, a diversidade disciplinar se manifestaria em organismos autocontidos: no campo científico, acade- mias e institutos; no campo educacional, faculdades e escolas superiores. Numa dupla perspectiva de mudança social e educacional, a Reforma Cabanis contribuiu para a extensão desse modo sistemático de organização do conhecimento científico e da formação/reprodução de quadros a outros campos de saberes e práticas. No ensino médico, propiciou a instituição de um regime linear de formação em que o conhecimento se transmite em unidades pedagógicas chamadas de “matérias”, e posteriormente de disciplinas, organizadas de modo sucessivo e gradual, do simples ao complexo, do básico ao aplicado, do teórico ao prático, do geral ao específico. Nesse contexto, com as reformas sociais bonapartistas, consolidou-se uma educação superior regida pelas profissões de saúde legitimadas pelo Estado pós-aristocrático 25,26. Em 12 anos, com avanços e recuos, mudanças profundas foram introduzidas na medicina francesa, culminando com a segmentação e elitização da prática médica mediante a obrigatoriedade da certificação profissional com diploma outorgado por faculdades credenciadas. Esse momento histórico crucial (circa 1800) permitiu a consolidação de um novo modelo de corporações médicas organicamente articuladas com a rede institucional republicana, resultando em um mandarinato 26 meritocrático promovido por um processo político no qual o próprio Cabanis se confirmou como liderança ativa e precoce. A Reforma Cabanis significou uma resposta à crise de identidade histórica da instituição universi- tária 27, todavia transformou essa instituição secular num organismo meramente coordenador de entes independentes: as faculdades. Em paralelo, a Reforma Humboldt fomentou a implantação da chamada universidade de pesquisa como gerador de desenvolvimento econômico e cultural da Alemanha e, pos- teriormente, dos países anglo-saxões. Desde então, os países industrializados paulatinamente abando- naram o modelo original da educação superior de recorte cabanisiano. Assim, em função de uma série de eventos encadeados, já no século XX, inicialmente a Reforma Flexner e recentemente o Processo de Bolonha, as universidades do Norte Global e suas ramificações cada vez mais evoluem para arquiteturas curriculares baseadas em educação geral, com foco na pesquisa e organizadas em ciclos de formação 27. Reforma Cabanis: o império das faculdades Ao extinguir as universidades francesas, a Revolução de 1789 produziu uma solução política radi- cal: o que era uma instituição de cultura e do conhecimento se tornou uma instituição promotora de formação técnica e profissional, ou seja, vocacional. Além da criação de “universidades nominais”, o sistema se caracteriza igualmente pela forte segmentação de qualidade entre universidades e Grandes Écoles e entre formação e investigação 7. Com esse regime institucional, dominado por faculdades e escolas isoladas e relativamente independentes das universidades, é justamente nessa fase histórica que se propõe uma arquitetura curricular linear e exclusiva para formação em carreiras profissionais mediante o ensino superior cada vez mais especializado, sobretudo na esfera da educação médica 25. Concretizada durante o período napoleônico, a Reforma Cabanis resultou enfim em faculdades dotadas de autonomia e capacidade para decidir sobre normas, disciplinas e cursos, bem como uma rede de escolas isoladas, tais como a Escola Politécnica e a Escola Normal Superior, exteriores à Uni- versidade, que tinham por objetivo atender às exigências da revolução industrial e às demandas por quadros dirigentes para a burocracia estatal. Cabanis confirma assim seu protagonismo num processo histórico rápido, contudo sinuoso e complexo, de criação de um sistema institucional de instrução pública pertinente à nova ordem econômica, social e política instaurada pela revolução burguesa na França. Cad. Saúde Pública 2017; 33(7):e00206416 CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 13 Referências Denise Coutinho, do Instituto de Psicologia da Universidade Federal da Bahia, revisou conteúdo e escrita, e contribuiu com rico diálogo sobre con- ceitos básicos deste ensaio. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico pela Bolsa de Produtividade em Pesquisa nível 1-A (pro- cesso: 306341/2011-5). 1. Almeida-Filho N. Reconhecer Flexner: in- quérito sobre produção de mitos na educação médica no Brasil contemporâneo. Cad Saúde Pública 2010; 26:2234-49. 1. Almeida-Filho N. Reconhecer Flexner: in- quérito sobre produção de mitos na educação médica no Brasil contemporâneo. Cad Saúde Pública 2010; 26:2234-49. 2. Almeida-Filho N. Nunca fomos Flexnerianos: Anísio Teixeira e a educação superior em saúde no Brasil. Cad Saúde Pública 2014; 30:2531-53. 3. Foucault M. O nascimento da clínica. Rio de Janeiro: Forense Universitária; 2011. 3. Foucault M. O nascimento da clínica. Rio de Janeiro: Forense Universitária; 2011. 4. Flexner A. Medical education in the United States and Canada. New York: Carnegie Foun- dation for the Advancement of Science; 1910. 5. Almeida-Filho N. Higher education and health care in Brazil. Lancet 2011; 377:1898-900. 6. Crosland M. The officiers de santé of the French Revolution: a case study in the chang- ing language of medicine. Med Hist 2004; 48:229-44. 7. Charle C, Verge J. História das universidades. São Paulo: Editora Unesp; 1996. 8. Cunha LA. A universidade temporã: o ensino superior da colônia à era Vargas. São Paulo: Editora Unesp; 2007. 9. Tosi L. Ciência e a mulher. Ciênc Hoje 1991; 13:26. 10. Freire-Costa J. A face e o verso. Estudos sobre o homoerotismo II. São Paulo: Escuta; 1995. 11. Caponi S. Clima, cerebro y degeneración em Cabanis. Hist Ciênc Saúde-Manguinhos 2009; 16:961-79. 12. Santos-Sousa RA. A extinção dos brasileiros segundo o conde Gobineau. Revista Brasileira de História da Ciência 2013; 6:21-34. 13. Picavet F. Les idéologues. Essai sur l’histoire des idées et des théories scientifiques, philo- sophiques, religieuses, etc. en France depuis 1789. New York: Burt Franklin; 1971. 14. Staum M. Cabanis: enlightenment and medical philosophy in the French Revolution. Princ- eton: Princeton University Press; 1980. 15. Role A, Boullet L. Georges Cabanis, médecin de Brumaire. Paris: Éditions Fernand Lanore; 1994. 16. Gourdol JY. Pierre-Jean-Georges Cabanis (1757-1808), médecin, philosophe et homme politique français. Paris: Medarus; 2010. 17. Mrozovski R. Médecine et politique dans l’œuvre de Pierre-Jean-Georges Cabanis [Tese de Doutorado]. Paris: Université Paris 1 – Pan- théon-Sorbonne; 2013. 18. Pouliquen Y. Cabanis, um idéologue de Mira- beau à Bonaparte. Comentários finais Não obstante, o sistema francês de ensino serviu de exemplo para outros povos submetidos à poderosa influência ideológica e cultural da França, particularmente na Europa meridional e nos países recém-saídos da condição de colônia. Com base nessa matriz ideológica, a partir de 1835, implantam-se faculdades de medicina no Rio de Janeiro e na Bahia, seguidas pelas faculdades de direi- to de São Paulo e do Recife, juntamente com escolas politécnicas e escolas de minas 11. Desse modo, em meados do século XIX, consolida-se no Brasil o império das faculdades, um modelo de educação superior em que as universidades têm papel secundário na formação profissional. Considerando as bases históricas acima expostas, pode-se enfim formular de modo mais preciso a hipótese objeto deste ensaio: a educação superior brasileira exibe um curioso traço arqueológico, pois, de modo hegemônico, conserva o regime institucional de faculdades e cultiva o conceito nominal de universidade, com entrada de estudantes diretamente em cursos de graduação profissional fundados sobre estruturas curriculares tendencialmente fixas, organizadas por blocos de conteúdos denomina- dos matérias ou disciplinas, ainda dentro do modelo institucional e político-pedagógico estabelecido na Reforma Cabanis. Na área da Saúde, essa hipótese pode ser empiricamente testada com a avaliação comparativa da estrutura dos currículos e dos modelos de ensino-aprendizagem praticados nas escolas médicas bra- sileiras. O objetivo de tal pesquisa será recolher, nos programas curriculares de formação profissional em saúde, indícios, resíduos e traços identificadores do modelo cabanisiano, assim reconhecido: ensi- no estruturado em cursos lineares, ministrados por faculdades, com currículos fechados, modelados para memorização, baseados em disciplinas (ou matérias), operando modelos de pedagogia analítica para graduar especialistas legitimados por diplomas licenciadores de profissões ditas imperiais, remi- niscência do modelo vocacional bonapartista que influenciou, de modo indelével, a organização do campo da educação nos países mediterrâneos e suas ex-colônias 28. Cad. Saúde Pública 2017; 33(7):e00206416 Almeida-Filho N 14 14 Almeida-Filho N Por ironia histórica, o Brasil de início do século XXI, corre o risco de se tornar o “último bastião” do modelo de ensino que, no início do século XIX, o revolucionário Cabanis inspirou ao reformador Bonaparte e que, duzentos anos depois, até mesmo a nação francesa que o originou busca hoje superá-lo. Cad. Saúde Pública 2017; 33(7):e00206416 Resumen Georges Cabanis fue un reformista de la práctica clínica y enseñanza médica que, durante la Revo- lución Francesa, lanzó las bases conceptuales del modelo de educación implantado en Francia a lo largo del siglo XIX. Ese modelo, a su vez, marcó la organización de los sistemas educacionales de mu- chos países latinoamericanos. El objetivo de este artículo es presentar y justificar una hipótesis: el modelo de educación médica hegemónico en Brasil, todavía hoy en día, se basa en la reforma caba- nisiana, desde una perspectiva conceptual lineal y cartesiana, con una matriz curricular disciplina- ria, formatos tradicionales de práctica pedagógi- ca y sumisión a la lógica profesional corporativa. Para una mejor comprensión del contexto social y político de los procesos históricos generadores de este anacronismo, en primer lugar, expongo un resumen de la biografía y del conjunto del pensa- miento-obra de Georges Cabanis, presentándolo como un personaje central en su contexto históri- co. En segundo lugar, destaco los principales ele- mentos estructurales del modelo cabanisiano, cen- trándome especialmente en la enseñanza médica, tomando algunos de los textos de su autoría como fuente documental. Finalmente, de forma sucinta, presento el proyecto de reforma de la enseñanza médica, elaborado por Cabanis y sus colaborado- res, como apoyo para la evaluación preliminar de la hipótesis propuesta. Georges Cabanis was a reformer of clinical prac- tice and medical education who laid the concep- tual foundations during the French Revolution for the development of education in France over the course of the 19th century. The model, in turn, marked the organization of educational systems in many Latin American countries. The objective of this article is to present and justify a hypothesis: the model of medical education still hegemonic in Brazil is still based upon the Cabanisian re- form, holding a linear and Cartesian conceptual perspective, with a discipline-based curriculum, traditional teaching formats, and submission to corporate professional logic. In order to better understand the social and political context of the historical processes that generated this anachro- nism, I begin with a summary of the biography and thinking of Georges Cabanis, introducing him as a central character in his historical context. Next, I highlight the main structural elements in the Cabanis model, with a special focus on medi- cal teaching, drawing on some of his writings as the documental source. Referências Paris: Odile Jacob; 2013. 19. Cabanis PJG. Sur un mode provisoire de police médicale. Paris: [publisher unknown]; 1798. http://www.academie-francaise.fr. CABANIS: RAÍZES DA EDUCAÇÃO MÉDICA NO BRASIL 1 15 20. Cabanis PJG. Coup d’oeil sur les révolutions et la réforme de la médecine. Paris: [publisher unknown]; 1804. http://www.academie-fran caise.fr. 25. Weisz G. The emergence of medical specializa- tion in the nineteenth century. Bull Hist Med 2003; 77:536-75. 26. Weisz G. The medical mandarins: the French Academy of Medicine in the nineteenth and early twentieth centuries. Oxford: Oxford University Press; 1995. 21. McGinnis J. Avicenna. Oxford: Oxford Uni- versity Press; 2010. y 22. Anstey P. The creation of the English Hip- pocrates. Med Hist 2011; 55:457-78. 27. Seabra-Santos F, Almeida-Filho N. A quarta missão da universidade: internacionalização universitária na sociedade do conhecimento. Coimbra: Imprensa da Universidade de Coim- bra/Brasília: Editora UnB; 2012. 23. Williams E. The physical and the moral: an- thropology, physiology, and philosophical medicine in France, 1750-1850. Cambridge: Cambridge University Press; 1994. 28. Coelho EC. As profissões imperiais: medicina, engenharia e advocacia no Rio de Janeiro, 1822- 1930. Rio de Janeiro: Editora Record; 1999. 24. Clavreuil J. A ordem médica. Poder e impotên- cia do discurso médico. São Paulo: Brasiliense; 1983. Health Education; Teaching; Universities; History of Medicine Recebido em 20/Dez/2016 Versão final reapresentada em 25/Mai/2017 Aprovado em 13/Jun/2017 Cad. Saúde Pública 2017; 33(7):e00206416 Resumen Finally, in broad strokes, I present the project for reform of medical teach- ing elaborated by Cabanis and his collaborators as backing for a preliminary evaluation of the pro- posed hypothesis. Health Education; Teaching; Universities; History of Medicine Educación en Salud; Enseñanza; Universidades; Historia de la Medicina Recebido em 20/Dez/2016 Versão final reapresentada em 25/Mai/2017 Aprovado em 13/Jun/2017 Cad. Saúde Pública 2017; 33(7):e00206416 Cad. Saúde Pública 2017; 33(7):e00206416
https://openalex.org/W4365448512	https://www.mdpi.com/2076-2615/13/8/1325/pdf?version=1681349232	English	null	Microsatellite Instability Assay as a Potential Approach to Evaluate Genotoxicity: Lead Exposure in a Nestling Passerine Bird at the Stage of Intensive Erythropoiesis	Animals	2,023	cc-by	9,511	n Markowski * , Adam Kali´nski, Jarosław Wawrzyniak, Michał Gl ˛adalski, Joanna Skwarska ´b Marcin Markowski and Jerzy Ba´nbura Department of Experimental Zoology and Evolutionary Biology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland * Correspondence: marcin.markowski@biol.uni.lodz.pl Simple Summary: This is the ﬁrst case of an experimental study with a novel approach to analyze microsatellite instability (MSI) as a detection tool for genetic alterations in a wild population of birds, in this case for great tits (Parus major) being exposed to lead compounds. Although this preliminary study did not ﬁnd any MSI in the analyzed microsatellite markers, it contributes to the examination of this molecular technique as a potentially applicable bioindication tool in ecotoxicological bird studies. We believe that certain issues should be considered in ﬁnding an explanation for our result. First, the exposure to lead was too low to induce genetic alterations. Second, the panel of microsatellite markers studied may have been unsusceptible to lead genotoxicity. Third, the time interval (5 days) between lead exposure and blood sampling for genetic analyses were too short to trigger genotoxicity. Further analyzes are needed to verify these ﬁndings and to evaluate the scope of application of the MSI analysis in wild bird population studies. animals animals Abstract: Although many avian studies have investigated the toxic effects of lead on important biochemical and physiological processes, organ and system function, and behavior, studies evaluating the speciﬁc genotoxic effects of exposure to lead are scarce. Nowadays, rapid technological advances can provide new molecular techniques in this regard. In this study, as a novel approach in bird studies, we used a panel of ten microsatellite loci to investigate the microsatellite instability (MSI) in response to experimental lead intoxication in a common hole-nesting species, the great tit Parus major. For this purpose, an experiment based on an intentional single supplementation of a lead (II) acetate trihydrate compound was conducted, with the use of two different doses, applied to randomly chosen great tit nestlings from randomly selected broods, being at the stage of intensive erythropoiesis. Although this preliminary study did not ﬁnd any MSI in the seven microsatellite markers retained for the ﬁnal comparison, it contributes to the examination of this molecular technique in ﬁeld conditions as being potentially applicable in ecotoxicological bird studies. We believe that certain issues should be considered in ﬁnding an explanation for our result. First, the single doses of lead used in this study may have been too weak to induce genetic instability. Second, the panel of microsatellite markers studied may have been unsusceptible to lead genotoxicity in general. Third, the relatively short time interval (5 days) between the experimental procedure (lead exposure) and the sampling of post-exposure material (blood) for genetic analyses could have limited the effect of lead genotoxicity. Further analyzes are needed to verify these ﬁndings and to evaluate the scope of application of the MSI analysis in wild bird population studies. Citation: Markowski, M.; Kali´nski, A.; Wawrzyniak, J.; Gl ˛adalski, M.; Skwarska, J.; Ba´nbura, J. Microsatellite Instability Assay as a Potential Approach to Evaluate Genotoxicity: Lead Exposure in a Nestling Passerine Bird at the Stage of Intensive Erythropoiesis. Animals 2023, 13, 1325. https://doi.org/ 10.3390/ani13081325 Academic Editor: Agnieszka Tomza-Marciniak Received: 10 March 2023 Revised: 6 April 2023 Accepted: 11 April 2023 Published: 12 April 2023 1. Introduction In numerous avian studies, lead (Pb) intoxication has been proven to cause a wide range of negative effects, starting from slight disruptions to physiological and biochemical processes, through behavioral alterations, to substantial damage or functioning impairment of meaningful organs and systems. As a consequence, individual conditions evaluated using various environment–sensitive physiological and biochemical indicators, as well as the survival of birds, have been signiﬁcantly affected in many cases [1–7]. Lead and lead compounds have also been studied for many years in various biological systems as potential mutagenic, clastogenic, and carcinogenic agents [8]. In most clinical and/or laboratory studies of the genotoxic consequences of lead exposure conducted so far, the focus has been on several genetic endpoints as sorts of bioindicator [8–12]. Interestingly, relatively little attention has been paid so far to wild bird populations in this regard. Although several molecular techniques have been successfully employed in avian studies to evaluate molecular alterations induced by various genotoxic contaminants [13–30], there is a paucity of work on novel molecular techniques that would allow us to verify the genotoxic properties of lead. Microsatellite instability (MSI) could be potentially effective as such a novel method of detection of genotoxicity of various compounds present in the environment. Microsatellites, Short Tandem Repeats (STRs), or Simple Sequence Repeats (SSRs) are simple sequences that contain a series of repeats of one to six nucleotide base pair motifs. They are a characteristic component of the nuclear genome of most eukaryotic organisms and are characterized by a high level of polymorphism. At the same time, they are considered as important markers of genomic DNA damages, e.g., in deletions or insertions and/or point mutations [31]. Microsatellite instability analyses in a relatively small number of clinical and animal laboratory studies has conﬁrmed that exposure to selected heavy metals, including lead and its compounds, can induce MSI [31–38]. For example, the effect of cadmium on genomic stability was analyzed for laboratory outbred strain mouse models. Using a panel of six microsatellite markers, Oliveira et al. [36] tested whether a single subcutaneous injection of CdCl2 induces MSI in murine testes. It turned out that the lowest dose out of three used in this study triggered the size alteration of two microsatellite loci. In another similar study, Du et al. [37] tested the mutagenic effects of cadmium on 42 SSRs in murine liver, kidney, testis, leukocytes, bone marrow, and small intestine. Copyright: © 2023 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/). Keywords: microsatellite instability; lead intoxication; experimental exposure; passerines; nestlings; biomarkers https://www.mdpi.com/journal/animals Animals 2023, 13, 1325. https://doi.org/10.3390/ani13081325 Animals 2023, 13, 1325 2 of 12 1. Introduction That study conﬁrmed the presence of MSI at three microsatellite loci in the kidney and in leukocytes of individuals exposed to lower doses of cadmium chloride. y p Genomic instability is also important from the viewpoint of human exposure to heavy metals, as it may lead to the acquisition of cancer hallmarks. Zienolddiny et al. [32], using two lines of human lung tumor cells, studied the potential genotoxic effects of nickel sulfate. As a result of the conducted analysis, the authors found that nickel can promote SSRs mutations in the majority of cell line samples. A correlation between heavy metal exposure and MSI has also been evaluated in traditional clinical studies. As one of the aims, Wang et al. [38] conducted such an analysis on 105 recruited patients with pathologically conﬁrmed gastric cancer and 62 health controls. MSI was detected in 18 patients with cancer. The concentration of 13 out of 18 heavy metals differed signiﬁcantly between healthy and cancer patients. Among the trace elements, gallium was found in signiﬁcantly higher amounts in the group of patients with conﬁrmed MSI compared to the patients with stable microsatellites, and only stibium had a positive correlation with MSI. Wang et al. [38] concluded that stibium has the potential to induce MSI in humans with gastric cancer. In another study, Hirose et al. [33] analyzed genomic instability using a panel of six microsatellite loci for chromate-exposed workers with lung cancer. The authors found that an increase in the time of exposure to the chromate was signiﬁcantly associated with a higher frequency of MSI. Their ﬁndings suggest that MSI could be relevant in chromium-induced carcinogenesis. To the best of our knowledge, the effect of any toxic heavy metal including lead on microsatellite instability has not yet been examined for wild bird species. As birds are Animals 2023, 13, 1325 3 of 12 characterized by a short lifespan of red blood cells in general—and in the case of developing nestlings, erythrocytes are being intensively produced from stem cells—the detection of replication errors, including those in microsatellites, seems likely. Therefore, avian blood has been recently considered to be a good matrix to assess genotoxic alterations reﬂecting recent exposure effects [28]. Assuming that lead exposure may cause replication errors in erythrocyte stem cells in the bone marrow of birds, newly emerging erythrocyte lines may show altered microsatellite genotypes as indicators of the MSI. 2. Materials and Methods g ) ( ) exposure to toxic a Study Site, Experiment Layout, Sampling, Isolation, Genotyping, and Statistical Analysis 2. Materials and Methods This experimental study was carried out as part of our long-term research concerning the breeding biology of hole-nesting passerines in central Poland [39]. The study was conducted in accordance with recommendations reported in the ARRIVE guidelines. All performed procedures in the study fulﬁlled the criteria of Polish legislation and were approved by the Local Bioethical Commission for Experiments on Animals, Medical Uni- versity in Lodz (No. 70/ŁB07/2015 and 11/ŁB07/2016) and the Regional Directorate for Environmental Protection (WPNII.6401.13.2016.MS, WPNII.6401.122.2016.KW2). 2. Materials and Methods Study Site, Experiment Layout, Sampling, Isolation, Genotyping, and Statistical Analysis This experimental study was carried out as part of our long-term research concerning the breeding biology of hole-nesting passerines in central Poland [39]. The study was con- ducted in accordance with recommendations reported in the ARRIVE guidelines. All per- formed procedures in the study fulﬁlled the criteria of Polish legislation and were ap- proved by the Local Bioethical Commission for Experiments on Animals, Medical Univer- sity in Lodz (No. 70/ŁB07/2015 and 11/ŁB07/2016) and the Regional Directorate for Envi- ronmental Protection (WPNII.6401.13.2016.MS, WPNII.6401.122.2016.KW2). ( ) During the 2021 breeding season, an experiment was carried out on 20 randomly selected broods of great tit, which included 10 broods from two study sites representing two types of contrasting habitats around Lodz, central Poland (urban park and mature deciduous forest; Figure 1). A more detailed description of the study sites can be found in [40]. ronmental Protection (WPNII.6401.13.2016.MS, WPNII.6401.122.2016.KW2). During the 2021 breeding season, an experiment was carried out on 20 randomly se- lected broods of great tit, which included 10 broods from two study sites representing two types of contrasting habitats around Lodz, central Poland (urban park and mature decid- uous forest; Figure 1). A more detailed description of the study sites can be found in [40]. Figure 1. Study area with the spatial location of the study sites (1—urban parkland; 2—mature deciduous forest). The map of Poland was created with STATISTICA software [41] using the Maps add-on (v. 3.1.85). The map with the pointed study sites was made by applying QGIS 3.16.3 Hannover software (https://www.qgis.org/, accessed on 1 September 2022). Both maps were merged and modiﬁed in GIMP v. 2.10.30 (GIMP Development Team 2022: http://gimp.org, accessed on 1 September 2022). Figure 1. 1. Introduction , x FOR PEER REVIEW 3 of 12 characterized by a short lifespan of red blood cells in general—and in the case of develop- ing nestlings, erythrocytes are being intensively produced from stem cells—the detection of replication errors, including those in microsatellites, seems likely. Therefore, avian Hence, as a novel approach, we decided to verify the possibility of implementing the MSI method to examine the genotoxic effect of lead intoxication in a common hole-nesting species, the great tit Parus major, at the stage of intensive erythropoiesis. For this purpose, we conducted an experiment on nestlings, based on a single supplementation of lead acetate solution, with two dose levels. We applied a set of microsatellite markers to examine and compare the allele size among individuals exposed to the speciﬁed doses of lead and those that were assigned to the control group. Our basic goals are (i) to conduct a preliminary assessment of the application of the MSI analysis for a wild passerine species (the great tit) and (ii) to verify if the selected microsatellite loci could be used as markers of exposure to toxic agents (in this particular case, of exposure to lead). p g y blood has been recently considered to be a good matrix to assess genotoxic alterations reﬂecting recent exposure eﬀects [28]. Assuming that lead exposure may cause replication errors in erythrocyte stem cells in the bone marrow of birds, newly emerging erythrocyte lines may show altered microsatellite genotypes as indicators of the MSI. Hence, as a novel approach, we decided to verify the possibility of implementing the MSI method to examine the genotoxic eﬀect of lead intoxication in a common hole-nesting species, the great tit Parus major, at the stage of intensive erythropoiesis. For this purpose, we conducted an experiment on nestlings, based on a single supplementation of lead ac- etate solution, with two dose levels. We applied a set of microsatellite markers to examine and compare the allele size among individuals exposed to the speciﬁed doses of lead and those that were assigned to the control group. Our basic goals are (i) to conduct a prelim- inary assessment of the application of the MSI analysis for a wild passerine species (the i ) d (ii) if if h l d i lli l i ld b d k f 2. Materials and Methods g ) ( ) exposure to toxic a Study area with the spatial location of the study sites (1—urban parkland; 2—mature deciduous forest). The map of Poland was created with STATISTICA software [41] using the Maps add-on (v. 3.1.85). The map with the pointed study sites was made by applying QGIS 3.16.3 Hannover software (https://www.qgis.org/, accessed on 1 September 2022). Both maps were merged and modiﬁed in GIMP v. 2.10.30 (GIMP Development Team 2022: http://gimp.org, accessed on 1 September 2022). Animals 2023, 13, 1325 4 of 12 The experiment was based on a previously designed and described setup [6], in which the nestlings were intentionally exposed through the gape to a solution of lead (II) acetate trihydrate, in doses which simulated increased levels of lead in the environment but which did not have a lethal effect. From each brood included in this experiment, 6 individuals on the 10th day of their lives were randomly selected and individually banded by using unique numbered aluminum rings. Samples of ca. 10 µL of blood per nestling were collected from the ulnar vain and applied onto FTA MiniCards, which after drying were stored at room temperature ready for the subsequent genetic analyses. Then, four randomly selected nestlings were administered a single dose of lead acetate solution: two of them received a dose of 15 µg Pb/g of body weight and the other two were administered a dose of 30 µg Pb/g of body weight. The remaining two nestlings were treated as controls and received deionized water as a placebo. On the 15th day of the nestlings’ lives, blood was again sampled onto the FTA MiniCards in the same layout. The lead doses used in this study were selected on the basis of our previous research [6,42] and following the relevant literature [43]. It was assumed that the lead doses applied simulated the exposure to lead and corresponded to lead burdens for great tits in areas considered to be highly contaminated (a dose of 30 µg Pb/g body weight) and minorly contaminated (a dose of 15 µg Pb/g body weight) [43]. All individuals included in this experiment were genotyped at ten microsatellite loci (PmaC25, PmaD22, PmaD130, PmaGAn27, PmaGAn30, PmaGAn40, PmaTAGAn71, PmaTAGAn86, PmaTGAn42, and PmaTGAn45), primarily developed for great tits, mainly for the purpose of use in population genetic and evolutionary studies [44]. To extract DNA from the gathered samples, the GeneMATRIX Bio-Trance DNA Pu- riﬁcation Kit was used. 3. Results and Discussion Exposure to widely distributed environmental pollutants, such as lead, adversely affects various groups of organisms. Lead has been proven to have toxic properties in most of its chemical forms, which have a wide range of detrimental effects on the physiological, behavioral, and biochemical functions of animals. Lead triggers deleterious effects on the haematopoietic, vascular, nervous, renal, reproductive, and immune systems, mainly through the increase in oxidative stress [46–53]. In ﬁsh, exposure to lead induces oxidative stress generated through the excessive production of reactive oxygen species (ROS). As a consequence, the neurotoxicity effect may occur, resulting in synaptic damage and the malfunction of neurotransmitters, leading to behavioral and cognitive dysfunction [53]. Lead was also described as an immune- toxicant factor inhibiting the immune response of ﬁsh. According to Dunier [54], Pb decreases haematopoietic activity in the spleen, as well as limiting phagocytic activity and antibody production. In the case of amphibians, several signs of lead intoxication have been reported. Although mostly concerning mortality after exposure [55], some studies have focused on alterations in development. Perez-Coll et al. [56] showed that in Pb-exposed embryos of the toad Bufo arenarum, stunted tails, underdeveloped gills, microcephaly, and increased mortality occurred. In another study, conducted on the tadpole Xenopus laevis [57], lead was also identiﬁed as a detrimental factor responsible for spinal deformities. Compared to other vertebrates, there are fewer toxicological studies on reptiles, par- ticularly those that analyze the toxic effect of lead. Nevertheless, there are some surveys that provide knowledge in this respect. Salice et al. [58] proved that the Western fence lizard Sceloporus occidentalis, when exposed to increased doses of lead, experienced reduced survival and decreased growth and food consumption. Hematological parameters such as haematocrit and haemoglobin concentration decreased signiﬁcantly with increased exposure to lead, which points to the classic signs of anemia that Pb induces. The toxic effects of lead poisoning have been widely studied in mammals. In this regard, studies on humans provide valuable data on lead toxic properties for this group of vertebrates. The main targets for Pb are mammals’ erythrocytes and their precursors, as well as the kidneys and the central and peripheral nervous system [59]. As a consequence, in many cases chronic exposure to lead has been reported to induce anemia and an increase in blood pressure. 2. Materials and Methods g ) ( ) exposure to toxic a In the ﬁrst step, a part of dried blood sample (ca. 2 mm2) was cut out from each FTA MiniCard by using a sterile cutter. Then, exact isolation procedure was conducted in accordance with the manufacturer’s instructions. PCR assay was performed in a 20 µL volume containing 10 µL of DreamTaq PCR MasterMix (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) and 0.5 µL of each primer. A total of 1 µL of the extracted DNA was added to each reaction sample. PCR ampliﬁcations followed the steps and conditions presented in the protocol developed by [44]. Forward primers used in PCR were labeled with 6-FAM ﬂuorescent dye. Microsatellite genotyping was performed in a commercial laboratory (Genomed, Warsaw, Poland). Sizes of the alleles were scored against an internal lane size standard (Genescan TM 600 LIZ, v2.0, Applied Biosystems, Foster City, CA, USA) by applying Geneious v11.1.5 software (Biomatters Ltd., Auckland, New Zeland). To examine any mistyped allele sizes and scoring errors due to large allele dropout or stuttering (i.e., the failure to discriminate similar-sized alleles in genotypes due to PCR artifacts), the Micro-Checker 2.2.3 software was used [45]. While for all individuals, most loci gave consistent and successful genotyping, three of them (PmaGAn30, PmaTAGAn86, and PmaTGAn45) were unreadable, mostly due to ampliﬁcation problems. Consequently, we retained a panel of seven out of ten microsatellite loci (Table 1) in the further analyses. Table 1. Summary of seven microsatellite loci characteristics for the great tits, including number of individuals (N), allele size ranges, motif of cloned alleles, and primer sequences. Table 1. Summary of seven microsatellite loci characteristics for the great tits, including number of individuals (N), allele size ranges, motif of cloned alleles, and primer sequences. Locus N Size Range (bp) Motif of Cloned Allele Primer Sequences PmaD130 119 376–452 (TAGA)17 F◦:TGAGTGGAAAGATGCTGGC R◦:CCCTATAAAAACCGAGGCTG PmaC25 120 303–330 (CAT)11 F◦:CGTCCTGCTGTTTGTATTTCTG R◦:CCATGAACCATTTTTAGGGTG PmaD22 120 376–448 (CTAT)15(CCAT)12 F◦:GATCAGAGCTTGCCTCAACAC R◦:TCTGGGCTGAAATACCTACCC PmaGAn27 120 198–253 (CAT)16 F◦:TATAAACCACAGCCACACGC R◦:CACAACCACAGAGGCATGAG PmaGAn40 120 406–422 (GA)10 F◦:CGTTCCTCCTTTGCTTTCTG R◦:AATGGCACAACACCTTCTCC Size Range (bp) Motif of Cloned Allele Primer Sequences 5 of 12 Animals 2023, 13, 1325 Table 1. Cont. Locus N Size Range (bp) Motif of Cloned Allele Primer Sequences PmaTAGAn71 120 173–209 (TAGG)6(TAGA)11 F◦:TCAGCCTCCAAGGAAAACAG R◦:GCATAAGCAACACCATGCAG PmaTGAn42 120 260–292 (TCCA)15 F◦:ACTTCCACATGCCAGTTTCC R◦:TGTTAAGGCAGAGAGGTGGG F: forward primer; R: reverse primer. Table 1. Cont. F: forward primer; R: reverse primer. Statistical analyses were performed using STATISTICA 13.0 software [41]. The results are reported as means ± standard error (S.E.). 2. Materials and Methods g ) ( ) exposure to toxic a Since all data did not meet the assumption of normal distribution, the Mann–Whitney U test was applied to examine the overall differences in allele sizes for each of the seven microsatellite loci of the same nestlings sampled and genotyped twice. These analyses were conducted separately for individuals assigned to one of the three experimental groups. Statistical signiﬁcance was deﬁned as p ≤0.05. 3. Results and Discussion High levels of lead were recorded as a signiﬁcant factor of severe damage to the brain and kidneys, regardless of age. Lead toxicity manifested also in the alteration of reproduction. Long-time exposure to high doses of lead caused miscarriages in pregnant woman, the low birth weights of infants, and reduced fertility in males. Intense exposure to lead also affects the central and peripheral nervous system, giving the symptoms of short-term memory loss, reduced concentration, depression, headaches, numbness, tingling Animals 2023, 13, 1325 6 of 12 in the arms and legs, and problems with coordination [60]. The neurotoxic effects of lead were also recorded in rats and monkeys exposed to lead during the prenatal, post-natal, and adult life stages. Studies on small mammals such as the bank vole, wood mouse, and shrews found that lead intoxication induced kidney damage [61]. Among all vertebrates, birds are pointed out as the most studied and very likely most affected taxon in regard to lead poisoning as an effect of exposure to contaminated food, mainly by lead shot pellets and/or lead ﬁshing sinkers [6,7]. The character and intensity of clinical signs observed in lead-intoxicated birds notably depend on the degree of exposure, with the time and quantity of absorbed lead being crucial [3]. Accordingly, typical symptoms of chronic exposure to lead include anemia, weakness, ataxia, weight loss, visible difﬁculties in ﬂying and walking, vomiting and green diarrhea, and convulsions [6,7,62]. According to Pain et al. [7], acute poisoning with high doses of lead can even induce lethal effects for birds without manifesting any of the mentioned signs of its toxicity. Toxicity of lead has been also investigated with regard to its mutagenic, clastogenic, and carcinogenic properties. However, the gathered data are still equivocal and many studies present contradictory conclusions. Moreover, they mostly concern human, animal, or cell-line studies conducted under clinical and/or laboratory conditions [8,63,64]. In this regard, there is a noticeable paucity of studies that concern investigation of the genotoxic effects of lead on wild living birds. We are aware of only a few that have analyzed the adverse effect of the heavy metal toxic spill from the Aznalcollar mine (southwestern Spain) on populations of two bird species that occupy a high level in the food chain, namely, the white stork Ciconia ciconia and black kite Milvus migrans [16–19]. 3. Results and Discussion In these studies, the authors applied the comet assay and showed that individuals of both species from the contaminated sites were characterized by an increased level of DNA damage measured in their lymphocytes in comparison to birds tested at the reference sites, although some inter- and intra-species differences were noted. According to Baos et al. [19], lead was not signiﬁcantly related to DNA damage in either nestling white storks or black kites during the conducted study. Presumably, this was caused by the low number of white stork and black kite nestlings exposed to doses of lead responsible for sublethal effects, 0.4% and 2.3%, respectively. In general, the paucity of bird studies that directly concern the detection and examination of genetic alterations induced by a single selected trace element such as lead offers an opportunity to ﬁll this gap and provide new knowledge in this respect. In this study, we did not ﬁnd any effect of experimental lead intoxication on genomic instability in nestling great tits by using peripheral blood erythrocytes. We found no change in allele sizes (microsatellite instability, MSI) in any of the seven tested loci (PmaC25, PmaD22, PmaD130, PmaGAn27, PmaGAn40, PmaTAGAn71, and PmaTGAn42) in the great tit nestlings that received a dose of 15 µg Pb/g of body weight (Figure 2; Supplementary Table S2; Table 2), nor in nestlings with a dose of 30 µg Pb/g of body weight (Figure 3; Supplementary Table S3; Table 2). Analogously, no alterations in allele sizes were observed for individuals from the control group (Figure 4; Supplementary Table S1; Table 2). Table 2. Results of the Mann–Whitney U test for the comparison of the mean allele sizes of SSRs genotyped twice for the studied individuals, with respect to the dose at which nestlings were supplemented with lead. Table 2. Results of the Mann–Whitney U test for the comparison of the mean allele sizes of SSRs genotyped twice for the studied individuals, with respect to the dose at which nestlings were supplemented with lead. 3. Results and Discussion Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. To our best knowledge, this is the ﬁrst case of an experimental study with a novel h t l MSI d t ti t l f ti lt ti i ild l ti f Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Birds age (days) for which SSRs were analyzed Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. Figure 2. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight. To our best knowledge, this is the ﬁrst case of an experimental study with a novel approach to analyze MSI as a detection tool for genetic alterations in a wild population of birds. The lack of bird studies on the application of the microsatellite instability assay to test the genotoxicity of heavy metals makes direct comparison of our results impossible. 3. Results and Discussion Dose of Lead (µg/g of Body Mass) Locus (Two Alleles) Control 15 30 PmaD130 Z = 0.013; p = 0.99 Z = 0.007; p = 0.99 Z = −0.007; p = 0.99 PmaD130 Z = -0.092; p = 0.93 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 PmaC25 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 PmaC25 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 PmaD22 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = 0.226; p = 0.82 PmaD22 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = 0.000; p = 1.00 PmaGAn27 Z = 0.005; p = 1.00 Z = 0.000; p = 1.00 Z = 0.005; p = 1.00 Dose of Lead (µg/g of Body Mass) Animals 2023, 13, 1325 7 of 12 0.99 = 1.00 Table 2. Cont. PmaD Table 2. Cont. 3. Results and Discussion Dose of Lead (µg/g of Body Mass) Locus (Two Alleles) Control 15 30 PmaGAn27 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaGAn40 Z = 0.006; p = 1.00 Z = −0.006; p = 1.00 Z = 0.006; p = 1.00 PmaGAn40 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTAGAn71 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTAGAn71 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTGAn42 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTGAn42 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaD22 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = 0.226; p = 0.82 PmaD22 Z = 0.005; p = 1.00 Z = 0.005; p = 1.00 Z = 0.000; p = 1.00 PmaGAn27 Z = 0.005; p = 1.00 Z = 0.000; p = 1.00 Z = 0.005; p = 1.00 PmaGAn27 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaGAn40 Z = 0.006; p = 1.00 Z = −0.006; p = 1.00 Z = 0.006; p = 1.00 PmaGAn40 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTAGAn71 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTAGAn71 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTGAn42 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 PmaTGAn42 Z = 0.005; p = 1.00 Z = −0.005; p = 1.00 Z = 0.005; p = 1.00 Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 2. 3. Results and Discussion Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Figure 4. 3. Results and Discussion However, we think that referring to some other study systems can be helpful in under- standing our negative results [31,35]. Although the selected microsatellite markers did not show any signs of signiﬁcant response to lead exposure (i.e., alterations in allele sizes), we still believe that such an effect is possible. At the same time, we think that the concept and approach of this study generally may direct attention to the microsatellite instability assay as a molecular technique that is potentially applicable in ecotoxicological bird studies. Animals 2023, 13, 1325 Animals 2023, 13, x FO Animals 2023, 13, x FOR 8 of 12 8 of 12 8 of 12 Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Locus PmaD130 PmaD130 PmaC25 PmaC25 PmaD22 PmaD22 PmaGAn27 PmaGAn27 PmaGAn40 PmaGAn40 PmaTAGAn71 PmaTAGAn71 PmaTGAn42 PmaTGAn42 10 15 Birds age (days) for which SSRs were analyzed 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 Allele sizes (mean ± S.E.) Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Birds age (days) for which SSRs were analyzed Birds age (days) for which SSRs were analyzed Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Figure 3. 3. Results and Discussion Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Birds age (days) for which SSRs were analyzed S E ) d d f SSR t d t i i Birds age (days) for which SSRs were analyzed Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5 day interval for nestlings, which were assigned to the control group. Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Figure 4. Allele sizes (mean ± S.E.) recorded for SSRs genotyped twice in 5-day interval for nestlings, which were assigned to the control group. Animals 2023, 13, 1325 9 of 12 9 of 12 We believe that the most likely reason for our negative experimental result was that the single doses of lead acetate administered to nestlings were too low to initiate any of the indirect mechanisms that could exert any modiﬁcation in the sizes of the SSRs we analyzed. Even though the biochemical and molecular mechanisms underlying the genotoxicity of lead are not yet fully elucidated, it has been reported that lead induces a genotoxic effect throughout indirect mechanisms, such as the inhibition of DNA repair, the excess production of free radicals contributing to DNA damage, and the regulation of suppressor and promotor genes in carcinogenesis [8,12]. Such conclusions were assumed in one of the experimental studies conducted by [35], in which MSI was evaluated using three microsatellite loci located within the genome of rats (Rattus norvegicus) exposed to various heavy metal compounds. The authors detected MSI in all of the loci tested for the rats exposed both to a single and to multiple doses of lead acetate and arsenic trioxide used in the study. El-Ghor et al. [35] suggested that oxidative stress or defects in mismatch repair genes could play a pivotal role in the genotoxicity of the metal compounds used, including lead acetate. The study by El-Ghor et al. [35] is the only study published to date to analyze MSI in a vertebrate species under exposure to lead acetate. The explanation of our result also seems to be reasonable because of another study, [31], which is conceptually and methodologically similar to [35], except for a cadmium compound being used instead of lead. El-Ghor et al. 3. Results and Discussion [31] found that in the case of single exposure to cadmium chloride, no microsatellite instability was observed in the three tested loci. In contrast, for rats that received multiple oral doses of cadmium, MSIs were recorded in three of the studied loci. To explain these results, the authors also invoke indirect mechanisms that may induce genotoxicity, in particular a mechanism based on cadmium chloride being able to inhibit DNA-mismatch repair. We are aware that, apart from the fact that the single doses of lead used in this study may have been too weak to induce genetic instability, some other issues should also be considered in ﬁnding an explanation for our result. First, the panel of microsatellite markers selected to determine MSI could be unsusceptible to lead genotoxicity in general. Second, the relatively short time interval (5 days) between the experimental lead exposure and the post-exposure blood sampling for genetic analyses could have largely limited the effect of lead genotoxicity. Consequently, our results indicate the need for further, more complex research to investigate whether the single doses of lead acetate as used in this study were too low to induce genetic instability and/or whether a selected panel of microsatellite markers characterize susceptibility to lead genotoxicity in general. In further research, blood lead levels should also be investigated to obtain additional signiﬁcant data about lead’s toxic effects and to examine how blood lead levels change with exposure time. Supplementary Materials: The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/ani13081325/s1, Table S1: Allele sizes recorded for SSRs genotyped twice in 5-day interval for nestlings assigned to the control group; Table S2: Allele sizes recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 15 µg Pb/g of body weight; Table S3: Allele sizes recorded for SSRs genotyped twice in 5-day interval for nestlings, which were supplemented with a dose of 30 µg Pb/g of body weight. Author Contributions: Conceptualization, M.M. and J.B.; methodology, M.M.; formal analysis, M.M.; investigation, M.M., A.K., J.W., M.G., J.S. and J.B.; resources, M.M.; writing—original draft preparation, M.M. and J.B.; writing—review and editing, M.M., J.B., A.K., J.W., M.G. and J.S.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript. Funding: The study was funded by the National Science Centre in Poland (grant no. DEC 2019/03/X/NZ8/01766). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the Supplementary Materials. Acknowledgments: We would like to thank E. Wróblewska, A. Jaksa, D. Ma´nkowska, and M. Janiszewska for their help and consent to conduct the study in the areas under their administration. We would also like to express our thanks to P. Minias for enabling us to use his lab as well as for his helpful comments. We are obliged to P. Procter for linguistic consultation. Conﬂicts of Interest: The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appeared to affect the work reported in this paper. References 1. Franson, J.C.; Pain, D. Lead in birds. 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https://openalex.org/W2567961709	https://europepmc.org/articles/pmc5225314?pdf=render	English	null	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion	MBio	2,017	cc-by	8,973	RESEARCH ARTICLE crossm RESEARCH ARTICLE Whole-Genome Sequencing of Invasion- Resistant Cells Identiﬁes Laminin 2 as a Host Factor for Bacterial Invasion Xander M. van Wijk,a Simon Döhrmann,b Björn M. Hallström,c,d Shangzhong Li,b,g,h Bjørn G. Voldborg,c Brandon X. Meng,a Karen K. McKee,e Toin H. van Kuppevelt,f Peter D. Yurchenco,e Bernhard O. Palsson,c,g,h Nathan E. Lewis,b,h Victor Nizet,b Jeffrey D. Eskoa Xander M. van Wijk,a Simon Döhrmann,b Björn M. Hallström,c,d Shangzhong Li,b,g,h Bjørn G. Voldborg,c Brandon X. Meng,a Karen K. McKee,e Toin H. van Kuppevelt,f Peter D. Yurchenco,e Bernhard O. Palsson,c,g,h Nathan E. Lewis,b,h Victor Nizet,b Jeffrey D. Eskoa Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USAa; Department of Pediatrics, University of California, San Diego, La Jolla, California, USAb; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmarkc; Royal Institute of Technology, Stockholm, Swedend; Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey, USAe; Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlandsf; Department of Bioengineering, University of California, San Diego, La Jolla, California, USAg; Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, La Jolla, California, USAh ABSTRACT To understand the role of glycosaminoglycans in bacterial cellular inva- sion, xylosyltransferase-deﬁcient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated pre- viously using chemical mutagenesis, an unexpected result was obtained. Bacterial in- vasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was pres- ent in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit 2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with hu- man LAMA2 cDNA signiﬁcantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-211/laminin-221 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvas- cular endothelial cells. Thus, the L4a domain in laminin 2 is important for cellular invasion by a number of bacterial pathogens. January/February 2017 Volume 8 Issue 1 e02128-16 Whole-Genome Sequencing of Invasion- Resistant Cells Identiﬁes Laminin 2 as a Host Factor for Bacterial Invasion Received 21 November 2016 Accepted 1 December 2016 Published 10 January 2017 Citation van Wijk XM, Döhrmann S, Hallström BM, Li S, Voldborg BG, Meng BX, McKee KK, van Kuppevelt TH, Yurchenco PD, Palsson BO, Lewis NE, Nizet V, Esko JD. 2017. Whole-genome sequencing of invasion-resistant cells identiﬁes laminin α2 as a host factor for bacterial invasion. mBio 8:e02128-16. https://doi.org/ 10.1128/mBio.02128-16. Received 21 November 2016 Accepted 1 December 2016 Published 10 January 2017 Cit ti Wijk XM Döh S H ll t ö Invited Editor Pyong Park, Childrens Hospital, Boston ® mbio.asm.org 1 RESULTS Bacterial invasion in XylT2 mutants generated by CRISPR-cas9 and in pgsA745 cells differs. Bacterial invasion of cells contributes to penetration of host barriers, a hallmark of pathogenicity, and provides an intracellular niche for bacterial survival and proliferation. To examine the role of GAGs in bacterial invasion, we inactivated Xylt2 in CHO-K1 cells using the CRISPR-cas9 system. Sequencing showed Xylt2 frameshift mu- tations in clonal lines 23A1 and 93A5, respectively, but not in control clonal lines 23A6 and 93A1 isolated from the same targeted cell pool (see Fig. S1 in the supplemental material). Inactivation of Xylt2 markedly reduced cell surface expression of HS as determined by ﬂow cytometry using the single-chain variable-fragment (scFv) antibody HS4C3 (Fig. 1a) and by the binding of an HS-dependent growth factor, ﬁbroblast growth factor 2 (FGF2) (Fig. 1b). Invasion of GBS was much lower in the Xylt2 mutants (Fig. 1c), in agreement with previous studies of mutant pgsA745 cells (9), which also carry a loss-of-function allele of Xylt2 (6). Group A Streptococcus (GAS) and Staphylo- coccus aureus can also bind to GAGs (10, 11), but their invasion was not compromised in the new XylT2 knockouts, suggesting that interaction with GAGs is not required for invasion (Fig. 1c). In contrast, invasion by all three pathogens was clearly reduced in strain pgsA745 (Fig. 1c), but there was no difference in invasion by methicillin-resistant S. aureus (MRSA) in wild-type and pgsA745 cells or CRISPR-cas9 control and knockout cells (data not shown). Stable transfection of pgsA745 cells with Xylt1 or Xylt2 cDNAs restored cell surface expression of HS (see Fig. S2a) but did not restore bacterial invasion (Table 1; see also Fig. S2b). Based on the resistance of XylT2 mutants derived by CRISPR-cas9, we concluded that GAGs are necessary for invasion by GBS but not by GAS or S. aureus. This is consistent with our previous observation that HS is important for the invasion of GBS in brain microvascular endothelial cells (2). The lack of resto- ration of GBS invasion in pgsA745 cells by transfection with xylosyltransferase led us to conclude that pgsA745 cells harbor an additional defect that alters susceptibility to infection by multiple bacterial species. Adhesion and endocytosis is normal in pgs745 cells. The ﬁrst event in bacterial invasion of cells requires attachment of the bacteria to adhesins on host cells. Invited Editor Pyong Park, Childrens Hospital, Boston Editor Gerald B. Pier, Harvard Medical School Copyright © 2017 van Wijk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Jeffrey D. Esko, jesko@ucsd.edu. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, lead- ing to invasion of epithelial and endothelial cells. Here, we show that cellular inva- sion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a speciﬁc domain of the laminin 2 subunit. This ﬁnding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs. G l s G lycosaminoglycans (GAGs) are long, polyanionic polysaccharides present on the surface of virtually all animal cells and in the extracellular matrix. GAGs, and in particular heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS), are involved in cellular adhesion and invasion by multiple pathogens (1). This long list of ® mbio.asm.org 1 ® mbio.asm.org 1 January/February 2017 Volume 8 Issue 1 e02128-16 ® van Wijk et al. pathogens includes viruses like herpes simplex virus, human immunodeﬁciency virus, and hepatitis C virus and bacteria like Listeria monocytogenes and Neisseria gonorrhoeae. We reported previously a role for GAGs in endothelial cell invasion by group B Streptococcus (GBS) during its penetration of the blood-brain barrier (2). The biosynthesis of HS and CS/DS starts with the formation of a linkage tetrasac- charide (xylose-galactose-galactose-glucuronic acid) attached to speciﬁc serine resi- dues in a small number of proteoglycan core proteins. Chinese hamster ovary (CHO) cell mutants deﬁcient in xylosyltransferase 2 (Xylt2), galactosyltransferase I (4galt7), and glucuronosyltransferase I (3gat3) were generated previously by chemical mutagenesis (3–5). The pgsA745 cell line harbors a nonsense mutation in Xylt2 (6), completely lacks HS and CS/DS, and has been used by many laboratories to assess the role of GAGs in various processes, including adhesion and invasion by pathogens (7). Genome editing has been simpliﬁed greatly by the introduction of the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) system (8). Here, we created new Xylt2-deﬁcient CHO mutants using this system and examined invasion by multiple bacterial pathogens. A discrepancy in infectivity became apparent when comparing bacterial invasion in pgsA745 cells to that in the new Xylt2-deﬁcient mutants. Invited Editor Pyong Park, Childrens Hospital, Boston Whole-genome sequencing and transcriptome sequencing (RNA-Seq) revealed that pgsA745 cells also contain a deletion in the gene encoding laminin subunit 2 (Lama2), which diminished bacterial invasion. The dele- tion removes much of domain L4a in the laminin 2 subunit, demonstrating the importance of this region in invasion by multiple bacterial species. January/February 2017 Volume 8 Issue 1 e02128-16 RESULTS Adhesion was unaffected in pgsA745 cells (Table 1) before and after transfection with Xylt, suggesting that bacterial resistance was due to a downstream factor involved in bacterial entry. A higher susceptibly of pgsA745 cells to cell death due to infection January/February 2017 Volume 8 Issue 1 e02128-16 mbio.asm.org 2 ® Laminin 2 Is a Host Receptor for Bacterial Invasion FIG 1 Resistance of pgsA745 cells to GAS and MSSA invasion does not correlate with lack of heparan sulfate expression. (a) Cell surface expression of heparan sulfate was reduced in Xylt2 knockout clonal lines 23A1 and 93A5 generated by CRISPR-cas9 targeting, as measured by ﬂow cytometry using scFv antibody HS4C3. (b) Binding of biotinylated FGF2 to cell surface heparan sulfate was affected similarly in the mutants. (c) Invasion by GBS was reduced in pgsA745 cells and Xylt knockout clonal lines 23A1 and 93A5. In contrast, GAS and MSSA invasion was altered in pgsA745 cells but normal in 23A1 and 93A5 cells compared to the levels of invasion in control clones 23A6 and 93A1. , P 0.05, *, P 0.001, and ns, not signiﬁcant versus results for wild-type cells using the two-tailed t test. Error bars indicate standard deviations (SD); n 3. FIG 1 Resistance of pgsA745 cells to GAS and MSSA invasion does not correlate with lack of heparan sulfate expression. (a) Cell surface expression of heparan sulfate was reduced in Xylt2 knockout clonal lines 23A1 and 93A5 generated by CRISPR-cas9 targeting, as measured by ﬂow cytometry using scFv antibody HS4C3. (b) Binding of biotinylated FGF2 to cell surface heparan sulfate was affected similarly in the mutants. (c) Invasion by GBS was reduced in pgsA745 cells and Xylt knockout clonal lines 23A1 and 93A5. In contrast, GAS and MSSA invasion was altered in pgsA745 cells but normal in 23A1 and 93A5 cells compared to the levels of invasion in control clones 23A6 and 93A1. , P 0.05, **, P 0.001, and ns, not signiﬁcant versus results for wild-type cells using the two-tailed t test. Error bars indicate standard deviations (SD); n 3. could falsely lower bacterial invasion levels, as the assay relies on the recovery of intracellular bacteria protected from membrane-impermeant antibiotics. However, the release of lactate dehydrogenase (LDH), a marker of cell lysis, did not differ between wild-type and pgsA745 cells, before or after infection and in the presence or absence of antibiotics (Fig. RESULTS (c) Intracellular bacterial growth/survival was unaltered in pgsA745 cells as measured by CFU count after 90 and 210 min of antibiotic protection. Bacterial counts at t 90 min were set to 100%. Error bars indicate SD; n 3. (d) Endocytosis and phagocytosis of ﬂuorochrome-labeled dextran, transferrin, zymosan, or beads were unaffected in pgsA745 cells as measured by ﬂow cytometry. (e) Actin relocalization probed using EPEC infection was unaltered in pgsA745 cells. Arrows point to typical actin “pedestals” induced by EPEC. Scale bar 20 m. defect in the host (see Fig. S2d). pgsA745 and wild-type cells also did not differ in extracellular and intracellular bacterial growth/survival (Fig. 2b and c). A general defect of pgsA745 cells in endocytosis/phagocytosis was also excluded, as uptake of ﬂuorochrome-labeled markers of macropinocytosis (dextran), clathrin-mediated endo- cytosis (transferrin), and phagocytosis (yeast cell wall zymosan and latex beads) was unaffected (Fig. 2d). To assess actin remodeling, a ﬁnal step in bacterial invasion (12), we infected the cells with enteropathogenic Escherichia coli (EPEC), which causes easily distinguishable actin pedestals (13). Similar numbers of actin pedestals were observed in response to EPEC infection of both wild-type and pgsA745 cells (Fig. 2e). Bacteria also exploit integrins for host invasion (14), but cell surface integrin expression as deter- mined by ﬂow cytometry appeared normal (see Fig. S3), with the exception of integrin 6 and 7 expression, which depended on GAG expression (i.e., integrin 6 and 7 expression is lost in pgsA745 cells but is restored in pgsA745-XylT cells). The reexpres- sion of these integrins in pgsA745-XylT cells did not restore susceptibility to infection, indicating that these integrins do not play a role in infection. pgsA745 cells contain a large deletion in Lama2. To identify the cause of reduced bacterial invasion in pgsA745 cells, we sequenced the entire genome and compared it to the sequence of a reference wild-type CHO genome. We also analyzed the entire transcriptome of pgsA745 cells by RNA-Seq and compared it to the transcriptomes of wild-type and pgsA745-XylT cells (see Data set S1 in the supplemental material). Numerous heterozygous mutations were present, most likely due to chemical mu- tagenesis and accumulation of mutations over time between the reference genome and the mutant, but none of these resulted in genetic changes deemed likely to affect infection. RESULTS 2a). In addition, an alternative bacterial invasion assay based on ﬂow cytometry also showed reduced bacterial invasion in pgsA745 cells with either live (see Fig. S2c in the supplemental material) or heat-killed GBS, indicating a cell-autonomous TABLE 1 Xylosyltransferase transfection does not restore bacterial invasion Bacterium (serotype) Mean CFU SD (%)a Wild-type cells pgsA745 cells pgsA745-XylT cells Adhesion Invasion Adhesion Invasion Adhesion Invasion GBS (III) 100 31 100 24 100 36 22 2b 122 30 17 3b GBS (Ia) 100 23 100 19 80 20 28 12c 107 12 22 14c GAS (M1T1) 100 19 100 14 109 10 16 5c 98 16 16 1c MSSA 100 29 100 6 104 25 27 8c 99 20 28 9c aPercentage of the results for wild-type cells; n 4 to 6. bP 0.001 versus wild type (two-tailed t test). cP 0.0001 versus Wild type (two-tailed t test). January/February 2017 Volume 8 Issue 1 e02128-16 TABLE 1 Xylosyltransferase transfection does not restore bacterial invasion mbio.asm.org 3 ® van Wijk et al. FIG 2 Viability, bacterial growth rates, endocytosis/phagocytosis, and actin relocalization are normal in pgsA745 cells. (a) LDH release was not affected by infection or treatment with antibiotics in pgsA745 cells. Error bars indicate SD; n 4. (b) Bacterial growth rate in pgsA745 cells was unaffected. Error bars indicate SD; n 3. (c) Intracellular bacterial growth/survival was unaltered in pgsA745 cells as measured by CFU count after 90 and 210 min of antibiotic protection. Bacterial counts at t 90 min were set to 100%. Error bars indicate SD; n 3. (d) Endocytosis and phagocytosis of ﬂuorochrome-labeled dextran, transferrin, zymosan, or beads were unaffected in pgsA745 cells as measured by ﬂow cytometry. (e) Actin relocalization probed using EPEC infection was unaltered in pgsA745 cells. Arrows point to typical actin “pedestals” induced by EPEC. Scale bar 20 m. FIG 2 Viability, bacterial growth rates, endocytosis/phagocytosis, and actin relocalization are normal in pgsA745 cells. (a) LDH release was not affected by infection or treatment with antibiotics in pgsA745 cells. Error bars indicate SD; n 4. (b) Bacterial growth rate in pgsA745 cells was unaffected. Error bars indicate SD; n 3. January/February 2017 Volume 8 Issue 1 e02128-16 mbio.asm.org 4 RESULTS However, a large deletion in the gene encoding the extracellular matrix protein laminin subunit 2 (Lama2) was identiﬁed by both methods. The deleted region consisted of 60 kbp at the genomic level, covering exons 9 to 16 (Fig. 3a; note that only the ﬁrst 28 of 61 exons are shown). This genomic deletion resulted in a corresponding loss of 1,074 bases (out of 9,558 bp) in the mRNA and 358 amino acids in a region that includes domains L4a and LEb of the laminin 2 chain (15). Interest- ingly, the number of RNA reads in the RNA-Seq data was reduced by approximately 50% in this region in the wild-type cells compared to the number of reads for other exons (Fig. 3a). Therefore, we concluded that parental CHO-K1 cells are heterozygous with respect to the deletion, or short allele, and heterozygosity was lost in pgsA745 cells. In other words, parental cells contain a long and a short form of Lama2, whereas January/February 2017 Volume 8 Issue 1 e02128-16 mbio.asm.org 4 ® Laminin 2 Is a Host Receptor for Bacterial Invasion FIG 3 RNA-Seq reveals a loss of 8 exons in Lama2 in pgsA745 cells. (a) RNA-Seq proﬁle of Lama2 showing exon reads in blue and exon-intron junctions in red. Note that only the ﬁrst 28 of 61 exons are shown. (b) pgsA745 cells express wild-type amounts of total (short long) Lama2 transcripts but lack expression of long Lama2 transcripts. Error bars indicate SD; n 3. (c) Immunocytochemistry using antibody 5H2 shows laminin 2 expression in both wild-type and pgsA745 cells. Scale bar 20 m. (d) Expression of the different laminin (Lama), (Lamb), and (Lamc) units as expressed in fragments per kilobase of transcript per million mapped reads (FPKM) measured by RNA-Seq. Error bars indicate SD; n 2. FIG 3 RNA-Seq reveals a loss of 8 exons in Lama2 in pgsA745 cells. (a) RNA-Seq proﬁle of Lama2 showing exon reads in blue and exon-intron junctions in red. Note that only the ﬁrst 28 of 61 exons are shown. (b) pgsA745 cells express wild-type amounts of total (short long) Lama2 transcripts but lack expression of long Lama2 transcripts. Error bars indicate SD; n 3. (c) Immunocytochemistry using antibody 5H2 shows laminin 2 expression in both wild-type and pgsA745 cells. Scale bar 20 m. January/February 2017 Volume 8 Issue 1 e02128-16 RESULTS (d) Expression of the different laminin (Lama), (Lamb), and (Lamc) units as expressed in fragments per kilobase of transcript per million mapped reads (FPKM) measured by RNA-Seq. Error bars indicate SD; n 2. pgsA745 cells contain only the short form. By calculating the read depth and percent- age of homozygosity for all DNA scaffolds of 100 kbp and by comparing the results to those of six other CHO genomes (16), including CHO-K1, we found that 45 of approximately 300 scaffolds with 80% homozygosity appeared to be uniquely mono- somic in pgsA745 cells. All 45 scaffolds mapped to either chromosome 2 or X of the Chinese hamster genome, suggesting partial monosomy of chromosome 2 and X for pgsA745. To conﬁrm the Lama2 deletion in pgsA745, we developed quantitative PCR (qPCR) primers speciﬁcally recognizing the long form of Lama2 and primers recognizing both the short and the long form. As expected, we did not observe expression of the long form in pgsA745 cells (Fig. 3b). Nevertheless, the short form of laminin 2 protein was expressed normally in the mutant, and immunocytochemistry showed extracellular localization similar to that in wild-type cells (Fig. 3c). Laminin 2 is important for bacterial invasion. Laminin consists of a trimer of different isoforms of , , and subunits. Inspection of the expression data from the RNA-Seq analysis showed that CHO cells express primarily subunits 2, 5, 1, 2, 1, and to a lesser extent, 2 (Fig. 3d). To assess whether bacteria bind to laminin, we mixed ﬂuorescently labeled laminin-211/221 (laminin-211/laminin-221) with different bacteria: two serotypes of GBS, GAS, S. aureus (methicillin-sensitive S. aureus [MSSA] and MRSA). Binding was measured using ﬂow cytometry gated on the bacteria. In all cases, there was a clear shift in ﬂuorescence after the addition of the labeled laminin, indicating laminin binding (Fig. 4a). In the second method, we noted signiﬁ- cantly enhanced binding of bacteria to a microtiter plate coated with laminin-211/221 (Fig. 4b). The addition of laminin-211/221 to a cellular assay also strongly enhanced the mbio.asm.org 5 January/February 2017 Volume 8 Issue 1 e02128-16 ® van Wijk et al. FIG 4 Laminin-211/221 facilitates bacterial invasion. (a) Flow cytometry results show that FITC-labeled laminin-211/221 binds to different bacteria. (b) FITC-labeled bacteria adhere to laminin-211/221-coated plates. RFU, relative ﬂuorescent units. RESULTS , P 0.05, and , P 0.01 versus the results for uncoated plates using a two-tailed t test. Error bars indicate SD; n 3 to 6. (c and d) Bacterial invasion is strongly enhanced in the presence of 5-g/ml laminin-211/221 in wild-type and pgsA745 cells (c), and this increase is independent of removal of HS by heparinase III (HepIII) (d). Removal of HS by heparinase III (HepIII) in wild-type cells reduced bacterial invasion without addition of laminin-211/221. (c) , P 0.001, and *, P 0.0001 versus the results for no addition of laminin-211/221 using the two-tailed t test. (d) , P 0.001 versus the results for untreated wild-type cells using a two-tailed t test. Error bars indicate SD; n 3 or 4. (e) Preincubation of GBS with 50-g/ml laminin-211/221 enhanced invasion in wild-type and pgsA745 cells. , P 0.05, and #, P 0.05 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (f) Bacterial invasion is strongly enhanced in the presence of 10-g/ml laminin-211/221 in human brain microvascular endothelial cells (hBMEC) and human alveolar epithelial cells (A549). , P 0.01, and , P 0.001 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (g) siRNA knockdown of the expression of long Lama2 reduces bacterial invasion. Inset, silencing of long Lama2 by siRNA reduces expression of long Lama2 transcripts relative to their expression when using negative-control siRNA (NC) or the buffer control (leftmost bar). , P 0.001, and *, P 0.0001 versus the results for buffer-treated cells using the two-tailed t test. Error bars indicate SD; n 3. FIG 4 Laminin-211/221 facilitates bacterial invasion. (a) Flow cytometry results show that FITC-labeled laminin-211/221 binds to different bacteria. (b) FITC-labeled bacteria adhere to laminin-211/221-coated plates. RFU, relative ﬂuorescent units. , P 0.05, and , P 0.01 versus the results for uncoated plates using a two-tailed t test. Error bars indicate SD; n 3 to 6. (c and d) Bacterial invasion is strongly enhanced in the presence of 5-g/ml laminin-211/221 in wild-type and pgsA745 cells (c), and this increase is independent of removal of HS by heparinase III (HepIII) (d). RESULTS Removal of HS by heparinase III (HepIII) in wild-type cells reduced bacterial invasion without addition of laminin-211/221. (c) , P 0.001, and *, P 0.0001 versus the results for no addition of laminin-211/221 using the two-tailed t test. (d) , P 0.001 versus the results for untreated wild-type cells using a two-tailed t test. Error bars indicate SD; n 3 or 4. (e) Preincubation of GBS with 50-g/ml laminin-211/221 enhanced invasion in wild-type and pgsA745 cells. , P 0.05, and #, P 0.05 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (f) Bacterial invasion is strongly enhanced in the presence of 10-g/ml laminin-211/221 in human brain microvascular endothelial cells (hBMEC) and human alveolar epithelial cells (A549). , P 0.01, and , P 0.001 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (g) siRNA knockdown of the expression of long Lama2 reduces bacterial invasion. Inset, silencing of long Lama2 by siRNA reduces expression of long Lama2 transcripts relative to their expression when using negative-control siRNA (NC) or the buffer control (leftmost bar). , P 0.001, and *, P 0.0001 versus the results for buffer-treated cells using the two-tailed t test. Error bars indicate SD; n 3. FIG 4 Laminin-211/221 facilitates bacterial invasion. (a) Flow cytometry results show that FITC-labeled laminin-211/221 binds to different bacteria. (b) FITC-labeled bacteria adhere to laminin-211/221-coated plates. RFU, relative ﬂuorescent units. , P 0.05, and , P 0.01 versus the results for uncoated plates using a two-tailed t test. Error bars indicate SD; n 3 to 6. (c and d) Bacterial invasion is strongly enhanced in the presence of 5-g/ml laminin-211/221 in wild-type and pgsA745 cells (c), and this increase is independent of removal of HS by heparinase III (HepIII) (d). Removal of HS by heparinase III (HepIII) in wild-type cells reduced bacterial invasion without addition of laminin-211/221. (c) , P 0.001, and *, P 0.0001 versus the results for no addition of laminin-211/221 using the two-tailed t test. (d) , P 0.001 versus the results for untreated wild-type cells using a two-tailed t test. Error bars indicate SD; n 3 or 4. January/February 2017 Volume 8 Issue 1 e02128-16 mbio.asm.org 6 RESULTS (e) Preincubation of GBS with 50-g/ml laminin-211/221 enhanced invasion in wild-type and pgsA745 cells. , P 0.05, and #, P 0.05 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (f) Bacterial invasion is strongly enhanced in the presence of 10-g/ml laminin-211/221 in human brain microvascular endothelial cells (hBMEC) and human alveolar epithelial cells (A549). , P 0.01, and , P 0.001 versus the results for no addition of laminin-211/221 using the two-tailed t test. Error bars indicate SD; n 3. (g) siRNA knockdown of the expression of long Lama2 reduces bacterial invasion. Inset, silencing of long Lama2 by siRNA reduces expression of long Lama2 transcripts relative to their expression when using negative-control siRNA (NC) or the buffer control (leftmost bar). , P 0.001, and *, P 0.0001 versus the results for buffer-treated cells using the two-tailed t test. Error bars indicate SD; n 3. invasion of GBS in both wild-type and pgsA745 cells (Fig. 4c). This increase was independent of HS, since the removal of HS by heparinase III in the presence of laminin-211/221 had no effect on invasion (Fig. 4d). In the absence of added laminin- 211/221, treatment with heparinase III reduced GBS invasion, consistent with our observation in CRISPR-cas9 mutants (Fig. 1c). Finally, preincubation of bacteria with laminin-211/221, followed by washing to remove unbound laminin, increased invasion as well (Fig. 4e). Laminin-211/221 also enhanced the invasion of GBS in human brain microvascular endothelial cells (hBMEC) and alveolar basal epithelial cells (A549) (Fig. 4f), both of which are susceptible to GBS invasion during experimental infection in vitro and in vivo (17). Long Lama2 isoform is important for bacterial invasion. To investigate the effect of the long Lama2 isoform in bacterial invasion, we speciﬁcally reduced its expression by using small interfering RNA (siRNA) directed to the long sequence (Fig. 4g, inset). Knockdown of the long form of Lama2 signiﬁcantly decreased bacterial invasion in wild-type cells but not in pgsA745 cells, as they lack the long form (Fig. 4g). Finally, mbio.asm.org 6 January/February 2017 Volume 8 Issue 1 e02128-16 ® Laminin 2 Is a Host Receptor for Bacterial Invasion FIG 5 Transfection of pgsA745 cells with human LAMA2 enhances bacterial invasion. (a) Bacterial invasion was restored in pgsA745 cells after transfection with human LAMA2 cDNA. RESULTS Untransfected, left 2 bars; transfected, right 2 bars. , P 0.05, , P 0.01, and *, P 0.0001 versus the results for untransfected cells using the two-tailed t test. Error bars indicate SD; n 2 to 6. (b) Relative expression of human LAMA2 determined by qPCR with primers speciﬁc for the human form of LAMA2, amplifying a region corresponding to CHO long Lama2. Error bars indicate SD; n 3. Note that human LAMA2 was not detected in untransfected cells. FIG 5 Transfection of pgsA745 cells with human LAMA2 enhances bacterial invasion. (a) Bacterial invasion was restored in pgsA745 cells after transfection with human LAMA2 cDNA. Untransfected, left 2 bars; transfected, right 2 bars. , P 0.05, , P 0.01, and **, P 0.0001 versus the results for untransfected cells using the two-tailed t test. Error bars indicate SD; n 2 to 6. (b) Relative expression of human LAMA2 determined by qPCR with primers speciﬁc for the human form of LAMA2, amplifying a region corresponding to CHO long Lama2. Error bars indicate SD; n 3. Note that human LAMA2 was not detected in untransfected cells. transfection of pgsA745 cells with the human form of full-length LAMA2 (hLAMA2) cDNA signiﬁcantly increased the invasion of GBS, GAS, and S. aureus, whereas trans- fection of wild-type cells had little if any effect (Fig. 5a). PCR analysis using primers speciﬁc for the human form of LAMA2 showed robust expression in both mutant and wild-type cells (Fig. 5b). January/February 2017 Volume 8 Issue 1 e02128-16 DISCUSSION Laminins are major components of basement membranes, a specialized extracellular matrix that underlies endothelium and epithelium (15). Different laminins are com- posed of different combinations of , , and subunits. Although theoretically more than 50 heterotrimers can exist, only a third of the possible combinations have been described (18). Laminin subunit 2 is found as part of laminin-211 (laminin-211), laminin-221 (laminin-221), and the less-abundant laminin-213 (laminin-213). Laminin-211 and -221 are primarily known for their function in basement membrane architecture. In this paper, we show that laminin 2 is also important for the cellular invasion of the bacterial pathogens GBS, GAS, and S. aureus. Consistent with this observation, proteins with laminin-binding properties have been described for these bacteria (19–24). However, the studies whose results are shown here are the ﬁrst to implicate a speciﬁc laminin isoform in the invasion of these bacteria. Interestingly, this isoform is also relevant for host-pathogen interactions of mycobacteria like Mycobac- terium leprae (25, 26). M. leprae, the causative agent of leprosy, can attach to Schwann cells via the interaction of laminin 2 with cellular laminin receptors, speciﬁcally integrin 4 (26) and -dystroglycan (27). In this context, laminin 2 acts as a bridge between the host cell and the pathogen, which may also pertain to the interaction of GBS, GAS, and S. aureus with host cells. The observation that incubation of host cells or GBS with laminin 2 enhances bacterial invasion supports a bridging function for laminins in infection. Other extracellular matrix proteins, such as ﬁbronectin, can function in a similar way, as has been reported for the invasion of S. aureus (28). The availability of pgsA745 cells lacking a speciﬁc part of laminin 2 maps a functional site for invasion by GBS, GAS, and S. aureus. This deleted region corresponds to an ~90% loss of the N-terminal globular domain L4a, as well as a loss of half of the mbio.asm.org 7 ® van Wijk et al. rodlike LEb domain. The function of these domains is largely unknown. One report indicated that the L4a domain, although only tested for the laminin 1 chain, binds to the extracellular matrix protein ﬁbulin-2 (29). The LE domains seem to function as spacers between the globular domains (30). Whether the L4a-LEb region serves as an attachment site for the bacteria or as a coreceptor for invasion remains unknown. DISCUSSION However, given that attachment of the bacteria evaluated here occurs normally in pgsA745 cells, it is conceivable that an interaction of laminin 2 with integrins or other receptors for internalization is impaired in pgsA745 cells. It is also possible that the deletion described above results in a global change in the laminin trimer, with a consequent loss in functionality as a portal of entry for bacteria. Thus, laminin 2 may contribute to colonization and the penetration of host cell barriers by providing an initial foothold for bacterial pathogens. The mutant pgsA745 cell line was identiﬁed by a forward screen seeking mutants that failed to incorporate radioactive sulfate into GAGs (3). It belongs to a collection of strains deﬁcient in speciﬁc biochemical steps involved in GAG biosynthesis (31). This particular mutant cell line has been used in a large number of studies that generally established the importance of GAGs in various systems. In most cases, the mutant phenotype correlates with a loss of GAGs, based on similar phenotypic changes in other GAG-deﬁcient CHO cell mutants and on correction of pgsA745 cells by transfection with xylosyltransferase isozymes (6, 32). However, as this paper illustrates, (chemically) mutagenized strains, such as pgsA745, may contain additional mutations that can inﬂuence experimental outcomes and can lead to incorrect interpretation of the data. Nevertheless, careful experiments to reconﬁrm initial ﬁndings using gene-targeting methods can help uncover interesting covert features of these original mutants. In conclusion, the combination of whole-genome sequencing and RNA-Seq led to the discovery that the pgsA745 cell line harbors an additional mutation in Lama2 that confers panresistance to bacterial invasion. pgsA745 cells are also resistant to infection by multiple viruses due to the lack of GAG receptors (e.g., see references 33 and 34). As CHO cells are used extensively for recombinant protein production, the mutant pgsA745 cell line is an excellent candidate for an infection-resistant expression system. January/February 2017 Volume 8 Issue 1 e02128-16 MATERIALS AND METHODS To determine the laminin-binding capacities of different bacteria, laminin-211/221 was concentrated using a 100-kDa-cutoff Amicon centrifugal ﬁlter unit (EMD Millipore) and FITC labeled using the FluoReporter FITC protein labeling kit (Life Technologies, Inc.) according to the manufacturer’s instruc- tions. Bacteria (3.75 107 CFU) were incubated with 50 g/ml of the labeled laminin for 30 min at 37°C, washed twice with PBS, and analyzed by ﬂow cytometry on a FACSCalibur instrument. Immunocytochemistry. For detection of laminin 2, 4.0 104 CHO cells per well were seeded in 8-well glass chamber slides (Lab-Tek). Two days later, cells were washed with PBS, ﬁxed with 4% paraformaldehyde (PFA) in phosphate buffer (PB-PFA) for 10 min, blocked with PBS containing 1% (wt/vol) BSA, and incubated with anti-laminin 2 antibody (1:500, clone 5H2; EMD Millipore), followed by Alexa Fluor 488-conjugated anti-mouse IgG (10 g/ml; Life Technologies, Inc.). For actin staining after incubation with EPEC, CHO cells (1.0 105 per well) were seeded in 4-well glass chamber slides (Lab-Tek). The next day, cells were incubated with or without ~2 105 CFU of log-phase-grown EPEC for 2.5 h at 37°C. Cells were washed twice with PBS, ﬁxed with 4% PB-PFA for 10 min, washed twice with PBS, permeabilized using 0.2% (vol/vol) Triton X-100 for 8 min, blocked with PBS containing 1% (wt/vol) BSA, and stained for actin using Alexa Fluor 488-conjugated phalloidin (1:100; Life Technologies, Inc.). The slides were mounted in SlowFade (Life Technologies, Inc.). Images were acquired using a Zeiss Axio Observer D1 microscope and Zen 2012 Blue software. siRNA and cDNA transfections. For siRNA transfections, 5.0 104 CHO cells per well were seeded into 24-well plates. About 1 h after seeding, cells were transfected with a ﬁnal concentration of 5 nM siRNA using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions. Laminin 2 was targeted using the following duplex: sense, GGAACAAACUUACCAGUCAdTdT, and antisense, UGACUGGUAAGUUUGUUCCdTdT (Sigma-Aldrich). Universal negative control #1 siRNA (Sigma- Aldrich) was used as a negative control. Two days after transfection, RNA was isolated as described below (see “DNA and RNA isolation”), and a bacterial invasion assay was performed as described above (see “Bacterial adherence and invasion assays”). For hLAMA2 cDNA transfection, CHO cells (3.0 105 cells per well) were seeded into 6-well plates. January/February 2017 Volume 8 Issue 1 e02128-16 MATERIALS AND METHODS Detailed materials and methods are provided in Text S1 in the supplemental material. Animal cells. CHO-K1 cells were obtained from the American Type Culture Collection (ATCC) (CCL-61). The mutant pgsA745 cell line was described previously (3) and subsequently shown to contain a point mutation in XylT2. Simian virus 40 (SV40) large T antigen-immortalized human brain microvas- cular endothelial cells (hBMEC) were obtained from Kwang Sik Kim (Johns Hopkins University, Baltimore, MD). A549 human alveolar basal epithelial cells were obtained from the ATCC (CCL-185). The culture conditions can be found in the supplemental materials and methods in Text S1 in the supplemental material. Bacterial strains. GBS strain COH1 is a serotype III isolate from an infant with bacteremia (35). GBS strain A909 is a serotype Ia neonatal isolate (36). GAS strain 5448 is an M1T1 serotype isolate from a patient with necrotizing fasciitis and toxic shock syndrome (37). Methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 TCH1516 is an isolate from an adolescent patient with severe sepsis syndrome (38). Staphylococcus aureus Newman (39) is a methicillin-sensitive (MSSA) strain. Generation of XylT2 knockouts using the CRISPR-cas9 system. Xylosyltransferase 2 (Xylt2) mutant cell lines CHO-23A1 and CHO-93A5 were generated using the Edit-R CRISPR-cas9 gene engineering system (Dharmacon GE Healthcare) according to the manufacturer’s instructions. In short, CHO-K1 cells were cotransfected with a cas9 expression vector containing a blasticidin resistance marker (pHCSVBlast- Cas9, catalog number U-001000-120), trans-activating CRISPR RNA (tracrRNA) (catalog number U-002000- 120), and CRISPR RNA (crRNA) speciﬁc for CHO Xylt2 (crRNA-107962; 5= GAGGCACUAAUGGGCGCUGCG UUUUAGAGCUAUGCUGUUUUG 3=). The target sequence (GAGGCACTAATGGGCGCTGCTGG, target identiﬁer (ID) 791342), found in exon 1, was determined using CRISPy, a web-based target-ﬁnding tool for CHO-K1 cells (40). Two days after transfection, cells were selected by incubation with 10-g/ml blasticidin S (Thermo Fisher Scientiﬁc) for 5 days, and cells were then seeded in 96-well plates to obtain single-cell clones. The clonal lines obtained were screened for heparan sulfate expression by ﬂow cytometry using the anti-heparan sulfate single-chain antibody HS4C3 (41) as described below (see “Flow cytometry”). Bacterial adherence and invasion assays. Adherence and invasion assays were performed essen- tially as described previously (2). In one experiment, wild-type cells were pretreated with or without 10 mIU/ml heparinase III in Ham’s F-12 growth medium supplemented with 0.5% (vol/vol) FBS for 30 min at 37°C before the addition of laminin-211/221 and GBS. MATERIALS AND METHODS In another experiment, GBS was preincubated mbio.asm.org 8 ® Laminin 2 Is a Host Receptor for Bacterial Invasion with or without 50-g/ml laminin-211/221 in phosphate-buffered saline (PBS) for 30 min at room temperature and washed with PBS before being added to CHO cells. To ensure that differences in bacterial invasion were not due to differences in cell viability, the release of lactate dehydrogenase (LDH) into the medium was quantiﬁed during bacterial incubation and antibiotic protection. The LDH in 50 l of culture supernatant or in 50 l of cells lysed with 0.025% (vol/vol) Triton X-100 was quantiﬁed using the CytoTox 96 nonradioactive cytotoxicity assay (Promega) according to the manufacturer’s instructions. Bacterial adhesion to laminin. Laminin-211/221 (10 g/ml, human merosin CC085; EMD Millipore) in 50 mM carbonate-bicarbonate buffer (product number C3041; Sigma-Aldrich) was used to coat a 96-well plate (Costar 9018, enzyme immunoassay/radioimmunoassay [EIA/RIA] high binding). The plate was covered with Paraﬁlm and incubated overnight at 4°C. The plate was washed 4 times with PBS and blocked with PBS containing 1% (wt/vol) bovine serum albumin (BSA) for 1 h at room temperature. Bacteria were labeled with ﬂuorescein isothiocyanate (FITC) as described in Text S1 in the supplemental material (see “Invasion assay with FITC-labeled bacteria”). The plate was washed 4 times with PBS, and ~107 CFU of bacteria in 100 l was added per well. The plate was centrifuged at 500 g for 10 min and incubated for 30 min at 37°C. Fluorescence (excitement/emission, 485/538) was measured on a plate reader (SpectraMax M3; Molecular Devices) before and after washing 5 times with PBS containing 1% (wt/vol) BSA. Flow cytometry. Flow cytometry was performed essentially as described previously (42). CHO cells were detached by using a Versene solution (Life Technologies, Inc.), incubated with the antibodies/ proteins described in Text S1 in the supplemental material, and analyzed on a FACSCalibur instrument (BD Biosciences). For detection of HS, cells were incubated sequentially with the vesicular stomatitis virus (VSV)-tagged single-chain variable-fragment (scFv) antibody HS4C3 (1:100 [41]), mouse anti-VSV IgG (1:10; P5D4), and Alexa Fluor 488 ﬂuorochrome-conjugated anti-mouse IgG (20 g/ml; Life Technologies, Inc.). For detection of FGF2 binding, cells were incubated sequentially with biotinylated FGF2 (1:250 [43]) and DyLight 488 ﬂuorochrome-conjugated streptavidin (10 g/ml; Thermo Scientiﬁc). Endocytic/phago- cytic assays are described in Text S1. MATERIALS AND METHODS The following day, cells were transfected with 2 g human LAMA2 cDNA in a pcDNA3.1 vector that was modiﬁed to contain a puromycin resistance gene instead of the zeocin resistance gene. Cells were transfected using Lipofectamine 3000 transfection reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Cells were reseeded the next day, and selection using 10 g/ml puromycin was started one day later. Selection pressure was maintained for two weeks, after which cells were used for bacterial invasion assays and isolation of RNA as described in the corresponding sections herein. pgsA745-XylT1 cells were generated by stable transfection with human XylT1 cDNA in pcDNA3.1, and a single-cell population was generated. pgsA745-XylT2 cells were a gift from Cuellar et al. (6). DNA and RNA isolation. For DNA isolation, cells were detached using 0.05% trypsin–0.53 mM EDTA and pelleted. DNA was isolated using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. For RNA isolation, cells were lysed directly using Trizol, after which chloro- form was added. Following centrifugation at 12,000 g for 15 min at 4°C, the transparent upper phase was transferred and an equal volume of 70% ethanol was added. This mixture was applied to an RNeasy mini-spin column (Qiagen), and the manufacturer’s instructions were followed. Quantitative PCR analysis. cDNA was synthesized from total RNA using the SuperScript III ﬁrst- strand synthesis system (Life Technologies, Inc.) according to the manufacturer’s instructions. Quantita- tive PCR (qPCR) was performed using the Power SYBR green PCR master mix (2; Life Technologies, Inc.) mbio.asm.org 9 ® van Wijk et al. on a CFX96-C1000 real-time PCR detection system (Bio-Rad) according to the manufacturers’ instructions. Primer sequences can be found in Text S1 in the supplemental material. Whole-genome/RNA sequencing. Whole-genome sequencing libraries were prepared using the TruSeq DNA sample prep kit (Illumina, San Diego, CA, USA), and RNA libraries were prepared for sequencing using the TruSeq stranded mRNA sample preparation kit (Illumina) according to the manufacturer’s instructions, with the following change: poly(A) enrichment was used to eliminate rRNA transcripts from RNA-Seq libraries. The libraries were clustered using cBot and sequenced on a HiSeq 2500 system (HiSeq Control Software version 2.2.38/RTA version 1.18.61) with a 2 101 setup. Bcl-to-Fastq conversion was performed using bcl2Fastq version 1.8.3 from the CASAVA software suite. The data analysis methods are described in Text S1 in the supplemental material. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/ mBio.02128-16. Supplemental material for this article may be found at https://doi.org/10.1128/ mBio.02128-16. TEXT S1, DOCX ﬁle, 0.04 MB. 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https://openalex.org/W4316506889	https://jnanobiotechnology.biomedcentral.com/counter/pdf/10.1186/s12951-022-01756-4	English	null	Deposition of hydrophilic Ti3C2Tx on a superhydrophobic ZnO nanorod array for improved surface-enhanced raman scattering performance	Journal of nanobiotechnology	2,023	cc-by	7,570	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Superhydrophobic substrate modifications are an effective way to improve SERS sensitivity by concen- trating analyte molecules into a small surface area. However, it is difficult to manipulate low-volume liquid droplets on superhydrophobic substrates. Results To overcome this limitation, we deposited a hydrophilic Ti3C2Tx film on a superhydrophobic ZnO nanorod array to create a SERS substrate with improved analyte affinity. Combined with its interfacial charge transfer proper- ties, this enabled a rhodamine 6G detection limit of 10−11 M to be achieved. In addition, the new SERS substrate showed potential for detection of biological macromolecules, such as microRNA. sion Combined with its facile preparation, the SERS activity of ZnO/Ti3C2Tx suggests it may provide e environmental pollutant-monitoring and effective substrate for biological analyte detection. Keywords SERS, Superhydrophobic substrate, Ti3C2Tx, DFT calculations, miRNA detection Correspondence: Ying Sun ysun@shsci.org Yaogang Li yaogang_li@dhu.edu.cn Yourong Duan yrduan@shsci.org Full list of author information is available at the end of the article Deposition of hydrophilic Ti3C2Tx on a superhydrophobic ZnO nanorod array for improved surface‑enhanced raman scattering performance Zhihua Wu1, De Zhao1, Xin Han3, Jichang Liu3, Ying Sun1, Yaogang Li2* and Yourong Duan1 Abstract Background Superhydrophobic substrate modifications are an effective way to improve SERS sensitivity by concen- trating analyte molecules into a small surface area. However, it is difficult to manipulate low-volume liquid droplets on superhydrophobic substrates. Results To overcome this limitation, we deposited a hydrophilic Ti3C2Tx film on a superhydrophobic ZnO nanorod array to create a SERS substrate with improved analyte affinity. Combined with its interfacial charge transfer proper- ties, this enabled a rhodamine 6G detection limit of 10−11 M to be achieved. In addition, the new SERS substrate showed potential for detection of biological macromolecules, such as microRNA. Conclusion Combined with its facile preparation, the SERS activity of ZnO/Ti3C2Tx suggests it may provide an ultra- sensitive environmental pollutant-monitoring and effective substrate for biological analyte detection. Keywords SERS, Superhydrophobic substrate, Ti3C2Tx, DFT calculations, miRNA detection Journal of Nanobiotechnology Journal of Nanobiotechnology Wu et al. Journal of Nanobiotechnology (2023) 21:17 https://doi.org/10.1186/s12951-022-01756-4 © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wu et al. Journal of Nanobiotechnology (2023) 21:17 Page 2 of 11 Wu et al. Journal of Nanobiotechnology Graphical Abstract Graphical Abstract of nanostructures [28]. Moreover, it can be easily deco- rated on the surface of different substrates, such as glass slides [29] and polydimethylsiloxane [30]. Although supe- rhydrophobic SERS substrates can increase the Raman intensity remarkably, their poor affinity for aqueous ana- lytes makes these molecules difficult to attach, which reduces the efficiency of the detection process. Introduction Surface-enhanced Raman scattering (SERS) is a widely used and powerful tool for analyzing molecules and biomarkers [1], in vivo imaging [2], and environmen- tal pollutants [3–6]. Recent development of new sur- face-enhancing materials and methods has markedly improved SERS detection limits [7–9]. Notably, the com- bination of nanostructured plasmics and Raman spec- troscopy has enabled single molecule detection [10–12]. However, the surface enhancement is restricted to “hot spots” with an area of a few nm2 [13–16]. The detection or identification of low-concentration analytes remains challenging because random diffusion of molecules on the hydrophilic substrate results in poor spatial locali- zation [17]. This random diffusion makes it time-con- suming to search for molecules of interest within the enhanced area [18, 19]. fi Herein, we report a facile method to fabricate a SERS substrate comprising a two-dimensional (2D) Ti3C2Tx monolayer on a supporting superhydrophobic ZnO nanorod array (Fig. 1). The hydrophilicity of the Ti3C2Tx surface increases the affinity of aqueous analytes and its intrinsic metallic conductivity and plasmon resonance enhance Raman scattering intensity [31–33]. We pre- pared a small-area Ti3C2Tx spot on a superhydrophobic ZnO nanorod array and demonstrated that the hydro- phobic substrate concentrates the analyte within the enhancement area and thus greatly improves the Raman scattering intensity. Density functional theory (DFT) calculations further showed that the interfacial charge transfer also contributes to the intensity enhancement. The ZnO/Ti3C2Tx substrate provides a cost-effective SERS platform for ultrasensitive analyte detection and identification. Various strategies have been used to overcome the above problems [20–22], such as the use of superhydro- phobic substrates to markedly enhance SERS intensity [17, 23–26]. Such substrates overcome the diffusion limit by concentrating molecules into a small area, resulting in a threefold increase in Raman intensity compared with ordinary hydrophilic substrates [23]. Superhydrophobic substrates can be fabricated using photolithography and those noble metal nanoparticle decorated SERS platform can achieve a detection limit of 10−18 M using rhodamine 6G (R6G) as a SERS probe [17]. However, the fabrication processes are sophisticated and the required equipment is expensive. Thus, more facile methods are required to prepare SERS substrates that confine the analyte mole- cule. Manipulating the morphology of inorganic nanoar- rays may lead to new superhydrophobic substrates [27]. Among these inorganic materials, ZnO is an ideal candi- date because it enables the facile synthesis and regulation DFT simulations and calculations First-principles calculations of the enhancing mechanism in our systems used the DFT-based projector augmented- wave method implemented in the Materials Studio soft- ware [40]. The exchange–correlation energy was treated using the generalized gradient approximation of Perdew, Burke, and Ernzerhof [41, 42]. The energy cutoff for the plane wave basis expansion was set to 400 eV during the geometry relaxation. The 3 × 3 × 1 Monkhorst–Pack k-point sampling grid was used to calculate the sur- face structures. The self-consistent calculations used an energy convergence threshold of 10−5 eV. The equi- librium lattice constants were optimized using a maxi- mum stress constraint of 0.03 eV/Å on each atom. Spin polarization was allowed in all calculations. The Ti3C2 Fabrication of monolayer Ti3C2Tx Monolayer Ti3C2Tx was obtained as previously described [34, 35]. Typically, 1.6 g LiF was added in 20 mL 9 M HCl in 5 min and magnetic stirred for another 5 min. Then, 1 g Ti3AlC2 was added over the course of 5 min in case of overheat. Etching was performed at room tem- perature for 24 h. The acidic mixture was centrifugated at 3500 rpm until pH ≥ 5 (each cycle for 5 min). Sedi- ment was re-dispersed by hand-shaking every time. The Ti3C2Tx colloid was collected when the supernatant turned stable dark-green. After that, monolayer Ti3C2Tx was collected after centrifugation at 3500 rpm for 1 h by collecting the supernatant. Material and methods Material LiF was purchased from Aladdin Reagents (Shanghai), 200 mesh MAX Ti3AlC2 was purchased from 11 technol- ogy co.,LTD, hydrochloric acid (HCl), Zinc nitrate hexa- hydrate (Zn(NO3)2), Hexamethylenetetramine (HMTA), potassium permanganate were purchased from Sinop- harm Chemical Reagent Co. Ltd. (China), Rhodamine 6G (R6G), methylene blue (MB), acid blue (AB), crys- tal violet (CV) were purchased from MACKLIN. All Wu et al. Journal of Nanobiotechnology (2023) 21:17 Wu et al. Journal of Nanobiotechnology Page 3 of 11 Fig. 1 Illustration of the fabrication of a ZnO/Ti3C2Tx substrate and the mechanism of its surface-enhanced Raman scattering (SERS) activity. The Ti3C2Tx monolayer is prepared using a selective etching method and then dispersed in deionized water. The ZnO/Ti3C2Tx substrate is fabricated by depositing the Ti3C2Tx solution on a superhydrophobic ZnO nanorod array Fig. 1 Illustration of the fabrication of a ZnO/Ti3C2Tx substrate and the mechanism of its surface-enhanced Raman scattering (SERS) activity. The Ti3C2Tx monolayer is prepared using a selective etching method and then dispersed in deionized water. The ZnO/Ti3C2Tx substrate is fabricated by depositing the Ti3C2Tx solution on a superhydrophobic ZnO nanorod array glass slide was thoroughly washed by distilled water. The ZnO decorated glass slide was then dried in oven at 90 ℃ for further use. ZnO film was prepaered as previously described[39]. chemicals were used as received without further purifica- tion. miRNA with sequence of UUUGUACUACACAAA AGUACUG (sense(5’-3’)) was purchased from RIBOBIO co.,LTD. To fabricate ZnO/Ti3C2Tx SERS substrate, 4 µL 1–2 mg/mL monolayer Ti3C2Tx aqueous solution was dropped onto ZnO decorated glass slide and dried at room temperature for further use. Before test, 4 µL 10–6 to 10–10 M R6G was dropped on the ZnO/Ti3C2Tx SERS substrate and dried at room temperature. Laser of 532 nm was used in the experiments at power of 5 mW with a static mode and 20 s exposure time to reduce background noise. As for Raman mapping, step of 1 µm and 2 s exposure time at 532 nm were adopted. Preparation of ZnO/Ti3C2Tx SERS substrate and enhancement performance Glass slides were pretreated using potassium perman- ganate and n-butanol as described previously [36]. To successfully construct ZnO nanorods on the glass slide surface, the pretreated glasses were firstly encapsulated by PDMS to form a 75 mm × 4 mm × 1 mm microchan- nel. Then, reaction solutions of 0.05 M Zn(NO3)2 and 0.05 M HMTA were injected by pump in two Teflon tubes at 50 µL/min for 2 h at 90 ℃ [37, 38]. After that, PDMS was removed and the obtained ZnO decorated Wu et al. Journal of Nanobiotechnology (2023) 21:17 Wu et al. Journal of Nanobiotechnology Page 4 of 11 Fig. 2 A X-ray diffraction (XRD) patterns of pristine Ti3AlC2 and Ti3C2Tx. B Scanning electron microscopy (SEM) image of a Ti3C2Tx monolayer under low magnification. C High-resolution transmission electron microscopy image of a Ti3C2Tx nanosheet and the corresponding selected area electron diffraction pattern. D Scanning TEM (STEM) coupled with energy dispersive X‐ray spectroscopy (EDX) of a Ti3C2Tx nanosheet. E XRD patterns of ZnO nanorod arrays with hexagonal wurtzite structure (Joint Committee on Powder Diffraction Standards card no. 36–1451). F SEM image of the ZnO nanorod array without Ti3C2Tx, and SEM images after drop-casting 4 µL of G 1 mg/mL, H 1.5 mg/mL, and I 2 mg/mL Ti3C2Tx solutions on the ZnO nanorod array, the inset is a sectional view of ZnO/Ti3C2Tx. J Illustration of the ZnO/Ti3C2Tx substrate fabrication process Fig. 2 A X-ray diffraction (XRD) patterns of pristine Ti3AlC2 and Ti3C2Tx. B Scanning electron microscopy (SEM) image of a Ti3C2Tx monolayer under low magnification. C High-resolution transmission electron microscopy image of a Ti3C2Tx nanosheet and the corresponding selected area electron diffraction pattern. D Scanning TEM (STEM) coupled with energy dispersive X‐ray spectroscopy (EDX) of a Ti3C2Tx nanosheet. E XRD patterns of ZnO nanorod arrays with hexagonal wurtzite structure (Joint Committee on Powder Diffraction Standards card no. 36–1451). F SEM image of the ZnO nanorod array without Ti3C2Tx, and SEM images after drop-casting 4 µL of G 1 mg/mL, H 1.5 mg/mL, and I 2 mg/mL Ti3C2Tx solutions on the ZnO nanorod array, the inset is a sectional view of ZnO/Ti3C2Tx. J Illustration of the ZnO/Ti3C2Tx substrate fabrication process the interface, molecule, and interface with molecules adsorbed. (001) and ZnO (101) surfaces were obtained from their corresponding bulk supercell structures. Preparation of ZnO/Ti3C2Tx SERS substrate and enhancement performance To describe the charge transfer at the interface of these structures, lattice parameters of a = 12.384727 and b = 19.073 were estab- lished. Finally, the molecular adsorption energy (Eb) was calculated using the expression [43] Eb = Etotal − E1 − E2, where E1, E2, and Etotal are the respective energies of Characterization of the ZnO/Ti3C2Tx SERS substrateh The ZnO nanorod arrays were prepared using a micro- fluidic chemical reaction. As shown in Fig. 2E, the XRD spectrum of the prepared ZnO was the same as that of ZnO with hexagonal wurtzite structure. Field emis- sion SEM showed that the inclined ZnO nanorods with a length of ≈2.5 µm and a uniform hexagonal structure Results and discussion Characterization of Ti3C2Tx nanosheets Two-dimensional Ti3C2Tx nanosheets were prepared by selectively etching the Al layer from Ti3AlC2. As shown Wu et al. Journal of Nanobiotechnology (2023) 21:17 Wu et al. Journal of Nanobiotechnology Page 5 of 11 Fig. 3 Characterization of water droplet contact angles on A pristine Ti3C2Tx film, B the ZnO nanorod array, and C ZnO/Ti3C2Tx ig. 3 Characterization of water droplet contact angles on A pristine Ti3C2Tx film, B the ZnO nanorod array, and C ZnO/Ti3C2Tx roplet contact angles on A pristine Ti3C2Tx film, B the ZnO nanorod array, and C ZnO/Ti3C2Tx were successfully decorated on the glass substrate (Fig. 2F). by the X-ray powder diffraction (XRD) patterns (Fig. 2A), the main peaks corresponding to MAX-phase Ti3AlC2 disappeared after etching, and the (002) diffraction angle decreased from 9.5° to 7.5°. Large Ti3C2Tx nanosheets were obtained using an in situ HCl/LiF etching method (Fig. 2B). High-resolution transmission electron micros- copy and selected area electron diffraction showed that monolayered Ti3C2Tx sheets were successfully derived from MAX-phase Ti3AlC2 (Fig. 2C). Elemental composi- tion maps obtained using X-ray energy-dispersive spec- troscopy further proved that the Al layer was completely removed from the Ti3C2Tx surface (Fig. 2D, Additional file 1: Figure S1). These results demonstrate the success- ful etching of the atomic Al layer in Ti3AlC2. Small-area ZnO/Ti3C2Tx spots were prepared by depositing Ti3C2Tx solutions on a ZnO nanorod- modified glass substrate. To prevent oxidation of the Ti3C2Tx, the spots were dried at room temperature. As shown in Fig. 2G–I, the coverage area of the 2D Ti3C2Tx nanosheets was improved by increasing the concentra- tion of the Ti3C2Tx solution. Furthermore, Fig. 2H shows that the Ti3C2Tx nanosheets were supported by the tips of ZnO nanorods and closely attached to the inclined nanorods. With the increasing concentration of Ti3C2Tx, a thin film gradually formed on the ZnO nanorod array (inset of Fig. 2I). Therefore, ZnO/Ti3C2Tx was fabricated by concentrating Ti3C2Tx nanosheets on ZnO nanorod arrays surface (Fig. 2J). Hydrophilicity of prepared substratesh The hydrophilicities of the Ti3C2Tx film, ZnO nanorod array, and ZnO/Ti3C2Tx were assessed by measuring the surface contact angles of water droplets. The Ti3C2Tx film was hydrophilic, having contact angles of 77.6 ± 2° and 72.5 ± 3° for droplet volumes of 1 and 4 μL, respectively Wu et al. Journal of Nanobiotechnology (2023) 21:17 Wu et al. Journal of Nanobiotechnology Page 6 of 11 Fig. 4 SERS performance of A the superhydrophobic ZnO nanorod array, B Ti3C2Tx film, C ZnO/Ti3C2Tx (R6G analyte concentration ranging from 10−6 to 10−11 M), and D ZnO film, E ZnO(film)/Ti3C2Tx (R6G analyte concentration ranging from 10−6 to 10−8 M) Fig. 4 SERS performance of A the superhydrophobic ZnO nanorod array, B Ti3C2Tx film, C ZnO/Ti3C2Tx (R6G analyte concentration ranging from 10−6 to 10−11 M), and D ZnO film, E ZnO(film)/Ti3C2Tx (R6G analyte concentration ranging from 10−6 to 10−8 M) SERS performance of the ZnO/Ti3C2Tx substrate Amorphous ZnO nanocages have remarkable SERS activity [44]. Here, crystalline ZnO also showed SERS activity, with a hexagonal wurtzite ZnO nanorod array yielding an R6G detection limit of 10−6 M. As indicated in Fig. 4A, characteristic peaks of R6G can be clearly identified. However, the characteristic Raman scattering peaks at 614, 773, 1360, 1500, 1573, and 1650 cm−1 were almost undetectable at an R6G concentration of 10−7 M. (Fig. 3A). The ZnO nanorod array surface was super- hydrophobic (Fig. 3B), having a low affinity for droplet volumes less than 4 µL. Even 4 μL droplets maintained a contact angle of 150.2 ± 2° and a relatively spheri- cal shape. Under gravity, droplet volumes of 6 and 8 µL made greater contact with the substrate surface and had an ellipsoidal shape. These results proved that the supe- rhydrophobic substrate was successfully fabricated by decorating the glass slide with the ZnO nanorod array. Coating the ZnO with Ti3C2Tx resulted in a contact angle of 130.7 ± 4° for 4 µL droplets (Fig. 3C). Smaller contact angles were observed for droplet sizes less than 4 µL, which may be explained by the micro/nanostruc- ture of ZnO/Ti3C2Tx. For example, if the droplet is larger than the hydrophilic modification area, the edge of ZnO/ Ti3C2Tx substrate can still be superhydrophobic. Impor- tantly, ZnO/Ti3C2Tx showed a higher hydrophilicity than the ZnO nanorod array, as evidenced by the smaller con- tact angles and easier droplet attachment. Hydrophilicity of prepared substratesh These results are con- sistent with UV–vis spectra of Ti3C2Tx-R6G (Fig. 5E), which show difference in the peak position between 500 and 550 nm that could be explained by the chemisorp- tion of Ti3C2Tx and R6G. Moreover, the calculated den- sity of states of ZnO/Ti3C2Tx-R6G increased toward the Femi level after adsorption of R6G (Fig. 5F), which again indicates there is interfacial charge transfer between the substrate and R6G. Chemisorption was likewise observed between Ti3C2Tx with MB, together with an obvious absorption peak shift (Fig. 5g), suggesting charge trans- fer also occurs between Ti3C2Tx and MB and leads to high SERS activity (detection limit of 10−10 M). However, because of the physisorption of the AB and CV molecules with the Ti3C2Tx surface, no obvious absorption peak shift was observed (Fig. 5H, I), which could explain why the SERS enhancement of AB and CV is limited (Addi- tional file 1: Figure S7b, c). Considering that the group of ZnO(film)/Ti3C2Tx with a larger contact area between Ti3C2Tx and ZnO film did not show more significant enhancement performance, this suggests that the hydro- phobic structure plays a more important role in Raman enhancement. These results suggest that the observed SERS performance of the ZnO/Ti3C2Tx substrate may result from a synergy between its analyte-concentrating ability and interfacial charge transfer properties. i g The SERS performance of the ZnO substrate may be influenced by the ZnO/Ti3C2Tx interfacial properties. A ZnO film and a ZnO(film)/Ti3C2Tx substrate were fabri- cated to assess the contribution of hydrophobic micro/ nanostructure to the SERS activity (Additional file 1: Fig- ure S3a and b). The ZnO film substrate had a higher SERS sensitivity compared with that of pristine ZnO, enabling detection of 10−6 M R6G (Fig. 4D). The ZnO(film)/ Ti3C2Tx substrate had an even higher SERS sensitivity, achieving an R6G detection limit of 10−8 M (Fig. 4E). Contact angle measurements indicated both ZnO film and ZnO(film)/Ti3C2Tx were hydrophilic, displaying 4 µL droplet contact angles of 37.1 ± 1.2° and 77.5 ± 1°, respec- tively (Additional file 1: Figure S4a and b). The above results demonstrate that the hydrophobic micro/nano- structure of ZnO/Ti3C2Tx may contribute more to the Raman scattering intensity than the hydrophilic micro/ nanostructure of ZnO (film)/Ti3C2Tx. y g p p To evaluate the spatial distribution of the SERS intensity, the Raman intensity from 10−6 M R6G drop- cast on the ZnO/Ti3C2Tx substrate was mapped at 1360 cm−1. Hydrophilicity of prepared substratesh Journal of Nanobiotechnology and 1360 cm−1, respectively (Additional file 1: Table S1, calculation details were presented in supporting informa- tion). The EF of ZnO/Ti3C2Tx is 2.10fold of Ti3C2Tx and 2.73fold of ZnO at peak of 614 cm−1. At 1360 cm−1, the EF of ZnO/Ti3C2Tx is 1.65fold and 2.64fold of Ti3C2Tx and ZnO, respectively. As can be seen in Additional file 1: Figure S6, with the decrease of analyte concentration, the distribution of Raman signal becomes uneven. This indi- cates that the distribution of molecules on the substrate is not uniform with decreasing concentration. According to the Raman mapping, 10–11 M R6G could be detected by mapping method. Thus, the combination of ZnO and Ti3C2Tx enhanced the R6G detection sensitivity by five orders of magnitude compared with that of pristine ZnO and four orders of magnitude compared with that of pris- tine Ti3C2Tx. The ZnO/Ti3C2Tx substrate was also highly sensitive to methylene blue (MB), yielding a detection limit of 10−10 M (Additional file 1: Figure S7a). and 1360 cm−1, respectively (Additional file 1: Table S1, calculation details were presented in supporting informa- tion). The EF of ZnO/Ti3C2Tx is 2.10fold of Ti3C2Tx and 2.73fold of ZnO at peak of 614 cm−1. At 1360 cm−1, the EF of ZnO/Ti3C2Tx is 1.65fold and 2.64fold of Ti3C2Tx and ZnO, respectively. As can be seen in Additional file 1: Figure S6, with the decrease of analyte concentration, the distribution of Raman signal becomes uneven. This indi- cates that the distribution of molecules on the substrate is not uniform with decreasing concentration. According to the Raman mapping, 10–11 M R6G could be detected by mapping method. Thus, the combination of ZnO and Ti3C2Tx enhanced the R6G detection sensitivity by five orders of magnitude compared with that of pristine ZnO and four orders of magnitude compared with that of pris- tine Ti3C2Tx. The ZnO/Ti3C2Tx substrate was also highly sensitive to methylene blue (MB), yielding a detection limit of 10−10 M (Additional file 1: Figure S7a). calculated energy of R6G absorption on the ZnO/Ti3C2Tx interface is −2.36 eV, indicating that the interface has a large adsorption capacity for R6G molecules. g p p y DFT simulations were also carried out to investigate charge transfer phenomena at the interface between Ti3C2Tx and R6G and found that charge can be further transferred from Ti3C2Tx to R6G molecules (Fig. 5D, top view Additional file 1: Figure S5b). Hydrophilicity of prepared substratesh These results demonstrate that the ZnO/Ti3C2Tx substrate greatly improved the water droplet attachment and maintained high contact angles after Ti3C2Tx modification. (Fig. 3A). The ZnO nanorod array surface was super- hydrophobic (Fig. 3B), having a low affinity for droplet volumes less than 4 µL. Even 4 μL droplets maintained a contact angle of 150.2 ± 2° and a relatively spheri- cal shape. Under gravity, droplet volumes of 6 and 8 µL made greater contact with the substrate surface and had an ellipsoidal shape. These results proved that the supe- rhydrophobic substrate was successfully fabricated by decorating the glass slide with the ZnO nanorod array. Coating the ZnO with Ti3C2Tx resulted in a contact angle of 130.7 ± 4° for 4 µL droplets (Fig. 3C). Smaller contact angles were observed for droplet sizes less than 4 µL, which may be explained by the micro/nanostruc- ture of ZnO/Ti3C2Tx. For example, if the droplet is larger than the hydrophilic modification area, the edge of ZnO/ Ti3C2Tx substrate can still be superhydrophobic. Impor- tantly, ZnO/Ti3C2Tx showed a higher hydrophilicity than the ZnO nanorod array, as evidenced by the smaller con- tact angles and easier droplet attachment. These results demonstrate that the ZnO/Ti3C2Tx substrate greatly improved the water droplet attachment and maintained high contact angles after Ti3C2Tx modification. An R6G detection limit of 10−7 M was achieved using Ti3C2Tx film (Fig. 4B), which is consistent with a previ- ously reported value [32]. Different Ti3C2Tx films were prepared by filtering Ti3C2Tx nanosheets (0.5–2.0 mg in 30 mL water) on a polytetrafluoroethylene mem- brane (50 mm of diameter). At an R6G concentration of 10−6 M, all films had slight difference of SERS activity (Additional file 1: Figure S2). However, reducing the R6G concentration from 10−6 to 10−7 M resulted in mark- edly lower intensities of the Raman peaks at 773 and 1500 cm−1 and rendered the characteristic peaks at 614, 1360, 1573, and 1650 cm−1 undetectable. The SERS activ- ity of the ZnO/Ti3C2Tx substrate was much higher than that of pristine ZnO and Ti3C2Tx, resulting in an R6G detection limit of 10−11 M (Fig. 4C) with enhancement factors (EFs) of 1.49 × 107 and 7.88 × 106 at peaks of 614 Page 7 of 11 Wu et al. Journal of Nanobiotechnology (2023) 21:17 Page 7 of 11 Wu et al. Hydrophilicity of prepared substratesh The signal distribution on the substrate did not exhibit a coffee ring effect, and its signal was slightly stronger in the depressions than in the pro- trusions (Fig. 6A and B). To investigate the reason for this phenomenon, we took SEM images of the distri- bution of gold nanoparticles on the substrate to simu- late the distribution of R6G (Fig. 6C). According to the image, gold nanoparticles tend to aggregate more in the depression than elsewhere, so the intensity of the Raman signal in the depression is also slightly stronger than elsewhere. Despite the slightly different molecular distributions, the overall signal intensity remained rel- atively uniform. Statistical analysis of 1360 cm−1 peak intensity in Fig. 6D yielded a relative standard deviation of only 6.23%, which indicates the platform is highly homogeneous and thus capable of providing reproduc- ible signals. Area mapping of 10–6 M to 10–11 M was DFT calculation of charge transfer properties f h l h h h g p p To further explore the enhancement mechanism, first- principles DFT calculations were employed. Using the optimized ZnO/Ti3C2Tx (Fig. 5A) and ZnO/Ti3C2Tx- R6G (Additional file 1: Figure S5a) interface struc- tures, the charge density difference was calculated. The results clearly identified a charge transfer mechanism at the interface, enabling electron transfer from the ZnO nanorod surface to Ti3C2Tx nanosheet (Fig. 5B). The work of adhesion (Wad) describes the relationship between the interface distance and system energy in the interface. The DFT calculations showed that Wad is lowest at an interface distance of ≈2.0 Å (Fig. 5C). This suggests that the distance between ZnO and Ti3C2Tx sur- faces is ≈2.0 Å, which results in their chemisorption. The Wu et al. Journal of Nanobiotechnology Wu et al. Journal of Nanobiotechnology (2023) 21:17 (2023) 21:17 Page 8 of 11 Fig. 5 Density functional theory calculations of molecular properties. A Optimized interface structure of ZnO/Ti3C2Tx. B Calculated charge density difference of ZnO/Ti3C2Tx. C Work of adhesion at the ZnO/Ti3C2Tx interface. D Calculated charge density difference of ZnO/Ti3C2Tx-R6G. E UV–vis spectra of R6G, Ti3C2Tx, and Ti3C2Tx-R6G measured by Ultraviolet–visible Spectrophotometer. F Density of states of ZnO/Ti3C2Tx and ZnO/ Ti3C2Tx-R6G. G–I UV–vis spectra of (G) MB, Ti3C2Tx, and Ti3C2Tx-MB, (H) AB, Ti3C2Tx, and Ti3C2Tx-AB, and (I) CV, Ti3C2Tx, and Ti3C2Tx-CV measured by Ultraviolet–visible Spectrophotometer Fig. 5 Density functional theory calculations of molecular properties. A Optimized interface structure of ZnO/Ti3C2Tx. B Calculated charge density difference of ZnO/Ti3C2Tx. C Work of adhesion at the ZnO/Ti3C2Tx interface. D Calculated charge density difference of ZnO/Ti3C2Tx-R6G. E UV–vis spectra of R6G, Ti3C2Tx, and Ti3C2Tx-R6G measured by Ultraviolet–visible Spectrophotometer. F Density of states of ZnO/Ti3C2Tx and ZnO/ Ti3C2Tx-R6G. G–I UV–vis spectra of (G) MB, Ti3C2Tx, and Ti3C2Tx-MB, (H) AB, Ti3C2Tx, and Ti3C2Tx-AB, and (I) CV, Ti3C2Tx, and Ti3C2Tx-CV measured by Ultraviolet–visible Spectrophotometer of 10−6 M was obtained and characteristic peaks at 925 cm−1 and 1040 cm−1 [45] were clearly observed at microRNA concentrations from 10−4 to 10−6 M. The measured Raman intensities show a certain linear rela- tionship at the two characteristic peaks of 925 cm−1 and 1040 cm−1(R2 925 cm −1 = 0.9858, R2 1040 cm −1 = 0.9865) with a detection limit of 10–6 M. We speculate that there are two possible causes of this phenomenon. Author details 1 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital School of Medicine, Shanghai Jiao Tong University, Shanghai 200032, China. 2 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, International Joint Laboratory for Advanced Fiber and Low‑Dimension Materials, College of Materials Science and Engi- neering, Donghua University, Shanghai 201620, China. 3 State Key Laboratory of Chemical Engineering, School of Chemical Engineering, East China Univer- sity of Science and Technology, Shanghai 200237, China. Received: 11 October 2022 Accepted: 22 December 2022 Received: 11 October 2022 Accepted: 22 December 2022 Author contributions YS, YL and YD worked together in designing the conceptualization of this project. YD participated in the guidance of the work throughout the whole process. ZW performed the experiments, drew schematics and wrote the manuscript. DZ assisted in evaluating the results, discussion and editing the manuscript. XH and JL provided the software and calculation. All authors read and approved the final manuscript. 12. Le Ru EC, Etchegoin PG. Single-molecule surface-enhanced Raman spectroscopy. Annu Rev Phys Chem. 2012;63:65–87. 13. Michaels AM, Nirmal M, Brus LE. Surface enhanced Raman spectroscopy of individual rhodamine 6G molecules on large Ag nanocrystals. J Am Chem Soc. 1999;121:9932–9. 14. Stöckle RM, Suh YD, Deckert V, Zenobi R. Nanoscale chemical analysis by tip-enhanced Raman spectroscopy. Chem Phys Lett. 2000;318:131–6. Competing interests Th h d l h The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1. Liang J, Teng P, Xiao W, He G, Song Q, Zhang Y, et al. Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identifica- tion of SARS-CoV-2 from clinical samples. J Nanobiotechnol. 2021;19:273. 1. Liang J, Teng P, Xiao W, He G, Song Q, Zhang Y, et al. Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identifica- tion of SARS-CoV-2 from clinical samples. J Nanobiotechnol. 2021;19:273. 2. Bock S, Choi YS, Kim M, Yun Y, Pham XH, Kim J, et al. Highly sensitive near-infrared SERS nanoprobes for in vivo imaging using gold-assembled silica nanoparticles with controllable nanogaps. J Nanobiotechnol. 2022;20:130. 2. Bock S, Choi YS, Kim M, Yun Y, Pham XH, Kim J, et al. Highly sensitive near-infrared SERS nanoprobes for in vivo imaging using gold-assembled silica nanoparticles with controllable nanogaps. J Nanobiotechnol. 2022;20:130. 3. Cardinal MF, Vander Ende E, Hackler RA, McAnally MO, Stair PC, Schatz GC, et al. Expanding applications of SERS through versatile nanomaterials engineering. Chem Soc Rev. 2017;46:3886–903. 3. Cardinal MF, Vander Ende E, Hackler RA, McAnally MO, Stair PC, Schatz GC, et al. Expanding applications of SERS through versatile nanomaterials engineering. Chem Soc Rev. 2017;46:3886–903. 4. Ding S-Y, You E-M, Tian Z-Q, Moskovits M. Electromagnetic theories of surface-enhanced Raman spectroscopy. Chem Soc Rev. 2017;46:4042–76. 5. Zong C, Xu M, Xu L-J, Wei T, Ma X, Zheng X-S, et al. Surface-enhanced Raman spectroscopy for bioanalysis: reliability and challenges. Chem Rev. 2018;118:4946–80. 6. Langer J, Jimenez de Aberasturi D, Aizpurua J, Alvarez-Puebla RA, Auguié B, Baumberg JJ, et al. Present and future of surface-enhanced Raman scattering. ACS Nano. 2020;14:28–117. 6. Langer J, Jimenez de Aberasturi D, Aizpurua J, Alvarez-Puebla RA, Auguié B, Baumberg JJ, et al. Present and future of surface-enhanced Raman scattering. ACS Nano. 2020;14:28–117. Declarations resulting in different signal intensities that enhanced by ZnO/Ti3C2Tx substrate. Besides, the ZnO/Ti3C2Tx sub- strate yielded a much lower Raman intensity than that obtained using a SERS substrate composed of Au nano- particles (Additional file 1: Figure S8a and b). However, the signal stability of the SERS performance of ZnO/ Ti3C2Tx was higher, especially at the peaks of 925 cm−1 and 1040 cm−1, characteristic peaks can be clearly iden- tified even at 10–6 M, which may result from its greater analyte-concentrating ability compared with that of the hydrophilic Au nanoparticles. The stable SERS perfor- mance of ZnO/Ti3C2Tx suggests it may provide a new substrate for biological analyte detection. resulting in different signal intensities that enhanced by ZnO/Ti3C2Tx substrate. Besides, the ZnO/Ti3C2Tx sub- strate yielded a much lower Raman intensity than that obtained using a SERS substrate composed of Au nano- particles (Additional file 1: Figure S8a and b). However, the signal stability of the SERS performance of ZnO/ Ti3C2Tx was higher, especially at the peaks of 925 cm−1 and 1040 cm−1, characteristic peaks can be clearly iden- tified even at 10–6 M, which may result from its greater analyte-concentrating ability compared with that of the hydrophilic Au nanoparticles. The stable SERS perfor- mance of ZnO/Ti3C2Tx suggests it may provide a new substrate for biological analyte detection. DFT calculation of charge transfer properties f h l h h h As can be seen in Additional file 1: Figure S7, the distribu- tion of analytes was uneven at low concentration, which result in the uneven distribution of Raman signal. Another possibility is that different nucleobase pairs (U and A) adsorb differently to the substrate [46–48], performed as can be seen in Additional file 1: Figure S6, with the decrease of analyte concentration, the distri- bution of Raman signal becomes uneven. This indicates that the distribution of molecules on the substrate is not that uniform with decreasing concentration, which result in the signal variation. A more reasonable sam- pling method is to first find the location of the analyte by point measurement, and then do the area mapping based on this, then analyze the area where the signal is more concentrated. Finally, the ability of the ZnO/Ti3C2Tx substrate to detect biological macromolecules was assessed. Using microRNA as an analyte (Fig. 6e), a detection limit Wu et al. Journal of Nanobiotechnology (2023) 21:17 Page 9 of 11 Wu et al. Journal of Nanobiotechnology Fig. 6 A SEM image of the ZnO/Ti3C2Tx substrate. B Spatial map of the SERS intensity (at 1360 cm−1) of 10−6 M R6G drop-cast on ZnO/Ti3C2Tx (n = 16 × 18). C SEM images of Au nanoparticles distributed on the ZnO/Ti3C2Tx substrate, inset provides the enlarged image of selected section. D The SERS intensity distribution of the 1360 cm−1 peak across all mapping sites in panel b. E SERS spectra of 10−4 to 10−6 M microRNA on ZnO/ Ti3C2Tx. F Linear fit plots of peaks of 925 cm−1 and 1040 cm.−1, respectively (n = 9) Fig. 6 A SEM image of the ZnO/Ti3C2Tx substrate. B Spatial map of the SERS intensity (at 1360 cm−1) of 10−6 M R6G drop-cast on ZnO/Ti3C2Tx (n = 16 × 18). C SEM images of Au nanoparticles distributed on the ZnO/Ti3C2Tx substrate, inset provides the enlarged image of selected section. D The SERS intensity distribution of the 1360 cm−1 peak across all mapping sites in panel b. E SERS spectra of 10−4 to 10−6 M microRNA on ZnO/ Ti3C2Tx. F Linear fit plots of peaks of 925 cm−1 and 1040 cm.−1, respectively (n = 9) Page 10 of 11 Page 10 of 11 Page 10 of 11 Wu et al. Journal of Nanobiotechnology (2023) 21:17 Wu et al. Journal of Nanobiotechnology (2023) 21:17 Acknowledgements 9. Chen R, Du X, Cui Y, Zhang X, Ge Q, Dong J, et al. 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https://openalex.org/W2024796943	http://www.journal-imab-bg.org/statii-10/vol16_b4_p14-17.pdf	English	null	INTERDISCIPLINARY APPROACH IN COMPEX TREATMENT OF ORAL LICHEN RUBER PLANUS /Review and a Case report/	Journal of IMAB	2,010	cc-by-sa	2,551	DOI: 10.5272/jimab.1642010_14-17 DOI: 10.5272/jimab.1642010_14-17 Journal of IMAB - Annual Proceeding (Scientific Papers) vol. 16, book 4, 2010 triggered by: triggered by: • Drugs - often due to gold therapy, some antibiotics, non steroidal anti-inflammatory drugs (NSAIDs), sulfonylureas, beta-blockers, and some angiotensin- converting enzyme (ACE) inhibitors. These drugs more commonly cause a skin eruption with the mouth being affected less often. Lichen planus(LP)is a common chronic mucocuta- neous disease of unknown etiology. Usually, it presents with lesions on the flexor surfaces of the upper extremities, genitalia and mucous membranes. Oral lesions can be the initial as well as the only manifestations of this condition. Patients with LP can present first to the general dental practitioner so sufficient knowledge on this condition is important for the proper diagnosis, treatment and prevention of further complications. The clinical management of LRP requires liaison between dental and medical specialists. • Contact allergens in dental restorative materials or toothpastes. 6 • Contact allergens in dental restorative materials or toothpastes. 6 • Mechanical trauma (Koebner phenomenon) 7 10 • Viral infection, particularly Hepatitis C 7, 10 10 This article provides a review of the current literature on LP and a case report of a 55 years old female suffering with this condition. A multidisciplinary approach in the diagnosis and management of this case is described. • In people who habitually chew betel quid.10 Oral lichenoid lesions are also part of the spectrum of chronic graft-versus-host disease that occurs after bone marrow transplantation.11 Key words: oral lichen planus, corticosteroids, retinoids, oral cavity, precancerous conditions/pathology In many patients, the onset of oral lichen planus is insidious, and patients are unaware of their oral condition. In such instances the General dental practitioner identifies the clinical changes in the oral mucosa. Dinkova At., D. Gospodinov, G. Gavasova, R. Cholakova, H. Daskalov, Iv. Chenchev Oral surgery department, Faculty of Dental Medicine, Medical University - Plovdiv, Bulgaria Oral surgery department, Faculty of Dental Medicine, Medical University - Plovdiv, Bulgaria 14 / JofIMAB; Issue: vol. 16, book 4, 2010 / INTERDISCIPLINARY APPROACH IN COMPEX TREATMENT OF ORAL LICHEN RUBER PLANUS /REVIEW AND A CASE REPORT/ Dinkova At., D. Gospodinov, G. Gavasova, R. Cholakova, H. Daskalov, Iv. Chenchev Dinkova At., D. Gospodinov, G. Gavasova, R. Cholakova, H. Daskalov, Iv. Chenchev INTRODUCTION Other patients report sensitivity of the oral mucosa to hot or spicy foods or oral hygiene products, painful oral mucosa, sore gums, red or white patches on the oral mucosa, red gums, or oral ulcerations. A broad range of systemic diseases may have oral lesions, including lichen planus. Lichen planus (LP) is a pruritic, papular eruption characterized by its violaceous color; polygonal shape; and, sometimes, fine scale. Approximately two thirds of patients report oral discomfort, especially in association with atrophic and erosive lesions. The oral pain exacerbated by trauma and hot spicy, or acidic foods. It was first described by Wilson in 1869 and is thought to affect 0.5–1% of the world’s population.9 The condition is a cell-mediated immune response of unknown origin and can affect either the skin or mucosa or both. Oral lesions and present as white striations (Wickham striae), white papules, white plaques, erythema (mucosal atrophy), erosions (shallow ulcers), or blisters. The lesions predominantly affect the buccal mucosa, tongue, and gingivae and usually are bilateral.They are classified as reticular, plaquelike, atrophic, papular, erosive, and bullous. Lichen planus is most commonly found on the flexor surfaces of the upper extremities, scalp, nails, on the genitalia, and on the mucous membranes. About half of the patients with skin lesions have oral lesions, whereas about 25% present with oral lesions alone.1, 2, 4, 9 In contrast to cutaneous lichen planus, the oral form may persist for up to 25 years with periods of exacerbation and quiescence. During periods of exacerbation (linked to psychological stress and anxiety), the area of erythema or erosion increases, with increased pain and sensitivity. During periods of quiescence, the area of erythema or erosion It affects woman more often than men in a ratio 2:3 and predominantly occurs in adults older than 40 years. 9 In most cases appears unexpectedly and is called idiopathic oral lichen planus. In other cases oral lichenoid reactions may be 14 / JofIMAB; Issue: vol. 16, book 4, 2010 / 14 decreases, with decreased pain and sensitivity. 1 decreases, with decreased pain and sensitivity. 1 The patient reports aggravation of the discomfort and sensitivity in the oral lesion from acidic or spicy food and drinks. The patient also suffered with skin eruptions on the flexor surfaces of the upper extremities twice in the past year. These were treated with Clobetasol propionate ungvent. INTRODUCTION Up to 44% of patients with oral lichen planus develop coincident skin lesions: flexor aspects of the wrists or ankles, the extensor aspects of the lower legs, the skin of the lower central part of the back, and the natal cleft. The genitals might be involved as well as the nails and scalp. Rarely, laryngeal, esophageal, and conjunctival involvement occur.1, 2 9 The past medical history includes a hepatitis A infection at the age of 8 and an allergic dermatitis associated with chemicals used at her work place 20 years ago. The patient is otherwise fit and healthy and is not taking any medication. She is a non-smoker does not use alcohol. A specialist in oral pathology or a dermatologist typically makes the primary diagnosis of oral lichen planus.7 Histopathologic examination of lesional tissue is the most relevant investigation in cases of oral lichen planus. There was no family history with similar findings. The general physical examination revealed a moderately built patient with satisfactory vital signs. There were no apparent skin lesions at the time of the assessment. g p Opinions may be sought from the following specialists if patients have relevant signs or symptoms: Right submandibular lymph nodes with soft consistency were palpable on the extra-oral examination. Dermatologist - For the diagnosis, treatment, and review of skin, nail, genital, and scalp lesions Intraorally, there were white hyperkeratotic white striations (Wickham striae) bilateral on the buccal mucosa with normal surrounding mucosa without any clinical manifestation of inflammation or ulceration. An ovoid ulceration (5mm ´ 15mm) on the right lateral margin of the tongue was also present. On palpation, the lesion of the tongue was slightly painful without oozing. The surrounding mucosa was of normal consistence without signs of induration. The bottom of the ulceration was brown in color. White streaks at the dorsum of the tongue were also present. The patient also had chronic generalized gingivitis and erithema on the hard palate. Otolaryngologist - For the diagnosis, treatment, and review of laryngeal and esophageal lesions Ophthalmologist - For the diagnosis, treatment, and review of conjunctival lesions Gynecologist - For the diagnosis, treatment, and review of vulval and vaginal lesions Clinically, the differential diagnosis should include lichenoid reactions, leukoplakia, squamous cell carcinoma, pemphigus, mucous membrane pemphigoid, and candidiasis. INTRODUCTION Medical treatment of oral lichen planus (OLP) is essential for the management of painful, erythematous, erosive, or bullous lesions.Topical corticosteroids are the mainstay of medical treatment.1, 2, 5, 8,10 Picture 1. Picture 1. Other potential therapies include systemic corticosteroids, antihistamines, hydroxychloroquine, azathioprine, mycophenolate, dapsone, and topical and systemic retinoids. 1, 2, 5, 8, 10 If systemic drug therapy (eg, treatment with NSAIDs, antimalarials, or beta-blockers) is suspected as the cause of oral lichenoid lesions, changing to another drug may be worthwhile. However, the switch rarely resolves the erosions, and almost never resolves the white patches of oral lichen planus. Because patients with OLP may be at an increased risk for the development of squamous cell carcinoma, periodic follow-up is mandatory to detect malignant transformation.4, 7, 9, 10 CASE REPORT: A 55-year-old female presented at the Oral surgery department, Medical university, Plovdiv, complaining of a painful ulceration on the right margin of the tongue with a 6 months history. Picture 1. Picture 1. The patient relates the onset of the symptoms with a local trauma. Her general medical practitioner advised application of Gerytamine (Vit. A, Vit. E, Soya butter). Although the patient noticed certain improvement and reduction in the lesion size after this treatment, the symptoms reoccurred following consumption of spicy food. / JofIMAB; Issue: vol. 16, book 4, 2010 / 15 The treatment in this case was carried out by a consultant dermatologist. The therapeutic regimen included Depo-Medrol® 0,04 (methylprednisolone acetate) Injectable Suspension, USP, 1 injection; Xyzal tabl. 0,02; 1 tabl. per day For 20 days; Elocom solution - for topical treatment, Mometasone furoate for topical treatment of the skin lesions once per day. The patient was advised to avoid spicy food, comply with a healthy diet rich in fresh fruit and vegetables and visit her dentist regularly for ongoing prevention. In the re-examine after a year the lesions in the oral cavity persists, but there aren’t any new ulcerations. White striations are present on the lips. The patient hasn’t any complaints. 6 / JofIMAB; Issue: vol. 16, book 4, 2010 / Picture 2. The initial diagnosis based on the clinical exam was oral lichen planus. Differential diagnosis was made with Pemphigus Vulgaris, lichenoid reactions, leukoplakia, squamous cell carcinoma, , mucous membrane pemphigoid, and candidiasis. The diagnosis of Lichen planus mucosae oris (Lichen erosivus region gingivae superior et inferior) was also confirmed by specialist dermatologist. Laboratory tests were undertaken to confirm the definite diagnosis. Candida albicans test was negative and the blood test was normal. A test for bimetallism was found positive. Nevertheless this finding is rarely directly related to the etiology of this condition. Tests for contact allergy to dental amalgam and its ingredients have proven negative. The ulcerative lesion on the tongue was excised along with an area of the surrounding mucosae. Picture 3. The histological findings (¹ 87, 88, 89 / 13.03-09) showed subepithelial mononuclear infiltrate consisting of lymphocytes and histiocytes, parakeratosis and acanthosis. This led to the definite diagnosis of Lichen rubber planus. Picture 4. Picture 5. Picture 3. The histological findings (¹ 87, 88, 89 / 13.03-09) showed subepithelial mononuclear infiltrate consisting of lymphocytes and histiocytes, parakeratosis and acanthosis. This led to the definite diagnosis of Lichen rubber planus. Picture 3. The histological findings (¹ 87, 88, 89 / 13.03-09) showed subepithelial mononuclear infiltrate consisting of lymphocytes and histiocytes, parakeratosis and acanthosis. This led to the definite diagnosis of Lichen rubber planus. Picture 4. Picture 5. Picture 5. Picture 3. The histological findings (¹ 87, 88, 89 / 13.03-09) showed subepithelial mononuclear infiltrate consisting of lymphocytes and histiocytes, parakeratosis and acanthosis. This led to the definite diagnosis of Lichen rubber planus. Picture 5. Picture 5. 16 / JofIMAB; Issue: vol. 16, book 4, 2010 / 16 Picture 6. condition. Biopsy and histological examination are essential for obtaining a definitive diagnosis. Therefore the management of OLP requires liaison between dental and medical specialists. The principal aims of current oral lichen planus therapy, which remain symptomatic, are the resolution of painful symptoms, the resolution of oral lesions, the reduction of the risk of malignant transformation, and the maintenance of good oral hygiene. In patients with recurrent painful disease, another goal is the prolongation of their symptom-free intervals. Systemic corticosteroids must be used cautiously and with discretion. Picture 6. 2. The general dental practitioner can be the first to detect Oral Lichen Ruber planus, especially where the oral lesions are the single manifestation of the condition. Adequate approach in these cases and prompt referral to a specialist dermatologist is necessary. DISCUSSION The incidence of oral lichen planus ranges between 0.5%-1% among the populations. About half of the patients with skin lesions have oral lesions, whereas about 25% present with oral lesions alone. 9 3. The malignant potential of lichen planus is still controversial and it has been commonly associated with the atrophic and erosive forms. Therefore patients should be observed periodically, particularly those with the erosive or atrophic forms and asked to report any changes in their lesions and/or symptoms. The existence of skin lesions may be helpful to confirm the diagnosis of oral lichen planus. However in the cases with oral manifestations only the general dental practitioner can be the first to detect the 9. N. Mollaoglu; Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey; Oral lichen planus: a review; British Journal of Oral and Maxillofacial Surgery (2000) 38, 370–377; doi:1054/bjom.2000.0335 the rationale for treating oral lichen planus?. Oral Dis. Jul 1999;5(3):181-2. 11. Sugerman PB, Savage NW, Walsh LJ, et al. The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med. 2002; 13(4): 350-65. 10. Scully C, Beyli M, Ferreiro MC, et al. Update on oral lichen planus: etiopathogenesis and management. Crit Rev Oral Biol Med. 1998;9(1):86-122 7. Lodi G, Scully C, Carrozzo M, Griffiths M, Sugerman PB, Thongprasom K. Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etio- pathogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. Jul 2005; 100(1): 40-51. CONCLUSIONS 1. An interdisciplinary approach is essential in the diagnosis, treatment, management and ongoing care for the patients suffering with Oral Lichen Ruber planus. Picture 6. REFERENCES: the rationale for treating oral lichen planus?. Oral Dis. Jul 1999;5(3):181-2. the rationale for treating oral lichen planus?. Oral Dis. Jul 1999;5(3):181-2. 4(2): 141-58. 1. Áîòóøàíîâ. Ï., Äèàãíîñòèêà è ëå÷åíèå íà çàáîëÿâàíèÿòà íà óñòíàòà ëèãàâèöà, 1997 ã.; 56-58 6. Koch P, Bahmer FA. Oral lesions and symptoms related to metals used in dental restorations: a clinical, allergological, and histologic study. J Am Acad Dermatol. Sep 1999; 41(3 Pt 1):422- 30. 9. N. Mollaoglu; Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey; Oral lichen planus: a review; British Journal of Oral and Maxillofacial Surgery (2000) 38, 370–377; doi:1054/bjom.2000.0335 2. Óãðèíîâ Ð., Ëèöåâî-÷åëþñòíà è Îðàëíà õèðóðãèÿ, 2006 ã.; 476-477 3. Axell T, Rundquist L. Oral lichen planus-a demographic study. Community Dent Oral Epidemiol. Feb 1987;15(1):52- 6. 7. Lodi G, Scully C, Carrozzo M, Griffiths M, Sugerman PB, Thongprasom K. Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etio- pathogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. Jul 2005; 100(1): 40-51. 7. Lodi G, Scully C, Carrozzo M, Griffiths M, Sugerman PB, Thongprasom K. Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etio- pathogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. Jul 2005; 100(1): 40-51. 10. Scully C, Beyli M, Ferreiro MC, et al. Update on oral lichen planus: etiopathogenesis and management. Crit Rev Oral Biol Med. 1998;9(1):86-122 4. Eisen D. The clinical features, malignant potential, and systemic associations of oral lichen planus: a study of 723 patients. J Am Acad Dermatol. Feb 2002; 46(2):207-14. 11. Sugerman PB, Savage NW, Walsh LJ, et al. The pathogenesis of oral lichen planus. Crit Rev Oral Biol Med. 2002; 13(4): 350-65. 5. Eisen D. The therapy of oral lichen planus. Crit Rev Oral Biol Med. 1993; 5. Eisen D. The therapy of oral lichen planus. Crit Rev Oral Biol Med. 1993; 8. McCartan B, McCreary C. What is Address for correspondence: Atanaska Dinkova, DMD 3 Ledenuka str., Plovdiv 4002 , Bulgaria tel: +359/886 711 031 E-mail: dinkova_asia@yahoo.com Address for correspondence: Atanaska Dinkova, DMD 3 Ledenuka str., Plovdiv 4002 , Bulgaria tel: +359/886 711 031 E-mail: dinkova_asia@yahoo.com / JofIMAB; Issue: vol. 16, book 4, 2010 / 17
https://openalex.org/W4283575157	https://aisberg.unibg.it/bitstream/10446/226890/1/Gestalt%20Filmology_def.pdf	English	null	null	Cinema & cie/Cinéma & Cie	2,022	cc-by	79,342	vol. 22 · no. 38 38 · 2022 GESTALT FILMOLOGY GESTALT FILMOLOGY INSIGHTS ON FORM AND EMBODIMENT INSIGHTS ON FORM AND EMBODIMENT IN THE FILM EXPERIENCE IN THE FILM EXPERIENCE EDITED BY ADRIANO D’ALOIA AND IAN VERSTEGEN GESTALT FILMOLOGY GESTALT FILMOLOGY INSIGHTS ON FORM AND EMBODIMENT INSIGHTS ON FORM AND EMBODIMENT IN THE FILM EXPERIENCE IN THE FILM EXPERIENCE EDITED BY ADRIANO D’ALOIA AND IAN VERSTEGEN vol. 22 · no. 38 38 · 2022 GESTALT FILMOLOGY GESTALT FILMOLOGY INSIGHTS ON FORM AND EMBODIMENT INSIGHTS ON FORM AND EMBODIMENT IN THE FILM EXPERIENCE IN THE FILM EXPERIENCE EDITED BY ADRIANO D’ALOIA AND IAN VERSTEGEN GESTALT FILMOLOGY GESTALT FILMOLOGY INSIGHTS ON FORM AND EMBODIMENT INSIGHTS ON FORM AND EMBODIMENT IN THE FILM EXPERIENCE IN THE FILM EXPERIENCE EDITED BY ADRIANO D’ALOIA AND IAN VERSTEGEN Editorial Staff Giorgio Avezzù, Università degli Studi di Bergamo Nicholas Berrettini, Yale University Alice Cati, Università Cattolica del Sacro Cuore Anna Caterina Dalmasso, Università degli Studi di Milano Laura Cesaro, Università degli Studi di Udine Elena Gipponi, Università IULM Dominic Holdaway, Università degli Studi di Urbino “Carlo Bo” Gabriele Landrini, Università degli Studi di Bari “Aldo Moro” Alessandra Luciano, Centre National de l’Audiovisuel, Luxembourg Giovanna Maina, Università degli Studi di Torino Simona Pezzano, Università IULM Giuseppe Previtali, Università degli Studi di Begamo Marta Rocchi, Alma Mater Studiorum — Università di Bologna Simona Schneider, Università degli Studi di Udine Paolo Villa, Università degli Studi di Udine Nicolò Villani, Università degli Studi eCampus Diana Wade, Università Bocconi Giorgio Avezzù, Università degli Studi di Bergamo Nicholas Berrettini, Yale University Alice Cati, Università Cattolica del Sacro Cuore Anna Caterina Dalmasso, Università degli Studi di Milano Laura Cesaro, Università degli Studi di Udine Elena Gipponi, Università IULM Dominic Holdaway, Università degli Studi di Urbino “Carlo Bo” Gabriele Landrini, Università degli Studi di Bari “Aldo Moro” Alessandra Luciano, Centre National de l’Audiovisuel, Luxembourg Giovanna Maina, Università degli Studi di Torino Simona Pezzano, Università IULM Giuseppe Previtali, Università degli Studi di Begamo Marta Rocchi, Alma Mater Studiorum — Università di Bologna Simona Schneider, Università degli Studi di Udine Paolo Villa, Università degli Studi di Udine Nicolò Villani, Università degli Studi eCampus Diana Wade, Università Bocconi Editors Editors Valentina Re, Università degli Studi Link Campus University (coordinator) Adriano D’Aloia, Università degli Studi di Bergamo Francesco Di Chiara, Università degli Studi eCampus Simone Dotto, Università degli Studi di Udine Luisella Farinotti, Università IULM Barbara Grespi, Università degli Studi di Milano Veronica 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Mariagrazia Fanchi, Università Cattolica del Sacro Cuore André Gaudreault, Université de Montréal Malte Hagener, Philipps-Universität Marburg Vinzenz Hediger, Goethe-Universität Frankfurt am Main Erik Hedling, Lunds Universitet Mette Hjort, Hong Kong Baptist University François Jost, Université Sorbonne Nouvelle — Paris 3 Gertrud Koch, Freie Universität Berlin Gloria Lauri-Lucente, L-Università ta’ Malta Sandra Lischi, Università di Pisa Denilson Lopes, Universidade Federal de Rio de Janeiro Trond Lundemo, Stockholms Universitet Adrian Martin, Monash University Lisa Parks, Massachusetts Institute of Technology Guglielmo Pescatore, Alma Mater Studiorum — Università di Bologna Andrea Pinotti, Università degli Studi di Milano Francesco Pitassio, Università degli Studi di Udine Leonardo Quaresima, Università degli Studi di Udine Vicente Sánchez-Biosca, Universitat de València Bhaskar Sarkar, University of California, Santa Barbara Irmbert Schenk, Universität Bremen Petr Szczepanik, 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Italianistica e Culture Comparate, Università degli Studi di Bari ‘Aldo Moro’; Dipartimento di Lettere, Filosofia, Comunicazione, Università degli Studi di Bergamo; Dipartimento delle Arti — Visive Performative Mediali, Università di Bologna — Alma Mater Studiorum; Dipartimento di Scienze della Comunicazione e dello Spettacolo, Università Cattolica del Sacro Cuore; Università degli Studi eCampus (Novedrate, Italy); Dipartimento di Comunicazione, arti e media “Giampaolo Fabris”, Università IULM, Milano; Dipartimento di Civiltà e Forme del Sapere, Università di Pisa; Università degli Studi Link Campus University, Roma; Dipartimento di Studi Umanistici e del Patrimonio Culturale, Università degli Studi di Udine. Cinéma & Cie vol. 22, no. 38, 2022 Cinéma & Cie vol. 22, no. 38, 2022 DOI 10.54103/2036-461X/2022/38 ISSN 2036-461X (online) ISSN 2035-5270 (print) Cinéma & Cie vol. 22, no. 38, 2022 DOI 10.54103/2036-461X/2022/38 ISSN 2036-461X (online) ISSN 2035-5270 (print) Design and layout Francesca Grilli Design and layout Francesca Grilli Design and layout Francesca Grilli Cover image: Pages from Rudolf Arnheim’s notebook (Archives of the History of American Psychology – Akron, OH) 139 REVIEWS / COMPTES-RENDUS 151 PROJECTS & ABSTRACTS BEYOND CINEMA 139 REVIEWS / COMPTES-RENDUS Contents / Table des matières GESTALT FILMOLOGY INSIGHTS ON FORM AND EMBODIMENT IN THE FILM EXPERIENCE 9 Adriano D’Aloia, Ian Verstegen The Future of the Past. Arnheim and Film Today 19 Maarten Coëgnarts The Meaning Potential of Motion Vectors in Cinema 37 Philippe Bédard Points of Anchorage: Exo-centric Images and the Perceptual Relativity of Camera Movement 55 Maria Poulaki A Gestalt Theory for ‘Disorder’: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art 69 Ryan Pierson Gestalt, Animation, and the Culture of Design 83 Massimo Locatelli Paul Fraisse’s Psychology of Rhythm: A Case for Filmology? 97 Emilio Audissino The Aha, Ha! Moment: A Gestalt Perspective on Audiovisual Humour BEYOND CINEMA 121 Sharon Jane Mee Rhythm Beyond the Cinematic Medium/The Pixel Beyond the Movie Theatre 139 REVIEWS / COMPTES-RENDUS 151 PROJECTS & ABSTRACTS 160 Contributors / Collaborateurs 151 PROJECTS & ABSTRACTS 160 Contributors / Collaborateurs 5 The Future of the Past. Arnheim and Film Today Adriano D’Aloia, Università degli Studi di Bergamo Ian Verstegen, University of Pennsylvania1 Keywords Rudolf Arnheim Film as Art Gestalt Psychology Gestalt Filmology Neurofilmology DOI Keywords Rudolf Arnheim Film as Art Gestalt Psychology Gestalt Filmology Neurofilmology DOI 10.54103/ 2036-461X/17977 ARNHEIM, AGAIN Exactly fifteen years ago, on June 9, 2007, psychologist of art and media theorist Rudolf Arnheim died in Ann Arbor, Michigan, one month before his 103rd birthday (he was born in Berlin on July 15, 1904). He devoted his entire life to the study of the arts — starting with film in the 1920s — and is the author of Art and Visual Perception and Visual Thinking and many other masterpieces that continue to be essential points of reference for generations of students, scholars and professionals in the fields of analysis, criticism and the practice of the visual arts2. Arnheim is also considered one of the classic film theorists for his application of the assumptions of Gestalt psychology to film analysis presented for the first time 90 years ago in his essay Film als Kunst.3 His radical positions have been criticized in various eras and intellectual spheres, but they are in some respects still valid and are often unwittingly adopted by critics and scholars. As the essays of this special issue demonstrate, a recontextualization and revitalization of Arnheim’s film theory and, more generally, a Gestalt approach to the film experience, can be still productive today.4 Keywords Rudolf Arnheim Film as Art Gestalt Psychology Gestalt Filmology Neurofilmology DOI 10 54103/ is licensed under a Creative Commons Attribution 4.0 International License This task has been made easier in the past few years due to a series of publications that have clarified aspects of Arnheim’s theoretical project or else further specified some of his philosophical commitments.5 For years in film studies there was a standoff between psychoanalytic derived and more cognitive approaches. More recently, we have learned to nuance how to blend an approach that is not afraid of experimentation or quantification with one that can address the perennial problems of more speculative film studies like spectatorship, the gaze, enunciation and embodiment.6 The latter, the turn toward embodied approaches to the filmic experience, has largely made this new idea possible. Yet many writers would still be hesitant that there is contained in Arnheim’s work or Gestalt theory more largely the pieces of a kind of theory of Gestalt embodiment. In some ways, such a theory would clarify some of the more untenable elements of embodiment theories which in general have been 9 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X adopted as specular reversals of cognitive theory.7 The body is meant to erase representation, when properly elaborated a Gestalt embodiment might show how the body and its role in filmic experience is still self-conscious because even awareness of the body is a form of consciousness. The present special issue of Cinéma & Cie goes deeper into some of Arnheim’s theories, broadening the platform of his work into Gestalt psychology more generally, and addressing question of fit between older theories and newer tendencies. THE PARTS AND THE WHOLE A multifaceted and passionate thinker, throughout the twentieth century Arnheim turned his interest to a variety of media and arts — from cinema to photography, from radio to television, from painting to sculpture, from architecture to video art —, applying with rigor and extending with creativity the assumptions of the Gestalt psychology method he learned in Berlin in the 1920s directly from its founders Max Wertheimer and Wolfgang Köhler. In clear opposition to the psychological and philosophical currents dominant at the time, in particular behaviorism and idealism, Gestalttheorie theorized perception as an immediate act through which the mind, in a predominantly innate way, organizes sensible data according to certain laws of ‘unification’ of single parts into a whole other than their simple sum. Distinct elements of the visual field tend to be perceived as belonging to a single overall configuration by virtue of their similarity, closeness, common destiny, continuity of direction, closure, figure-ground relationship, meaningfulness. In short, the images — or rather some of their qualities — ‘communicate’ directly with the observer, eliciting a mediation and organization of the visible that the human eye and mind tend to intuitively grasp, supported by their physiology. Gestalt psychology cannot be reduced to a descriptive theory of optical phenomena, it is indeed a theory of mind based on the elective relationship between perception and cognition. Optical illusions and ambiguous figures (such as Kanizsa’s triangle, Rubin’s vase, or Jastrow’s duck-rabbit) are evidence of the discrepancy between the physical object and its phenomenal perception. For Gestalt psychology and in particular for Arnheim, who applied its laws to artistic phenomena, perceiving is always also thinking, reasoning is also intuition, observation is also invention. The image is traversed by a system of forces which, in the eyes of the observer, make it more or less dynamic, unstable, tending to a momentarily disrupted equilibrium. Thanks to its gestalt qualities, the image expresses some relevant aspects of human experience, including emotions. Grasping the meaning of an image therefore means actively participating in the adventure of its perceptual organization. An important element of properly understanding Arnheim — and one which is present in the following volumes — is the recognition that although moving forms are phenomenally immediate, it is the spatial, temporal and multi-medial 10 D’Aloia, Verstegen, The Future of the Past context that dynamically shifts their meaning. FORMATIVE FORMALISM At the turn of the 1920s and 1930s, Arnheim made an original application of Gestalt laws to cinema, a phenomenon of considerable aesthetic importance which, however, had not been studied with a scientific approach until then. There was no better laboratory than cinema theaters to test the principles according to which the eye captures the forces, the vectors, the dynamism, the intensity and all the expressive element of a work of art on the basis of the systemic relationships between the visual elements involved. Also based on a large number of examples collected during his activity as a film critic at Die Weltbühne (a leading periodical of the Weimar Republic), in 1932 Arnheim composed an impressive volume entitled Film als Kunst 8. The book was soon banned by the Nazi regime due to its author’s Jewish origins and a too direct allusion to the similarity between the mustache of Charlie Chaplin’s Charlot and those of Hitler in one of his review. Film als Kunst, however, was immediately translated into English and began to circulate in some Italian intellectual circles, in particular among the teachers and students of the Centro Sperimentale di Cinematografia in Rome. Having fled from Germany, Arnheim arrived in Rome in the summer of 1933 and for the following five years was one of the main editors of the magazines Intercine and Cinema. On the columns of these magazines he published a large number of articles on the psychological and aesthetic aspects of film, many of which were signed with various pseudonyms which have only recently been directly attributed.9 His Italian ‘idyll’ was destined to end soon: in 1938, the promulgation of the racial laws in Italy forced him to a new escape (first to London and then to New York), which corresponded to a sort of ‘denial’ of cinema, of which he will return to deal only sporadically or incidentally.10 of cinema, of which he will return to deal only sporadically or incidentally. Film als Kunst is a tormented and controversial book not only for the political context in which it hardly begins to circulate, but also for its content. It was at the end of the 1920s that the transition from silent to sound was taking place and Arnheim opposed this and other technical innovations — color, the panoramic format — by taking a defensive position on the specificity of cinema. THE PARTS AND THE WHOLE Therefore, there is an inherent provisional and fallible background to any observations made in an Arnheimian manner. His theory is the opposite of a dogmatic formalism and is therefore highly useful in our contemporary context because it links the ultimate meaning of the filmic work to analytic methods that are not reductive. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X FORMATIVE FORMALISM On the other hand, Arnheim considers inappropriate and gratuitous — formalist instead of formative — the abundant use of extreme close shots in Theodor Dreyer’s The Passion of Joan of Arc (1928), a solution that hides action and therefore prevents the viewer’s full understanding of the narrative. Second, the possibility of composing the shot by exploiting the ‘duplicity’ of the image, which gives both the impression of a real event thanks to the illusion of depth, but it is still a two-dimensional image. Thus, for example, in King Vidor’s The Crowd (1928) the shot of young John Sims climbing the stairs, already suspecting that he has lost his father, is not only a body that approaches the camera, but also a figure that enlarges by progressively widening towards the angles of the frame, physically and symbolically going towards his adult age. Third, the possibility of expressing inner states or symbolic meanings through the grayscale, lighting and contrast, without resorting to color, as in Walter Ruttmann’s Berlin Symphony of a Great City (1927). For Arnheim, the availability of the entire color range is by no means an advantage for the film artist. Indeed, it is the grayscale, in its complex essentiality, that offers the possibility of expressing symbolic contents. It is no coincidence that Arnheim was on the jury of the 1964 Venice Film Festival which awarded the Leone d’oro to Michelangelo Antonioni’s Red Desert, a film in which an non-naturalistic use of color is the reflection of the characters interiority. Fourth, the use of the limits of the frame to exclude and then reveal portions of the scene (off-screen space), to ‘cut out’ and bring a significant detail closer (close-up) or to play with the dimensions and proportions of objects placed at different depth levels (deep focus). The almost-squared aspect ratio in use in the 1910s and 1920s helped the viewer to have a comprehensive view of the action depicted on the screen, which is Yet precisely from its limitations with respect to reality, cinema has the possibility of being art, as the use of these ‘differentiating factors’ as ‘formative means’ allowed the ‘cinematographic artist’ to make a creative compensation of the gaps. Arnheim therefore describes the artistic use of each factor. First, the possibility of choosing a particular point of view, thus creating a surprise effect from a hiding/revelation dynamic. D’Aloia, Verstegen, The Future of the Past FORMATIVE FORMALISM As eminently a visual medium, in order to claim its artistic status, cinema had to avoid contamination with non-original means of expression and to remain autonomous in developing and using its own language artistically. These contaminations were in fact evident concessions to spectacle and commercial success, but above all a fatal reproductive approach to reality, from which the film had to keep away. This ‘purist’ position remained substantially unchanged over time despite the advances in the art of cinema. In the new edition of the book, 11 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X published in the United States in 195711, the entire second part was replaced by a series of writings dating back to the Italian period, including the well-known essay A New Laocoön (1938) which confirmed his opposition to talkies.12 At the turn of the 1950s and 1960s, however, in the wake of the success of Art and Visual Perception, the interest around Arnheim theory of cinema rekindled. The fundamental assumptions of Arnheimian film theory is that cinema is not a means of simple mechanical transcription of reality, but a reality in its own right which dialogues with the former by difference. The transposition of reality on the screen brings out the insuperable partiality of the human eye (and of the camera) in front of the natural world. Arnheim identifies and describes a series of ‘absences’: the absence of depth, of color, of off-screen space, of space-time continuity, of non-visual stimuli.13 of absences : the absence of depth, of color, of off screen space, of space time continuity, of non-visual stimuli.13 Yet precisely from its limitations with respect to reality, cinema has the possibility of being art, as the use of these ‘differentiating factors’ as ‘formative means’ allowed the ‘cinematographic artist’ to make a creative compensation of the gaps. Arnheim therefore describes the artistic use of each factor. First, the possibility of choosing a particular point of view, thus creating a surprise effect from a hiding/revelation dynamic. For example in the opening scene of Charles Chaplin’s The Immigrant, the rear angle suggests that the character, leaning on the railing of the ship, is feeling bad, while it is later revealed that he was only fishing. FORMATIVE FORMALISM For example in the opening scene of Charles Chaplin’s The Immigrant, the rear angle suggests that the character, leaning on the railing of the ship, is feeling bad, while it is later revealed that he was only fishing. On the other hand, Arnheim considers inappropriate and gratuitous yi g , pp p g — formalist instead of formative — the abundant use of extreme close shots in Theodor Dreyer’s The Passion of Joan of Arc (1928), a solution that hides action and therefore prevents the viewer’s full understanding of the narrative. Second, the possibility of composing the shot by exploiting the ‘duplicity’ of the image, which gives both the impression of a real event thanks to the illusion of depth, but it is still a two-dimensional image. Thus, for example, in King Vidor’s The Crowd (1928) the shot of young John Sims climbing the stairs, already suspecting that he has lost his father, is not only a body that approaches the camera, but also a figure that enlarges by progressively widening towards the angles of the frame, physically and symbolically going towards his adult age. Third, the possibility of expressing inner states or symbolic meanings through the grayscale, lighting and contrast, without resorting to color, as in Walter Ruttmann’s Berlin Symphony of a Great City (1927). For Arnheim, the availability of the entire color range is by no means an advantage for the film artist. Indeed, it is the grayscale, in its complex essentiality, that offers the possibility of expressing symbolic contents. It is no coincidence that Arnheim was on the jury of the 1964 Venice Film Festival which awarded the Leone d’oro to Michelangelo Antonioni’s Red Desert, a film in which an non-naturalistic use of color is the reflection of the characters interiority. Fourth, the use of the limits of the frame to exclude and then reveal portions of the scene (off-screen space), to ‘cut out’ and bring a significant detail closer (close-up) or to play with the dimensions and proportions of objects placed at different depth levels (deep focus). The almost-squared aspect ratio in use in the 1910s and 1920s helped the viewer to have a comprehensive view of the action depicted on the screen, which is 12 D’Aloia, Verstegen, The Future of the Past instead prevented by the horizontal expansion of the format (as in Abel Gance’s panoramic Napoléon [1927]). FORMATIVE FORMALISM Fifth, the possibility of unnaturally reconstituting the fragments of space and time through editing. Finally, the ability to arouse sound perceptions using only images (in Josef von Sternberg’s The Docks of New York [1928], the shot of a gun corresponds to the sudden flight away of a flock of birds). Arnheim accuses sound and in particular the dialogue of paralyzing the action, as it relieves the actor from the need to use the body and facial expressions as primary means of communication. Moreover, the absence of the nonvisual world of the senses, allows the film to arouse a sense of vertigo by exploiting the discrepancy between the viewer physical immobility and the camera movements. The artistic use of these means made it possible to combine form and content: simple aesthetic solutions could immediately express inner states and symbolic meanings. The set of these means constituted the specific aesthetic language of cinema — a system that today we take for granted, but which in the 1930s still had to be described systematically. Arnheim had already described the developed machine-like money profit orientation of the Berlin cinema industry, and in America saw this expanded in the Hollywood system.14 Once the talking film could no longer be dislodged from the tastes of the public and the production schedules of the studios, he immediately recognized the new importance of ideological analysis, which was developed in his friendship with Siegfried Kracauer. At the same time, as a keen observer of technological issues, he was considering the consequences to cinema of the improvement of film stocks. Sharper and better emulsions almost required a shift toward realism, while he predicted that the traditional aims of cinema could be satisfied through animation. A KINOGESTALTTHEORIE Film als Kunst was therefore a retrospective book, aimed at discussing the effect of technological innovations on the great age of cinema as art. However, the immutability of Arnheim’s position over the years, made his theory even more radical (as well as more criticized, especially in its method15). Despite the intrinsic limitations of Film as Art, in fact, the laws of Gestalt psychology continue to act in our perceptual experience of visual artifacts (as well as in their production), including films. Even without considering this approach as exclusive and exhaustive, but rather in integration with other models capable of explaining more rigorously the complexity of the viewing experience, a filmic Gestalttheorie can still be a productive means of access to the interpretation of film. The evolution of the history of visual media, characterized by the digitalization, the multiplication of screens and formats, the overabundance of images, and the multimedia contamination of languages, have changed the occasions and conditions of film viewing. However, these phenomena have not distorted the basic components of visual communication, nor do they suspend 13 13 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X the duty of a critic, a scholar or a student to describe the communicative and artistic effectiveness (or ineffectiveness) of audiovisual products (cinema, TV series, commercials, music videos...). The study of cinematographic formative means can still help us today. Film analysis could return to dwell more precisely on the compositional aspects of shots (e.g. off-screen space, camera angles and point of view, shots scale, editing and deep focus) and its dynamics (in terms of vectors, balance, configuration, etc.).16 Today eye-tracking tools allow us to register and study empirically the viewers’ gaze behavior and the dynamics of attention.17 This approach has the potential to enrich the analysis of the film experience with information on the quality and adequacy of stylistic choices. The way in which viewers process editing — or what Arnheim called the ‘space-time discontinuity’ — is inherently a gestaltic activity: in fact, the viewer mentally reconstructs the continuity behind the logic of events despite the fact that there are lacks in space and time, applying the law of ‘good continuity’, that is, filling the gaps and constituting a whole that does not derive from simple juxtaposition of the shots or narrative chunks. D’Aloia, Verstegen, The Future of the Past A KINOGESTALTTHEORIE In this sense, editing follows the functions of the mind in tending towards a ‘complete’ or ‘pregnant’ — that is, simple, coherent, logically structured — figure. On closer inspection, in drawing attention to the differences between reality and the film, Arnheim legitimized infringements of the rules of continuity (and, more generally, of Hollywood’s ‘ideology’).18 What matters is not balance per se, but the ability of the visual configurations to tend towards it, that is, to make themselves unstable, dynamic, in need of compensation.19 As Gestalt approach thus identifies and describes the expressive potential of dynamic tension, and even of discontinuity, if this stimulates the viewer’s propensity to comprehend the overall meaning of the narrative. Applied to the so-called ‘puzzle films’ or in general to complex storytelling, this approach allows us to conceive the film as a great mental game whose content is not the narration (or, the content), but the processes activated through the formal elements. An insight — another concept developed by Gestalt psychology — emerges as a reconfiguration of the relationship between the fragments in a new, sudden and intuitive way that allows a resolutive vision of a problem. If the film hides a real state of affairs until the last sequence, then there is no insight. If, on the other hand, it uses the space-time ‘folds’ of editing as formal means of expressing a momentarily incomprehensible state of affairs, then it involves the viewer actively in the comprehension of the content of the film. In short, it is likely that Arnheim would have loved Christopher Nolan’s Memento (2000) for its formalism in the use of editing (and also for the mixed use of cinematography), but not Bryan Singer’s The Usual Suspects (1995) or films that adopt the same type of ‘unreliable’ narrator. Among the formative means, the ability of the film to evoke non-visual sensory experiences is one of the most interesting. This affects primarily the negative effects of the dialogue on acting, that Arnheim conceived as mostly a pantomimic activity. Interestingly, contemporary filmmakers have spontaneously rediscovered the power of the absence of speech, when the predominantly 14 D’Aloia, Verstegen, The Future of the Past visual medium takes over. A KINOGESTALTTHEORIE For example, Brian De Palma — who had studied with Arnheim for a time at Sarah Lawrence College — remarked that he was ‘essentially a silent film director’, as he demonstrated with the abundant use of long-takes with little or no dialogue in his films.20 Furthermore, for Arnheim, the absence of non-visual stimuli affects not only the actor’s body, but also the viewer’s. The latter, in fact, not only watches the movement represented on the screen, but also experiences camera movements. Here, the relativity of film expression emerges non only in terms of visual perception, but of the relation, potentially conflictual, between the eye and the body, between visual perception and proprioception and equilibrioception. Contemporary cinema often uses this conflict in order to intensify the viewer’s perception. In this case, ‘embodied cognition’ is the result of a process that includes and strategically uses a ‘disembodying phase’ of viewer’s perception.21 In short, Arnheim could be considered as a classical theorist of modern and contemporary cinema. On the one hand, he explained for the first time in a systematic way the aesthetic implications of the film language at the time of its evolution. On the other hand, with his ‘differential’ theory, he has aesthetically legitimized infringements of the rules of continuity and balance, identifying precisely in the interference to physiological perceptual laws, a vast range of artistic potential that the cinema then naturally developed and that today we can return to observe with its far-sighted gaze. The orientation of much film scholarship to either avant-garde cinema or an inverted canon of popular culture and the condemnation of the elitist orientation of critique of the Frankfurt school caused Arnheim’s approach to appear hopelessly outdated. Yet his argument was intended to be a playbook for those seeking various artistic effects. In that sense, its recommendations were rather uncontroversial. Once one no longer believes in a positivist manner that a prediction of a theory is a law-like certainty, one can begin to see Gestalt principles as tendencies, in interaction with others for unpredictable results. One can almost express the necessity of an Arnheimian filmology as a tautology. It is simply the science of aesthetic effects, presumed by each and every filmmaker for whatever their purpose. FILM AS ART EXTENDED As the contributions to this special issue demonstrates, a Gestalt approach to film is still productive today. Indeed, the coupling of Arnheim to Gestalt psychology focuses the film theorist less on Arnheim the auteur than on him as a Gestalt psychologist. Consequently, each of the essays contained in this special issue in some sense extends Arnheim’s thought to new domains, brings in other aspects of Gestalt psychology that he may have neglected, and reconnects aspects of Gestalt filmological thinking to contemporary trends in thinking. The article by Maarten Coëgnarts, ‘Meaning Potential of Motion Vectors in 15 15 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Cinema’, connects the notion of vectors and directed tension — a prominent feature of Arnheim’s post-film writing to his earlier work, creating a useful consolidation of Arnheimian theory, moving vector discourse into film. Vectors for Arnheim create meaning by creating pictorial analogues of existence within artistic works. Following Herbert Zettl (a theorist whose relation to Arnheim would be useful to further explore), there are also motion vectors in cinema: primary motion, secondary motion, and tertiary motion. Armed with such concepts, Coëgnarts pushes Zettl’s discussion of motion vectors into meaningful expression with case studies of three films: Akira Kurosawa’s Sanshiro Sugata, Ridley Scott’s Blade Runner, and Brian De Palma’s The Untouchables. The next essay, Philippe Bédard’s ‘Points of Anchorage: Exo-centric Images and the perceptual Relativity of Camera Movement’, considers camera movement as a kind of illusion. The exo-centric image, wherein a fixed camera registers a character’s fixed face and body, while the world moves, are slightly jarring (see Darren Aronofsky’s Requiem for a Dream, or Todd Phillips’ The Hangover). Such subjective effects reverse body-space relations, denaturalizing the tacit acceptance of the camera as a fixed eye, recording events of the world. Bédard’s analysis usefully moves filmic movement back into the general psychology of ego-centric motion. Next Maria Poulaki, in ‘A Gestalt Theory for “Disorder”: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art’ returns to Arnheim’s comments in his 1971 book Entropy and Art. This book, an homage to Arnheim’s mentor Wolfgang Köhler, tried to place the physicist’s notion of entropy, which by then had been overtaken by humanistic popularization, and revisit its meaning for contemporary discourse. Poulaki brings Arnheim’s opposition of order and disorder into dialogue with newer approaches from dynamic systems theory (e.g. complexity theory) to the neurosciences. D’Aloia, Verstegen, The Future of the Past FILM AS ART EXTENDED By using the example of the digital endlessly transforming creations of artist and film director Marco Brambilla, Poulaki reminds us of the dynamic emergence and breaking of order in the spirit of the Gestalt theory inspiring Arnheim. Brambilla’s animations are a natural invitation to consider the topic more broadly, which is accomplished in Ryan Pierson’s ‘Gestalt, Animation and the Culture of Design’. Animation is a natural topic for Gestalt psychologists because experience itself is not regarded as a copy of reality. Accurately geometrically presented stimuli, for example, may not produce the most robust illusion. So too with animation, which though not live-action can be more ‘real’ than the former. For Pierson it is therefore fitting that Gestalt psychologists ought to have anticipated, and vice versa, been inspired by animators. Each also, for Pierson, participate in a culture of design, which can be construed as a parallel project of arranging senses and populations, a psychotechnics for a sociotechnics. In line with the reconnection of filmic ideas with larger traditions of Gestalt theory, Massimo Locatelli’s ‘Paul Fraisse’s Psychology of Rhythm: A Case for Filmology?’ connects Arnheim to the work on rhythm by Albert Michotte’s student, Paul Fraisse (in effect, Arnheim’s theoretical ‘cousin’). For Fraisse, rhythm follows Gestalt organization but also connects to sensory-motor 16 D’Aloia, Verstegen, The Future of the Past activation. It becomes a particularly important way to structure narrative and the comprehension of events, but in a way not even anticipated by Fraisse can also induce both bodily and neural entrainment. It therefore creates an informed link to recent neurofilmological approaches. Finally, Emilio Audissino addresses the specific case of humor as cognitive restructuring in ‘The Aha, Ha! Moment: A Gestalt Perspective on Audiovisual Humour’. Departing initially from observations on music, Audissino considers music to be a ‘part’ or micro-configuration of a ‘whole’ or macro-configuration. Incongruity not only among single modal elements but multi-modal elements can lead to humour. The restructuring related to an ‘aha’ moment can be reconfigured for humour as an ‘Aha, Ha!’ moment. Humour is indissolubly linked to problem-solving behaviour. It is the hope of the editors that readers will appreciate the bridges that have been built across the career of Rudolf Arnheim, and from there to broader elements of Gestalt psychology from which a Gestalt filmology can profit. Limited exegesis of Arnheim’s works will limit the appeal of his work and understanding of its potential breadth. Notes 1 This introduction is the product of work carried out in common by two authors, and the result of a single shared process of reflection. As far as the writing of the individual sections of the text is concerned, sections 1, 3, and 4 can be attributed to Adriano D’Aloia; 2, and 5 to Ian Verstegen. 2 Rudolf Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley-Los Angeles: University of California Press, 1954/1974); Visual Thinking (Berkeley: University of California Press, 1969). 3 See Dudley Andrew, ‘Rudolf Arnheim’, in Id., The Major Film Theories: An Introduction (London: Oxford University Press, 1976), 27–41. 2 Rudolf Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley-Los Angeles: University of California Press, 1954/1974); Visual Thinking (Berkeley: University of California Press, 1969). 2 Rudolf Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley-Los Angeles: University of California Press, 1954/1974); Visual Thinking (Berkeley: University of California Press, 1969). 3 See Dudley Andrew ‘Rudolf Arnheim’ in Id The Major Film Theories: An Introduction (London: Oxford 3 See Dudley Andrew, ‘Rudolf Arnheim’, in Id., The Major Film Theories: An Introduction (London: Oxford University Press, 1976), 27–41. 4 A previous attempt of revaluation of Arnheim’s contribution to cinema and media theory can be found in Arnheim for Film and Media Studies, ed. by Scott Higgings (New York: Routledge, 2011). 4 A previous attempt of revaluation of Arnheim’s contribution to cinema and media theory can be found in Arnheim for Film and Media Studies, ed. by Scott Higgings (New York: Routledge, 2011). 4 A previous attempt of revaluation of Arnheim’s contribution to cinema and media theory can be found in Arnheim for Film and Media Studies, ed. by Scott Higgings (New York: Routledge, 2011). 5 For example, Ian Verstegen, Arnheim, Gestalt and Media: An Ontological Theory (Cham: Springer, 2019). 6 See Robert Sinnerbrink, ‘Phenomenology Encounters Cognitivism’, Projections, 13(2), 2019, 1–19, <doi: 10.3167/proj.2019.130201>; and Adriano D’Aloia and Ruggero Eugeni, ‘Philo-, Neuro-, Post-. Che cos’è e cosa sarà la teoria del cinema’, in Teorie del cinema. Il dibattito contemporaneo, ed. by Idd. (Milano: Raffaello Cortina, 2017), 9–28.i 5 For example, Ian Verstegen, Arnheim, Gestalt and Media: An Ontological Theory (Cham: Springer, 2019). 6 See Robert Sinnerbrink, ‘Phenomenology Encounters Cognitivism’, Projections, 13(2), 2019, 1–19, <doi: 10.3167/proj.2019.130201>; and Adriano D’Aloia and Ruggero Eugeni, ‘Philo-, Neuro-, Post-. FILM AS ART EXTENDED We encourage scholars to continue to consolidate knowledge and fill in gaps to construct a vigorous Gestalt filmology. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 17 Rudolf Arnheim in The Responsive Eye (Brian De Palma, 1965) Rudolf Arnheim in The Responsive Eye (Brian De Palma, 1965) 17 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Notes 13 See Verstegen, Arnheim, Gestalt and Media, for an insertion of this argument into the Br ontological tradition and its affinity to Roman Ingarden’s ontology of the work of art. i 14 See Arnheim’s comments on the ‘Psychology of the Mass-Produced Film’, in Film als Ku rly English translation Film (170–181), but absent from the 1957 Film as Art. 14 See Arnheim’s comments on the ‘Psychology of the Mass-Produced Film’, early English translation Film (170–181), but absent from the 1957 Film as Art. 15 See Noël Carroll, Philosophical Problems of Classical Film Theory (Princeton: Princeton ress, 1988). 16 See for example Meraj Dhir, ‘A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies, 89–106. 16 See for example Meraj Dhir, ‘A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies, 89–106. 17 See for example the relevance of Arnheim’s The Power of the Center: A Study of Composition in the Visual Arts (Berkeley-Los Angeles: University of California Press, 1982) in Tim Smith’s ‘Watching You Watch Movies: Using Eye Tracking to Inform Cognitive Film Theory’, in Psychocinematics: Exploring Cognition at the Movies, ed. by Arthur P. Shimamura (Oxford: Oxford University Press, 2013), 165–190. 18 F hi P l ki 17 See for example the relevance of Arnheim’s The Power of the Center: A Study of Composition in the Visual Arts (Berkeley-Los Angeles: University of California Press, 1982) in Tim Smith’s ‘Watching You Watch Movies: Using Eye Tracking to Inform Cognitive Film Theory’, in Psychocinematics: Exploring Cognition at the Movies, ed. by Arthur P. Shimamura (Oxford: Oxford University Press, 2013), 165–190. 19 See Meraj Dhir, ‘A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies, 90–106. 20 On the influence of Arnheim’s theory and teaching on De Palma’s cinema, see Didier Truffo, ‘Brian de Palma avec Rudolf Arnheim’, La furia umana, 18, 2013, http://www.lafuriaumana.it/index.php/40-archive/ lfu-18/74-didier-truffot-brian-de-palma-avec-rudolf-arnheim#sdfootnote6sym [accessed 25 May 2022]. 21 See for example D’Aloia ‘Overturning Upside-down dissimulations’ in Id Neurofilmology of the Moving 19 See Meraj Dhir, A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies, 90–106. 20 On the influence of Arnheim’s theory and teaching on De Palma’s cinema, see Didier Truffo, ‘Brian de Palma avec Rudolf Arnheim’, La furia umana, 18, 2013, http://www.lafuriaumana.it/index.php/40-archive/ lfu-18/74-didier-truffot-brian-de-palma-avec-rudolf-arnheim#sdfootnote6sym [accessed 25 May 2022].i 21 See for example D’Aloia, ‘Overturning. Notes Che cos’è e cosa sarà la teoria del cinema’, in Teorie del cinema. Il dibattito contemporaneo, ed. by Idd. (Milano: Raffaello Cortina, 2017), 9–28.i 7 See for example Adriano D’Aloia, Neurofilmology of the Moving Image: Gravity and Vertigo in Contemporary Cinema (Amsterdam: University of Amsterdam press, 2021). For a discussion of Gestalt theory in the context of film psychology, see Maria Poulaki, ‘The “Good Form” of Film. The Aesthetics of Continuity from Gestalt Psychology to Cognitive Film Theory’, Gestalt Theory, 40, 1, 2018, 29–44, <doi: 10.2478/gth-2018-0004>. 7 See for example Adriano D’Aloia, Neurofilmology of the Moving Image: Gravity and Vertigo in Contemporary Cinema (Amsterdam: University of Amsterdam press, 2021). For a discussion of Gestalt theory in the context of film psychology, see Maria Poulaki, ‘The “Good Form” of Film. The Aesthetics of Continuity from Gestalt Psychology to Cognitive Film Theory’, Gestalt Theory, 40, 1, 2018, 29–44, <doi: 10.2478/gth-2018-0004>. 8 Arnheim, Film als Kunst (Berlin: Ernst Rowohlt, 1932), translated into English as Film (London: Faber & Faber, 1933). 8 Arnheim, Film als Kunst (Berlin: Ernst Rowohlt, 1932), translated into English as Film (London: Faber & Faber, 1933). 9 See Arnheim, I baffi di Charlot. Scritti italiani sul cinema 1932-1938, ed. by Adriano D’Aloia (Torino: Kaplan, 2009). See also Arnheim, Film Essays and Criticism (Madison: The University of Wisconsin Press, 1997), originally published as Kritiken und Ausfsätze zum Film (München-Wien: Carl Hanser Verlag, 1977). 9 See Arnheim, I baffi di Charlot. Scritti italiani sul cinema 1932-1938, ed. by Adriano D’Aloia (Torino: Kaplan, 2009). See also Arnheim, Film Essays and Criticism (Madison: The University of Wisconsin Press, 1997), originally published as Kritiken und Ausfsätze zum Film (München-Wien: Carl Hanser Verlag, 1977). 10 See D’Aloia, ‘Rodolfo Arnheim’, Comunicazioni Sociali on-line, 5, 2012, 29–44. ( ) 10 See D’Aloia, ‘Rodolfo Arnheim’, Comunicazioni Sociali on-line, 5, 2012, 29–44. 11 Arnheim, Film as Art (Berkeley-Los Angeles-London: University of California Press, 1957). 12 Arnheim, ‘A New Laocoön: Artistic Composites and the Talking Film’, in Id., Film as Art, 199–230. 12 Arnheim, ‘A New Laocoön: Artistic Composites and the Talking Film’, in Id., Film as Art, 199–230. 13 See Verstegen, Arnheim, Gestalt and Media, for an insertion of this argument ontological tradition and its affinity to Roman Ingarden’s ontology of the work of art. The Meaning Potential of Motion Vectors in Cinema Maarten Coëgnarts, Universiteit Antwerpen Maarten Coëgnarts, Universiteit Antwerpen Keywords Camera movement Editing Embodied cognition Film style Forces Vectors DOI 10 54103/ This essay examines the meaning potential of directed forces or vectors in cinema. The first part draws on the pioneering work of Rudolf Arnheim to highlight the prominent role of vectors in the visual structuring of meaning in paintings. In the second part, we move on to explore the semantic significance of motion vectors in cinema. To this aim we first define and diagram the filmic space in which vectors may articulate themselves visually. Having firmly grounded this spatial framework in film theory, we adopt the terminology of Herbert Zettl to further distinguish between three types of motion vectors: primary motion vectors (elicited by motion of visual objects), secondary motion vectors (elicited by camera movement) and tertiary motion vectors (elicited by editing). We conclude by applying the proposed conceptual tools of this essay to three filmic case-studies in which the relation between narrative meaning and motion vectors is further discussed and illustrated. This work is licensed under a Creative Commons Attribution 4.0 International License ive Commons Attribution 4.0 International License sed under a Creative Commons Attribution 4.0 International License DOI 10.54103/ 2036-461X/16785 In memory of Stephen Prince Notes Upside-down dissimulations’, in Id., Neurofilmology of the Moving Image, 143–163, <doi: 10.5117/9789463725255_ch05.> 21 See for example D’Aloia, ‘Overturning. Upside-down dissimulations’, in Id., Neurofilmology of the Moving Image, 143–163, <doi: 10.5117/9789463725255_ch05.> 18 18 D’Aloia, Verstegen, The Future of the Past Keywords Camera movement Editing Embodied cognition Film style Forces Vectors DOI 10.54103/ 2036-461X/16785 INTRODUCTION Research in cognitive science increasingly shows that meaning is not just a matter of a transcendent disembodied view of logic at the level of sentences (or propositions), nor is meaning a matter of truth-conditions or referential relations only. Rather, meaning is highly dependent upon our bodily disposition and the way we physically interact with the world.1 One way in which scholars have tried to make sense of this theme of embodied cognition is by showing how the semantics of certain linguistic concepts are metaphorically grounded in gestalt patterns, so called ‘image schemas’, that arise in and through our sensory- motor experience.2 This idea is central to the field of cognitive linguistics and the theory of Conceptual Metaphor Theory as it was first proposed by George Lakoff and Mark Johnson in 1980.3 One pattern in particular that has been argued to play a pivotal role in the structuring of meaning in language is the ‘force’ image schema.4 This schema arises in and through our physical interaction with objects and persons in our environment and functions as an important source domain for metaphorically structuring the abstract target domain of (emotional) 19 19 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X causality (‘The home run threw the crowd into a frenzy’, ‘He drove her crazy’, or ‘She pulled me out of my depression’).5 It might not come as a surprise that both Lakoff and Johnson took inspiration from the work of the famous theorist of visual art Rudolf Arnheim in order to develop the image schema of force and its structuring role in language.6 Even long before the embodied view gained foothold in the cognitive sciences, Arnheim believed that sensory perception structures and informs thinking. His notion of ‘visual thinking’ served as the key concept to signify this non-dualist interplay.7 Informed by the principles of Gestalt psychology, he considered visual percepts to be prominently dynamic and therefore as intrinsically meaningful and expressive. 8 Consequently, he extended this view to the realm of art by showing, through various inspiring case studies, how non-temporal media such as paintings and sculptures are able to portray the (abstract) dynamics of human life ‘as a closed system in which all relevant forces are shown together in configuration, each in its characteristic direction and appropriate strength’.9 in configuration, each in its characteristic direction and appropriate strength . THE MEANING POTENTIAL OF VECTORS When people talk about force they usually refer to a physical or gravitational manifestation of it. It is the literal sort that we all experience when our bodies causally interact with objects and persons in our environment.14 Forces, however, do not only manifest themselves in physical experience. We may also observe them, as Arnheim pointed out, as ‘directed tensions’ in our acts of perception.15 They constitute the ‘perceptual forces’ that are inherent in the shapes, configurations, colors and locomotion that inhabit the visual world. As Arnheim writes: ‘These dynamic properties, inherent in everything our eyes perceive, are so fundamental’, Arnheim argues, ‘that we can say: Visual perception consists in the experiencing of visual forces’.16 This applies to both natural objects (e.g., the highly dynamic curve that we perceive in an ocean wave) as well as works of art (e.g., a painting), but with this fundamental difference that the former were never intended to be seen as embodying an abstract pattern or configuration of forces. As Arnheim remarks: ‘They carry visual form only impurely and approximately’.17 By contrast, works of visual art are made for the exclusive purpose of being perceived, and therefore ‘the artist endeavours to create the strongest, purest, most precise embodiment of the meaning, that consciously or unconsciously, he intends to convey’.18 That is, in works of art forces or visual dynamic fulfil an important semiotic function as carriers of the meaning or theme of a work. The theme is the formal pattern that indicates what the work is about. It turns the visual pattern into a semantic statement on the human condition. This is also the original sense of the term vector. Combining the Latin verb, vehere (to carry) with the agentive suffix -tor, a vector literally means ‘carrier’. Vectors thus are the carriers of dynamics. And since dynamics are the ‘very basis of expression’, and expression is the manifestation of life, vectors become also the ‘carriers of meaning’.19 To illustrate how the meaning of a work may be conveyed through a dynamic configuration of vectors or directed forces, let us consider an excerpt from Arnheim’s brilliant analysis of Giotto’s depiction of the Lamentation.20 This fresco, as shown in fig. 1, depicts the subject matter of the story (death and resurrection) through a formal interplay between the horizontal and the vertical. INTRODUCTION One concept in particular that plays a highly significant role in Arnheim’s work on dynamics in the visual arts is the concept of a ‘vector’. Vectors are the ‘forces generated by the shapes and configurations of visual objects’.10 As such, they are the ‘carriers of dynamics’, that is, ‘the directed tension perceived in visual objects’.11 It is through the configuration of vectors in the composition of a work that Arnheim believed that artists are able to communicate themes and narrative meaning in a purely visual way. More recently, the semantic role of a vector has also become a crucial component in Kress and van Leeuwen’s account of the grammar of visual representation in which the authors merge concepts from Arnheim’s theory of visual dynamics in art with concepts from Halliday’s functional-semantic theory of language.12 In both the works of Arnheim and Kress and van Leeuwen, however, the meaning potential of vectors has been predominantly analysed with respect to fixed images such as photographs, sculptures and paintings. Only limited attention has been paid to moving images. It is the goal of this essay then to demonstrate the semantic potential of vectors in narrative cinema. To this aim the essay follows a threefold structure. In the first part we provide a brief case study from Arnheim to show how the link between vectors and meaning can be established visually in painting. In the second part, we move on to explore the semantic significance of motion vectors, a concept which we adopt from Herbert Zettl.13 To assess its use in cinema we first define the filmic space in which vectors may articulate themselves visually. Having firmly grounded this spatial framework in film theory, we extend Zettl’s terminology to further distinguish between three types of motion vectors: primary motion vectors (elicited by motion of visual objects), secondary motion vectors (elicited by camera movement) and tertiary motion vectors (elicited by editing). We conclude by applying the proposed conceptual tools of this essay to three filmic case-studies in which the relation between narrative meaning and motion vectors is further discussed and illustrated. 20 Coëgnarts, The Meaning Potential of Motion Vectors THE MEANING POTENTIAL OF VECTORS As Arnheim writes: ‘The horizontal of death is indicated but left behind by the body of Christ, which has been lifted and thus endowed with the dynamic quality of oblique position’.21 Obliqueness thus fulfils a fundamental role in the creation of directional tension (and hence meaning), something which Arnheim explicitly states: ‘Oblique orientation is probably the most elementary and effective means of obtaining directed tension’.22 Returning to the fresco, Arnheim draws the viewer’s attention to the arms which ‘are made to deviate obliquely from the body’. The author sees in this deviation ‘a motif of revival’ which then finds its continuation in the vector of the diagonal ridge of the hill: ‘Just broad enough for a man to walk upward, it leads through the entire picture, from the horizontal of death to the verticals of the two upright men, the vertical edge of the picture 21 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Fig. 1: Lamentation (The Mourning of Christ) by Giotto (c.1304 - c.1306) frame, and the three. The tree takes over where the diagonal of the hill is about to tend and turns the oblique climbing into straight rising’.23 It is only a brief excerpt, but it vividly illustrates how graphical vectors play a significant role in the shaping of visual meaning. Throughout his career Arnheim analyzed many other examples, albeit almost exclusively with an emphasis on the meaning potential of vectors in fixed images such as photographs, sculptures and painting.24 There is, however, no reason to assume that the vectors involved in motion do not play an equally important role in the structuring of visual meaning. Coëgnarts, The Meaning Potential of Motion Vectors THE MEANING POTENTIAL OF VECTORS One merely has to look at the experiments on so called ‘functional relations’, wherein one perceives such high-level properties as animacy and causality in simple animated movies.25 Evidence can also be found in an increasing number of publications on the subject of expression and cinematic movement.26 Moreover, Arnheim himself includes the notion of ‘locomotion’ in his definition of directed tension.27 The fundamental difference is that when actual motion is used, as in the dance, the theater, and the film, ‘direction is already indicated by movement’.28 This is also how Herbert Zettl defines a motion vector as ‘a vector created by an object that is actually moving or seen as moving-on screen’.29 He considers motion vectors generally to have a higher magnitude than index vectors, which in turn have a higher magnitude than graphic vectors. The latter ‘is created by a stationary element that guides our eyes in a certain albeit undefined direction’ (e.g., horizontal and vertical lines) whereas an index vector is created ‘by something that points undeniable in a specific direction’ (e.g., an arrow, or people point or looking in a particular direction). Not all vectors are explicitly 22 Coëgnarts, The Meaning Potential of Motion Vectors supported by visual objects that are represented in the visual field (e.g., shape, color, movement), what Arnheim, dubs ‘retinal presence’.30 For instance, the index vector created by a figure’s glance, what Kress and van Leeuwen call an ‘eyeline vector’, is not.31 The magnitude of a vector then can be defined as ‘a product of its relative strength, that is, its directional certainty and perceived directional force’.32 It expresses what the scholar labels the ‘aesthetic energy’, the energy that we perceive from aesthetic phenomena such as color, sound and motion. A high-vector magnitude is strong, a low vector magnitude is weak. Hence, because motion vectors always have a precise screen direction (contrary to graphic vectors) and because they have a perceived object speed, motion vectors are generally assumed to have a higher magnitude. Zettl’s definition of a motion vector, however, remains formal in the sense that it provides no indication of the meaning potential of motion vectors.33 For instance, he does not demonstrate, as Arnheim does with respect to graphical and index vectors, how motion vectors can convey story content. In other words, Zettl provides no semiotic definition. THE MEANING POTENTIAL OF VECTORS Moreover, his definition is strictly limited to primary motion (motion of objects) and does not take into account the structural importance of secondary motion (camera movement) and tertiary motion (editing). In the next section of this paper we address this issue by providing a tentative conceptual framework for analyzing motion vectors in cinema. Having provided a toolkit for doing so, we are conceptually equipped to lay bare its relation to the story content of three filmic cases. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X MOTION VECTORS IN CINEMA Before examining the question of motion vectors in cinema, we first have to define the space through and in which they may manifest themselves. A distinction made by film theorist Noël Burch seems to be a good point to start. In his classical book Theory of Film Practice, the author identifies two kinds of space in a film: the space included in the frame, the on-screen space, and the space included outside the frame, the off-screen space, which he further divides into six segments.34 The first four of these areas are the most obvious ones, and are determined by the four borders of the frame: the space left of the frame, the space right of the frame, the space below the frame, and the space above the frame. The fifth segment is a rather peculiar one as it cannot be defined with the same degree of geometric accuracy. It refers to the off-screen space ‘behind the camera’. Characters commonly reach this area by passing just to the right or left of the camera. Finally, there is the sixth zone of space behind the set which a character may reach by, for example, going out a door or going around a street corner, or by ‘disappearing behind a pillar or behind another person […]. The outer limit of this sixth segment of space is just beyond the horizon’.35 We may continue to further define the on-screen space, as Zettl does, by adapting the spatial coordinate system of the French philosopher René Descartes which consists of three axes: the horizontal x-axis (screen width), 23 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X the vertical y-axis (screen height) and the illusionary depth line or z-axis with ‘stretches from the screen (camera lens) to the horizon’.36 Whereas the former two axes have definite spatial limits, imposed by the boundary of the frame, screen depth is ‘virtually infinite’.37 Zettl considers the z-axis aesthetically the most flexible screen dimension for it allows for a staging or blocking in depth; a technique which was famously applauded by the French film critic André Bazin. However, as Wittusen has pointed out, Bazin’s interest lies not only in the z-axis as the single possible articulation of depth.38 While making reference to the films of Jean Renoir, he also attributes significance to another depth dimension, which he coins ‘the lateral depth’. Fig. 2: Diagramming the filmic space on the basis of film theory MOTION VECTORS IN CINEMA ‘In The Rules of the Game’, he writes, ‘Renoir actually uses simultaneous action on the same plane more than depth of field. This functions like a kind of lateral depth of field’.39 In other words, the screen may suggest the presence of a field of depth which continues in the off-screen space beyond the spatial limits of the x-axis and the y-axis. As Bazin writes, ‘The rest of the scene, while effectively hidden, should not cease to exist. The action is not bounded by the screen, but merely passes through it’.40 More recently, Julian Hanich has adopted Bazin’s concept of lateral depth for his own phenomenological analysis of complex mirror shots. Interestingly, he brings in two additional concepts to the discussion of depth in film: the ‘anterior depth’ which he uses in a similar sense as Zettl’s z-axis dimension, and the ‘posterior depth’, which he uses to signify the ‘depth’ of Burch’s sixth zone of off-screen space, that is, the depth ‘behind and next to the camera’.41 Whereas the anterior depth channels the viewer to look into the screen, the lateral and posterior depth dimensions make us look beyond the image. Taken together, these theoretical terms constitute a spatial framework, which we may diagram as in fig. 2, and which allows us to further explore any number of vectors and dynamic forces within the frame space including not only graphical and index vectors, but also motion vectors which we will look 24 Coëgnarts, The Meaning Potential of Motion Vectors Fig. 2: Diagramming the filmic space on the basis of film theory Fig. 2: Diagramming the filmic space on the basis of film theory 24 Coëgnarts, The Meaning Potential of Motion Vectors into now. As stated above, Zettl originally used the term only to refer to the movement of visual objects, what he elsewhere describes as ‘primary motion’.42 In this paper, however, we will expand the notion of motion vector to also include Zettl’s two other categories of motion, namely ‘secondary motion’ (or camera movement) and ‘tertiary motion’ (or editing). The latter further selects the primary and secondary motion vectors of the individual shots to shape what Karen Pearlman recently coined a ‘trajectory phrase’.43 In doing so, however, we should be aware of the phenomenological differences that still exist between the three types. Primary Motion By primary motion we mean movement of visual objects (e.g., characters) in the on-screen space. Since the movement trajectory in this space is always relative to the ‘container’ of the frame in which the movement takes place, we may further distinguish between four dynamic patterns of fixed-frame movement: ‘entry’, ‘exit’, ‘approaching’, and ‘distancing’.44 Entry is created by an object that enters the on- screen space. The starting point from which the movement begins, is always located in one of the six zones of off-screen space as defined by Burch. When that same object leaves the screen space again, we speak of ‘exit’. In this case one of the off-screen zones serves as the ending point of the moving object or trajectory. An object may also exit the frame along the z-axis. In such a case the pattern of exit is accompanied by a pattern of ‘approaching’. The visual object increases in graphic mass as it comes closer towards the camera. By contrast, when an object ‘distances’ itself from the camera, the graphic mass will gradually decrease. As Burch has argued, these patterns of ‘enters into’ and ‘exits from’ a frame are crucial for delineating or ‘defining’ the spatial segments of off-screen space. By this Burch means that ‘one or another of the spatial segments in question takes shape in the viewer’s imagination every time an entrance or exit occurs into or out of that segment’.45 This ‘shaping’ becomes especially vivid when a single primary motion vector starts to interact with another primary motion vector. Zettl goes further to delineate three such types of relationships that primary motion vectors can have within a single shot (and as we shall see later, also across a series of shots): ‘continuing’ (when two or more vectors point in the same direction), ‘converging’ (when they point to each other) and diverging (when they point away from each other).46 The converging type can be nicely illustrated with a brief example that Burch describes in his book and that occurs towards the beginning of Jean Renoir’s Nana (1926). It involves a static shot (see fig. 3) in which the paths of two men cross each other for a very brief moment. Young Georges has just left Nana’s dressing room. Enchanted by her beauty he enters the frame left, along the horizontal x-axis. MOTION VECTORS IN CINEMA As will become clear below, secondary and tertiary motion vectors seem more elusive and less definable than primary motion vectors. Fig. 3: Entries into and exits from the frame of Jean Renoir’s Nana (1926) Fig. 4: Primary motion change of eyeline vector in Alfred Hitchcock’s Notorious (1946) Secondary motion In cinema, however, we are not only dealing with motion vectors that have their origins in the actual on-screen movement of objects. In addition, we may also distinguish between motion vectors that emanate from the movement of the camera (e.g. dolly, zoom, tracking shots, pan shots). Naturally we do not see secondary motion vectors in the same way as we observe primary motion vectors. We do not literally see the camera moving from the right to the left as we see a character moving from the right to the left. What we do see is the on- screen effect of the mobile camera, that is, the component changes in what the camera records when its moves. For instance, it is well known that a camera pan to the left makes it seem as if a static object onscreen moves to the right.49 In contrast to primary motion vectors, secondary motion vectors usually do not interact with other secondary motion vectors in one and the same screen space unless the frame is a composite of multiple screens (e.g., split-screen). What the viewer perceives is the dynamic effect of the mobile frame as elicited by a single camera’s movement. But secondary motion vectors may engage with primary motion vectors in various aesthetic ways. For instance, we speak of motivated camera movements as when the secondary motion vector is in tune or continuing with a primary motion vector. This occurs, for example, when the camera follows a character from behind. In such a case, both camera and character share the same direction. By contrast, when the secondary motion vector detaches itself from the character, such as is often the case in modernist cinema (e.g., the cinema of Antonioni), it becomes a ‘wandering camera’.50 It reveals the presence of a filmic narrator distinct from any of the characters. In this case, the independent movement of the camera (independent of any character’s point of view) generates the spectators’ awareness of an ‘independent presence’, that of an omniscient narrator.51 In cinema, however, we are not only dealing with motion vectors that have their origins in the actual on-screen movement of objects. In addition, we may also distinguish between motion vectors that emanate from the movement of the camera (e.g. dolly, zoom, tracking shots, pan shots). Naturally we do not see secondary motion vectors in the same way as we observe primary motion vectors. Secondary motion We do not literally see the camera moving from the right to the left as we see a character moving from the right to the left. What we do see is the on- screen effect of the mobile camera, that is, the component changes in what the camera records when its moves. For instance, it is well known that a camera pan to the left makes it seem as if a static object onscreen moves to the right.49 In contrast to primary motion vectors, secondary motion vectors usually do not interact with other secondary motion vectors in one and the same screen space unless the frame is a composite of multiple screens (e.g., split-screen). What the viewer perceives is the dynamic effect of the mobile frame as elicited by a single camera’s movement. But secondary motion vectors may engage with primary motion vectors in various aesthetic ways. For instance, we speak of motivated camera movements as when the secondary motion vector is in tune or continuing with a primary motion vector. This occurs, for example, when the camera follows a character from behind. In such a case, both camera and character share the same direction. By contrast, when the secondary motion vector detaches itself from the character, such as is often the case in modernist cinema (e.g., the cinema of Antonioni), it becomes a ‘wandering camera’.50 It reveals the presence of a filmic narrator distinct from any of the characters. In this case, the independent movement of the camera (independent of any character’s point of view) generates the spectators’ awareness of an ‘independent presence’, that of an omniscient narrator.51 Primary Motion At the same time, the other guy, Muffat, rushes toward Nana’s dressing room by entering the frame from the right. Their motion vectors cross like two arrows, without their even glancing at each other, and they exit on 25 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X opposite sides of the screen. As Burch observes: ‘The essential part of the action in this shot (the trajectories of the two men) takes place off screen, although in such a brief span of time — the moment preceding and following each entrance and each exit — that it borders on the instantaneous; this action simultaneously defines the left and right segments of off-screen space’.47 In other words, primary motion vectors play a significant role in further establishing Bazin’s concept of ‘lateral depth of field’, as elucidated above. Primary motion vectors, however, should not be limited to the actual movement of a visual object from one location to another. A primary motion vector may also originate from a fixed position by a character that moves his eyes away from one spatial zone to another (comparable to the tilting or panning of a camera). In such a case the direction of the eyeline vector (as an instance of an index vector) changes: the primary motion vector is not supported by the retinal presence of a movement trajectory along one of the axes, but by the retinal presence of a change of gaze. In a much cited example from Alfred Hithcock’s Notorious (1946) there is a significant medium close-up in which Alicia (Ingrid Bergman) changes the direction of her glance from one zone (containing the reflection of Sebastian’s shadow on the bathroom door) to another (containing the keys on the desk) (see fig. 4). Through this primary motion vector which makes us look beyond the image into its lateral depth, the film already foretells the movement trajectories that will unfold in the subsequent next shots, first of the camera getting closer, then of the character itself.48 Coëgnarts, The Meaning Potential of Motion Vectors e Fig. 4: Primary motion change of eyeline vector in Alfred Hitchcock’s Notorious (1946) Fig. 4: Primary motion change of eyeline vector in Alfred Hitchcock’s Notorious (1946) 26 Coëgnarts, The Meaning Potential of Motion Vectors Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Tertiary motion Other filmmakers, however, will not strive for these subtle linkages, but for harsh collisions under Sergei Eisenstein’s dictum that ‘A smooth transition is an opportunity lost’. As Pearlman further writes, ‘a collision might be a cut that juxtaposes light and dark, close-up and wide shot, but also movement left to right with right to left, […] and so on’.58 Trajectory phrasing then is joining together movement trajectories in different shots to shape the flow of energy (movements) between and through them. This is done by choices of takes and positioning of cuts. Using the vector terminology of this paper, we might well speak of ‘vector phrasing’ with the primary motion vectors and secondary motion vectors constituting a significant part of the raw material from which the editor produces affective rhythms, that is, patterned movement over time. This makes vectors at the level of tertiary motion to function more like an overflow effect, a higher-order accumulation or interplay of other vectors — comparable to what Eisenstein would label ‘tonal’ and ‘over-tonal’ effects in montage. They become a good deal more indistinct and diffuse as a result of it. Depending on the intentions of the filmmaker, we may nevertheless distinguish between several styles of editing for putting these vectors together. In most cases of narrative cinema, the editor will keep the audience spatially oriented. For this purpose, the editor will adhere to a prescribed syntax of continuity editing.57 If for instance, a filmmaker shows a long shot of two people talking to each other (i.e., converging index vectors) and maintains these converging vectors in the subsequent separate close-ups of these people, there is still continuity across the converging vectors. Other filmmakers, however, will not strive for these subtle linkages, but for harsh collisions under Sergei Eisenstein’s dictum that ‘A smooth transition is an opportunity lost’. Tertiary motion As Pearlman further writes, ‘a collision might be a cut that juxtaposes light and dark, close-up and wide shot, but also movement left to right with right to left, […] and so on’.58 There is more to be said about vectors in cinema and of their possible relationships with each other and with other types of vectors (e.g., color vectors, sound vectors, musical vectors), but for now let us put some of the conceptual tools that we currently have into practice by exploring, as Arnheim and Kress and van Leeuwen did with respect to fixed images, the expressive and metaphorical potential of motion vectors in cinema. THREE CASE-STUDIES In the last section of this paper we briefly discuss three filmic cases for the purpose of showing how motion vectors contribute significantly to the conveyance of meaning in cinema. In doing so, we will first stress the importance of vectors in two existing film analyses from the literature. We conclude this section by providing a more elaborated case study of our own. Tertiary motion Once the primary and secondary vectors are created they can be further shaped into a temporal sequence by means of sequence motion or what Zettl terms ‘tertiary motion’. Through a change of shots (i.e., single runs of the camera), we perceive ‘a progression, a visual development’.52 As Zettl points out, ‘the important aspect of tertiary motion is not so much the vector field of the individual shot but the moment of change — the relationship of vector fields from shot to shot’.53 In her own research Karen Pearlman, who is an editor herself, has termed this assemblage of movements appropriately ‘trajectory phrasing’.54 Together with timing and pacing it is one of the tools at an editor’s disposal for shaping time, energy and movement for the purpose of creating cycles of tension and release. More specifically, trajectory phrasing ‘describes the manipulation of energy in the creation of rhythm’.55 The word ‘trajectory’ means ‘the path described by a body moving under the action of given forces’.56 27 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Trajectory phrasing then is joining together movement trajectories in different shots to shape the flow of energy (movements) between and through them. This is done by choices of takes and positioning of cuts. Using the vector terminology of this paper, we might well speak of ‘vector phrasing’ with the primary motion vectors and secondary motion vectors constituting a significant part of the raw material from which the editor produces affective rhythms, that is, patterned movement over time. This makes vectors at the level of tertiary motion to function more like an overflow effect, a higher-order accumulation or interplay of other vectors — comparable to what Eisenstein would label ‘tonal’ and ‘over-tonal’ effects in montage. They become a good deal more indistinct and diffuse as a result of it. Depending on the intentions of the filmmaker, we may nevertheless distinguish between several styles of editing for putting these vectors together. In most cases of narrative cinema, the editor will keep the audience spatially oriented. For this purpose, the editor will adhere to a prescribed syntax of continuity editing.57 If for instance, a filmmaker shows a long shot of two people talking to each other (i.e., converging index vectors) and maintains these converging vectors in the subsequent separate close-ups of these people, there is still continuity across the converging vectors. Case study 1: Sanshiro Sugata (Akira Kurosawa, 1943) 1943) In his brilliant treatment of Akira Kurosawa’s cinema, Stephen Prince is keen to point out the importance of movement patterns in the conveyance of themes in the oeuvre of the Japanese director.59 He states among others that the director frequently organizes movement to produce dialectical relationships often 28 Coëgnarts, The Meaning Potential of Motion Vectors in combination with the use of the telephoto lens. For instance, he describes one brief moment of converging (primary) motion vectors from Yojimbo (1961) in which the planes of movement are perpendicular to each other: the hero (Toshiro Mifune) crosses the frame along the horizontal x axis, from left to right in the foreground, while the villain advances from behind the setting (i.e., Burch’s sixth zone) towards the camera from the background (the z-axis), as the telephoto lens produces the illusion that both primary motion vectors ‘are about to crash into each other’.60 In other occasions, however, motion vectors serve a more important narrative function by helping to situate characters according to ‘important psychological and social relations prevailing along them’, an interest that according the author runs throughout Kurosawa’s oeuvre and reaches its climax in Seven Samurai (1954).61 One vivid example that Prince analyses appears already very early in the director’s career. It involves a scene near the beginning of Sanshuro Sugata (1943) in which the young pupil Sanshiro accompanies Momma and his mob to a late night ambush of the judo instructor Shogoro Yano (Denjiro Okochi). Kurosawa’ formalizes Sanshiro’s relation to Yano by introducing a lateral tracking shot and reduplicating its secondary motion vector three times. Each time Yano defeats one of Momma’s men, the motion vector ends by including Yano into the frame, while the film intercuts between the two characters, ‘each framed in isolation, in contrast to Momma’s group’.62 ‘Here we can see clearly’, as Prince argues, ‘Kurosawa’s injunction to use form in a meaningful manner: Yano is to be Sanshiro’s teacher and spiritual guide, and the bonds between them are set out in visual terms during this early sequence, in which Sanshiro functions as an observer, learning a first lesson by watching a master’. Case study 2: Blade Runner (Ridley Scott, 1982) Case study 2: Blade Runner (Ridley Scott, 1982) For our second example, let us consider a juxtaposition of images from Ridley Scott’s Blade Runner (1982) that recently gave rise to an interesting discussion among two cognitive film scholars: Tim J. Smith and Karen Pearlman whose work we already cited above.63 The first shot, as shown in fig. 5, involves a similar change of eyeline vectors that we already illustrated above with the example of Notorious. It shows how the character of Deckard (Harrison Ford) shifts his gaze from a horizontal x-axis index vector (pointing towards the second off screen space where the character of Rachel is located) to a z-axis index vector (pointing towards the sixth zone where an artificial owl is located). In the next shot the actual owl is shown as it gestures an opposite movement: the owl’s gaze shifts from right (a z-axis index vector) to left (horizontal x-axis index vector). The film then cuts back to Deckard in a trajectory that exactly copies the owl’s movement. Deckard now turns his head again from the owl in the direction of Rachel in the fourth off-screen zone (left of the frame). Smith considers this last cut to be ‘slightly off’, by which he means ‘that the owl’s gaze shift will cue viewer attention in the opposite direction to the saccade 29 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X required to shift back to Deckard’s face’.64 The owl looks over Deckard’s head and guides your eyes to slip over Deckard’s head and then you slip them back. ‘Such a mismatch’, he argues, ‘may result in a violation of a priori continuity’.65 In her own analysis of the same scene, however, Karen Pearlman, argues that this mismatch is not a mistake. As she argues, ‘it offers a different kind of editing, one that adds a visual rhythm, an embodied sense of alignment with character’ and an element of what she reveals as ‘a significant subtext’.66 ‘Its first creative purpose is to create a movement phrase. Like a dance phrase, a movement phrase created by the juxtaposition of two gestures is a statement of a rhythmic idea’.67 But more importantly, she also adds a semiotic purpose to the movement phrase created by editing. Fig.5: A trajectory phrase of eyeline motion vectors in Ridley Scott’s Blade Runner (1982) Coëgnarts, The Meaning Potential of Motion Vectors Case study 2: Blade Runner (Ridley Scott, 1982) As she writes: ‘By comparing the owl’s behaviour with Deckard’s behaviour, the cut subtextually suggests that they are alike’.68 She finds further evidence in the dialogue that immediately follows the comparison. Earlier Rachel has asked Deckard if he likes their owl. When Deckard moves his head away from the owl back to Rachel, he says ‘it’s artificial’ upon which Rachel responds ‘of course it is’. This is where the comparison takes on a semantic dimension. As Pearlman argues, it suggests that ‘if they are similar, the possibility Deckard is also artificial, something that (spoiler alert) we learn only much later in the film may be true’.69 From the terminology of this paper, we may further argue that this existential resemblance is pronounced visually by a formal similarity that is articulated at the level of primary motion eyeline vectors.70 Fig.5: A trajectory phrase of eyeline motion vectors in Ridley Scott’s Blade Runner (1982) 30 Coëgnarts, The Meaning Potential of Motion Vectors Case study 3: The Untouchables (De Palma, 1987) Our last example considers a case study that shows how vectors play an import role in the visual structuring of so called ‘flow-of-emotion scenarios’.71 A flow-of- emotion scenario conceives emotions to be embedded in a causal chain of three events: an emotion arousing event (the cause of event), an emotional state and a behavioural response (the effect of emotion). Such narratives of mental causation are not only ubiquitous in our ordinary lives, but also in films. To illustrate this, let us consider the first segment of the famous ‘Odessa Steps’ inspired staircase scene from Brian De Palma’s The Untouchables (1987). During the Prohibition- era of the nineteen twenties, Chicagoan cop Elliott Ness (Kevin Costner) along with his sharp-shooting partner George Stone (Andy Garcia) have arrived at the city’s Union Station in order to arrest bookkeeper Walter Payne (Jack Kehoe), the one man that could put Crime boss Al Capone in prison. As they wait for his arrival, a suspenseful chain of mental causation starts to unfold which we may analytically divide into a number of flow-of-emotion scenarios, the first of which may be described as follows: Ness, at the top of the stairs, sees a young mother appearing at the foot of the stairs, pushing a buggy while simultaneously carrying two large suitcases (the emotion arousing event). Perception in turn causes the first emotional state: Ness is nervously torn between helping the woman carry her buggy up the marble steps, and maintaining his watch for the accountant. The increase of emotional intensity results in a behavioural response: Ness gives into his good nature, and rushes down the steps to help the mother drag her buggy up the stairs (the effect of emotion). The first part of this flow-of-emotion scenario is visualised, as shown in fig. 6, by extending the principle of index-vector target continuity: shots of Eliot Ness looking off-screen alternate between POV shots of the woman and the baby (located downstairs, Burch’s second zone of off screen space) and shots of the station clock up the stairs. The emotional intensity increases as the clock almost reaches the pivotal moment of 12 am. To express this the film makes use of the Fig. 6: Visually structuring the flow-of-emotion scenario in Brian De Palma’s The Untouchables (1987) Fig. 6: Visually structuring the flow-of-emotion scenario in Brian De Palma’s The Untouchables (1987) Fig. Case study 3: The Untouchables (De Palma, 1987) 6: Visually structuring the flow-of-emotion scenario in Brian De Palma’s The Untouchables (1987) Fig. 6: Visually structuring the flow-of-emotion scenario in Brian De Palma’s The Untouchables (1987) 31 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X dynamic pattern of enclosure, here elicited by tertiary motion rather than by secondary motion: as the clock nears the arrival time and emotional intensity increases, the clock inside Nett’s visual field increases in graphical substance. This visual progression in time can be seen as a cinematic manifestation of an embodied metaphor, which in the literature is known as the metaphor ‘increase in emotional intensity is increase of substance in a container’, a subtype of the more general metaphor ‘emotions are forces’.72 As the theory behind this metaphor goes, humans have a tendency to conceptualize the rising of a strong emotion (e.g., joy, anger, fear) inside a person’s body in terms of the increase of a substance inside a container. When there is very little substance in the container, the pressure is low and thus emotion is at low intensity (there is enough breathing space).73 By contrast, with an increase of the substance, the pressure becomes higher, and thus also the intensity of the emotion.74 The increase of pressure, here visualized through the increase of the clock’s graphical mass, further triggers a behavioral response. This is also what happens next. The eyeline vector, the directed force of Elliot’s gaze, soon changes into a motion vector as our hero now hastens himself to go downstairs and help the woman. This further prompts the camera to move: as Ned lowers himself to approach the lady (and the location of the camera), the camera tracks backwards so as to include the woman and the baby inside the frame (i.e., Ness’ ‘container’). Another way of capturing this would have been to start with a fixed shot of the woman followed by a motion vector of fixed-frame movement as Ned enters the shot from above. But the effect of this would have been less dynamic and meaningful as it is Ness who ‘brings’ the woman and the baby safe into his ‘personal container’, not the other way around. Notes 7 Rudolf Arnheim Visual Thinking (Berkeley and Los Angeles: University of California Press 1969) 8 Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley and Los Angeles: University of California Press, 1974); Rudolf Arnheim, ‘Perceptual Dynamics’, American Scientist, 76 (1988), 585–591; Arnheim, The Power of the Center (Berkeley and Los Angeles: The University of California Press, 1988). 8 Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley and Los Angeles: University of California Press, 1974); Rudolf Arnheim, ‘Perceptual Dynamics’, American Scientist, 76 (1988), 585–591; Arnheim, The Power of the Center (Berkeley and Los Angeles: The University of California Press, 1988). 8 Arnheim, Art and Visual Perception: A Psychology of the Creative Eye (Berkeley and Los Angeles: University of California Press, 1974); Rudolf Arnheim, ‘Perceptual Dynamics’, American Scientist, 76 (1988), 585–591; Arnheim, The Power of the Center (Berkeley and Los Angeles: The University of California Press, 1988). 9 Arnheim, ‘Perceptual Dynamics’, 590. 10 Arnheim, The Power of the Center, 229. 12 Gunther Kress and Theo Van Leeuwen, Reading Images: The Grammar of Visual Design (London: Routledge, 2021); Morten Boeriis and Theo van Leeuwen, ‘Vectors’, in New Studies in Multimodality, ed. by Ognian Seizov and Janina Wildfeuer (London: Bloomsbury, 2017), 15–36. 12 Gunther Kress and Theo Van Leeuwen, Reading Images: The Grammar of Visual Design (London: Routledge, 2021); Morten Boeriis and Theo van Leeuwen, ‘Vectors’, in New Studies in Multimodality, ed. by Ognian Seizov and Janina Wildfeuer (London: Bloomsbury, 2017), 15–36. 13 Herbert Zettl, Sight, Sound, Motion: Applied Media Aesthetics (Boston: Cengage Learning, 2017), 127– 131. As will become clear below, we will use the concept of a ‘motion vector’ in a wider sense than originally proposed by Zettl. 13 Herbert Zettl, Sight, Sound, Motion: Applied Media Aesthetics (Boston: Cengage Learning, 2017), 127– 131. As will become clear below, we will use the concept of a ‘motion vector’ in a wider sense than originally proposed by Zettl. 14 Johnson, 43. 15 Arnheim, Art and Visual Perception, 416. 16 Ibidem, 412. 17 Arnheim, Visual Thinking, 270. 18 Ibidem, 270–271. 19 Arnheim, ‘Perceptual Dynamics’, 585. 20 Arnheim, Art and Visual Perception, 441–443. 21 Ibidem, 441. 22 Ibidem, 424. 23 Ibidem, 441. , 24 It should be stressed, however, that Arnheim also wrote extensively about film, especially at the beginning of his career. CONCLUSION The goal of this article was to draw attention to the meaning potential of motion vectors in cinema. To this aim we first provided a preliminary discussion of the concept of a vector in the visual arts by reviewing the pioneering work on visual dynamics by Rudolf Arnheim. This theorist has convincingly demonstrated and illustrated through various cases how vectors play an important role in the visual structuring of narrative meaning in fixed images. Subsequently, we extended the question of vectors not only to the domain of fixed images, but also to the domain of moving images by addressing the meaning potential of motion vectors. For this purpose, we first had to define the filmic space in which motion vectors may unfold themselves. Having done so we distinguished between three types of motion vectors: primary motion vectors, secondary motion vectors and tertiary motion vectors. Since the proof of the pudding is in the eating, we concluded this paper by illustrating the concepts proposed using three filmic case-studies. The insights offered in this paper will hopefully provide an impetus to further explore, in an interdisciplinary way, the role of motion vectors in the structuring of meaning in cinema. 32 Coëgnarts, The Meaning Potential of Motion Vectors Notes 1 Barbara Tversky, Mind in Motion: How Action Shapes Thought (New York: Basic Books, 2019); Varela, Francisco J., Evan Thompson and Eleanor Rosch, The Embodied Mind: Cognitive Science and Human Experience (Cambridge: The MIT Press, 1991); Raymond W. Gibbs Jr., Embodiment and Cognitive Science (Cambridge: Cambridge University Press, 2006); George Lakoff and Mark Johnson, Philosophy in the Flesh: The Embodied Mind and its Challenge to Western Thought (New York: Basic Books, 1999). 2 The concept of an ‘image schema’ is central in two classic 1987 monographs: Lakoff, Women, Fire, and Dangerous Things: What Categories Reveal About the Mind (Chicago: University of Chicago Press, 1987), and Johnson, The Body in the Mind: The Bodily Basis of Meaning, Imagination and Reason (Chicago: University of Chicago Press, 1987). 3 Lakoff and Johnson, Metaphors We Live By (Chicago: University of Chicago Press, 1980). 4 Johnson, 42–48; see also Leonard Talmy, ‘Force Dynamics in Language and Cognition’, Cognitive Scie 12 1 (1988) 49 100 3 Lakoff and Johnson, Metaphors We Live By (Chicago: University of Chicago Press, 1980). 4 ‘ 3 Lakoff and Johnson, Metaphors We Live By (Chicago: University of Chicago Press, 1980). 4 Johnson, 42–48; see also Leonard Talmy, ‘Force Dynamics in Language and Cognition’, Cognitive Science, 12.1 (1988), 49–100. 3 Lakoff and Johnson, Metaphors We Live By (Chicago: University of Chicago Press, 1980). 4 Johnson, 42–48; see also Leonard Talmy, ‘Force Dynamics in Language and Cognition’, Cognitive Science, 12.1 (1988), 49–100. 4 Johnson, 42–48; see also Leonard Talmy, ‘Force Dynamics in Language and Cognition’, Cognitive Science, 12.1 (1988), 49–100. 5 Lakoff and Johnson, Metaphors We Live By, 184–185; María Sandra Peña Cervel, ‘Subsidiarity Relationships Between Image-schemas: An Approach to the Force Schema’, Journal of English Studies, 1 (1999), 187–208. 5 Lakoff and Johnson, Metaphors We Live By, 184–185; María Sandra Peña Cervel, ‘Subsidiarity Relationships Between Image-schemas: An Approach to the Force Schema’, Journal of English Studies, 1 (1999), 187–208. 6 For Johnson’s discussion of the force schema, see Johnson, 76–79. 6 For Johnson’s discussion of the force schema, see Johnson, 76–79. 6 For Johnson’s discussion of the force schema, see Johnson, 76–79. 9 Arnheim, ‘Perceptual Dynamics’, 590. 6 For Johnson’s discussion of the force schema, see Johnson, 76–79. 10 Arnheim, The Power of the Center, 229. Notes 34 Noël Burch, Theory of Film Practice, trans. by Helen R. Lane (Princeton: Princeton University Press, 1981), 17. 35 Ibidem, 17. For a discussion, see also David Bordwell and Kristin Thompson, Film Art: An Introduction (New York: McGraw-Hill, 2004), 258; Maarten Coëgnarts, Film as Embodied Art: Bodily Meaning in the Cinema of Stanley Kubrick (Boston: Academic Studies Press, 2019), 75. 35 Ibidem, 17. For a discussion, see also David Bordwell and Kristin Thompson, Film Art: An Introduction (New York: McGraw-Hill, 2004), 258; Maarten Coëgnarts, Film as Embodied Art: Bodily Meaning in the Cinema of Stanley Kubrick (Boston: Academic Studies Press, 2019), 75. 61–163; see also Michael Frierson, Film and Video Editing Theory: How Editing Creates Meanin Routledge, 2018), 5. 38 Cato Wittusen, ‘Varieties of Temporal Overlapping and Depth’, New Review of Film and Television Studies, 12.2 (2014), 112–124 (118). 38 Cato Wittusen, ‘Varieties of Temporal Overlapping and Depth’, New Review of Film and Television Studies, 12.2 (2014), 112–124 (118). 39 André Bazin, What is Cinema?, trans. by Timothy Barnard (Montreal: Caboose, 2009), 69. 40 Bazin, Jean Renoir, trans. by W. W. Halsey II and William H. Simon (New York: Da Capo Press, 1992), 89. 39 André Bazin, What is Cinema?, trans. by Timothy Barnard (Montreal: Caboose, 2009), 69. 40 Bazin, Jean Renoir, trans. by W. W. Halsey II and William H. Simon (New York: Da Capo Pr Bazin, Jean Renoir, trans. by W. W. Halsey II and William H. Simon (New York: Da Capo Press, 1992), 89. 41 Julian Hanich, ‘Reflecting on Reflections: Cinema’s Complex Mirror Shots’, in Indefinite Visions: Cinema and the Attractions of Uncertainty, ed. by Martine Beugnet (Edinburgh: Edinburgh University Press, 2017), 131–156 (141). 42 Zettl 294 41 Julian Hanich, ‘Reflecting on Reflections: Cinema’s Complex Mirror Shots’, in Indefinite Visions: Cinema and the Attractions of Uncertainty, ed. by Martine Beugnet (Edinburgh: Edinburgh University Press, 2017), 131–156 (141). 43 For a discussion of the concept of ‘trajectory phrasing’, see Karen Pearlman, Cutting Rhythms: Intuitive Film Editing (Amsterdam: Focal Press, 2016); Pearlman, ‘Editing and Cognition Beyond Continuity’, Projections: The Journal for Movies and Mind, 11.2 (2017), 67–86; Pearlman, ‘On Rhythm in Film Editing’, in The Palgrave Handbook of the Philosophy of Film and Motion Pictures, ed. by Noël Carroll, Laura T. Di Summa and Shawn Loht (Cham: Palgrave Macmillan, 2019), 143–163. 44 Coëgnarts, Film as Embodied Art, 84–85. 51 Ibidem, 49. 52 Zettl, 299. 49 This apparently paradoxical phenomenon of the relativity of movement has been well described and illustrated by perceptual psychologists. See e.g., Julian Hochberg and Virginia Brooks, ‘Perception of Motion Pictures’, in Cognitive Ecology, ed. by Morton P. Friedman and Edward C. Carterette (San Diego: Academic Press, 1996), 205–292 (237–240). ( Notes Notable in this regard is his landmark book Film as Art (Berkeley and Los Angeles: University of California Press, 1957). However, since Arnheim never undertook any serious attempts to update or revise his position, and since his later work mostly focuses on static art media, the implications of vector dynamics for the study of meaning in film have been largely ignored in the literature.i i 25 Notable in this regard are two classic works in the field of perceptual psychology: Albert Michotte’s The Perception of Causality (New York: Basic Books, 1963), which is also cited and discussed by Arnheim, Art and Visual Perception, 388–395, and Fritz Heider and Marianne Simmel’s influential article on perceptual animacy, entitled ‘An Experimental Study of Apparent Behavior’, The American Journal of Psychology, 57 (1944), 243–249. i 25 Notable in this regard are two classic works in the field of perceptual psychology: Albert Michotte’s The Perception of Causality (New York: Basic Books, 1963), which is also cited and discussed by Arnheim, Art and Visual Perception, 388–395, and Fritz Heider and Marianne Simmel’s influential article on perceptual animacy, entitled ‘An Experimental Study of Apparent Behavior’, The American Journal of Psychology, 57 (1944), 243–249. ng others, Christina Schmitt, Sarah Greifenstein and Hermann Kappelhoff, ‘Expressive 26 See, among others, Christina Schmitt, Sarah Greifenstein and Hermann Kappelhoff, 33 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Movement and Metaphoric Meaning Making in Audio-Visual Media’, Body-Language-Communication, vol. 2, ed. by Cornelia Müller, Alan Cienki, Ellen Fricke, Silva Ladewig, David McNeill and Jana Bressem (Berlin, München and Boston: De Gruyter Mouton, 2014), 2092–2112; Jordan Schonig, The Shape of Motion: The Cinema and the Aesthetics of Movement (Oxford: Oxford University Press, 2021); and Patrick Keating, The Dynamic Frame: Camera Movement in Classical Hollywood (New York: Columbia University Press, 2019). Ibidem, 28. 29 Zettl, 128. Notice that Zettl does not make reference to camera movement and editing in his definition of a motion vector. 29 Zettl, 128. Notice that Zettl does not make reference to camera movement and editing in his definition of a motion vector. 30 Arnheim, The Power of the Center, 228. 30 Arnheim, The Power of the Center, 228. 31 Kress and van Leeuwen, 72. 33 See also the criticism of Boeriis and van Leeuwen, 16. 34 Noël Burch, Theory of Film Practice, trans. by Helen R. Lane (Princeton: Princeton University Press, 1981), 17. 53 Ibidem, 299. 50 Seymour Chatman, Antonioni or, The Surface of the World (Berkeley: University of California Press, 1985). See also Kenneth Johnson, ‘The Point of View of the Wandering Camera’, Cinema Journal, 32.2 (1993), 46–56. 47 Burch, 19. 48 For a discussion of this scene, see also Pearlman, ‘Editing and Cognition’, 79; Vittorio Gallese and Michele Guerra, ‘Embodying Movies: Embodied Simulation and Film Studies’, Cinema: Journal of Philosophy and the Moving Image, 3 (2012), 183–210 (200–201); Coëgnarts, ‘Cinema and the Embodied Mind: Metaphor and Simulation in Understanding Meaning in Films’, Palgrave Communications, 3.1 (2017): 1–15; and Mircea Valeriu Deaca, ‘The Control Cycle: Cognitive Model in Biology, Film and Culture’, Ekphrasis. Images, Cinema, Theory, Media, 20.2 (2018), 63–97 (88–89). y 27 Arnheim, Art and Visual Perception, 416. 46 Zettl, 129–130. 45 Burch, 18. 44 Coëgnarts, Film as Embodied Art, 84–85. 28 Ibidem, 28. Notes 48 For a discussion of this scene, see also Pearlman, ‘Editing and Cognition’, 79; Vittorio Gallese and Michele Guerra, ‘Embodying Movies: Embodied Simulation and Film Studies’, Cinema: Journal of Philosophy and the Moving Image, 3 (2012), 183–210 (200–201); Coëgnarts, ‘Cinema and the Embodied Mind: Metaphor and Simulation in Understanding Meaning in Films’, Palgrave Communications, 3.1 (2017): 1–15; and Mircea Valeriu Deaca, ‘The Control Cycle: Cognitive Model in Biology, Film and Culture’, Ekphrasis. Images, Cinema, Theory, Media, 20.2 (2018), 63–97 (88–89). 49 This apparently paradoxical phenomenon of the relativity of movement has been well described and illustrated by perceptual psychologists. See e.g., Julian Hochberg and Virginia Brooks, ‘Perception of Motion Pictures’, in Cognitive Ecology, ed. by Morton P. Friedman and Edward C. Carterette (San Diego: Academic Press, 1996), 205–292 (237–240). 50 Seymour Chatman, Antonioni or, The Surface of the World (Berkeley: University of California Press, 1985). See also Kenneth Johnson, ‘The Point of View of the Wandering Camera’, Cinema Journal, 32.2 (1993), 46–56. 34 Coëgnarts, The Meaning Potential of Motion Vectors 54 Pearlman, Cutting Rhythms, 60. 54 Pearlman, Cutting Rhythms, 60. 55 Pearlman, ‘On Rhythm’, 155. 55 Pearlman, ‘On Rhythm’, 155. 56 Ibidem, 155. 57 For a discussion of continuity principles in relation to vectors, see Zettl, 369–393. 58 Pearlman, Cutting Rhythms, 170. Pearlman, Cutting Rhythms, 170. 59 Stephen Prince, The Warrior’s Camera: The Cinema of Akira Kurosawa (Princeton: Princeton University Press, 1999). 59 Stephen Prince, The Warrior’s Camera: The Cinema of Akira Kurosawa (Princeton: Princeton University Press, 1999). 60 Ibidem, 172–173. 61 Ibidem, 42. 62 Ibidem, 41. 63 Tim J. Smith, ‘The Attentional Theory of Cinematic Continuity’, Projections: The Journal for Movies and Mind, 6.1 (2012), 1–27 (20); Pearlman, ‘Editing and Cognition’, 75. 64 S i h 20 63 Tim J. Smith, ‘The Attentional Theory of Cinematic Continuity’, Projections: The Journal for Movies and Mind, 6.1 (2012), 1–27 (20); Pearlman, ‘Editing and Cognition’, 75. 66 Pearlman, ‘Editing and Cognition’, 75. 67 Ibidem, 75. 68 Ibidem, 76. 69 Ibidem. 70 A similar interpretation may be given to the cutting of the red men’s bathroom in Stanley Kubrick’s The Shining (1980). This is well addressed by Frierson, 22: ‘Kubrick cuts across the 180° line to create a visual metaphor linking (Jack Nicholson) and Mr. Grady (Philip Stone) as socially isolated characters by flipping their position from shot to shot)’. 73 For a discussion of the container image schema, see Lakoff and Johnson, Philosophy in the Flesh, 31–32. 74 For a discussion of this metaphor in relation to the elevator scene from Francis Ford Coppola’s The Conversation (1974), see Coëgnarts, ‘Analyzing Metaphor in Film: Some Conceptual Challenges’, in Current Approaches to Metaphor Analysis in Discourse, ed. by Ignasi Navarro i Ferrando (Berlin: De Gruyter Mouton, 2019), 295–320 (314–316). 71 For a discussion, see Coëgnarts, Film as Embodied Art, 28. Keywords Camera movement Exo-centric images Relative movement Forbidden movements Phenomenology DOI Notes Here, we may speak of a flipping of eyeline vectors through editing or rather the flipping of the participants from which these vectors emanate. 72 Kövecses, 65–68; see also Coëgnarts, Film as Embodied Art, 63. 73 For a discussion of the container image schema, see Lakoff and Johnson, Philosophy in the Flesh, 31–32. 74 For a discussion of this metaphor in relation to the elevator scene from Francis Ford Coppola’s The Conversation (1974), see Coëgnarts, ‘Analyzing Metaphor in Film: Some Conceptual Challenges’, in Current Approaches to Metaphor Analysis in Discourse, ed. by Ignasi Navarro i Ferrando (Berlin: De Gruyter Mouton, 2019), 295–320 (314–316). g , , p y , 74 For a discussion of this metaphor in relation to the elevator scene from Francis Ford Coppola’s The Conversation (1974), see Coëgnarts, ‘Analyzing Metaphor in Film: Some Conceptual Challenges’, in Current Approaches to Metaphor Analysis in Discourse, ed. by Ignasi Navarro i Ferrando (Berlin: De Gruyter Mouton, 2019), 295–320 (314–316). 35 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Points of Anchorage: Exo-centric Images and the Perceptual Relativity of Camera Movement Philippe Bédard, Université de Montréal This essay analyzes a unique filmmaking technique to highlight the fact that camera movement is fundamentally an optical illusion based on a misinterpretation of visual cues. The unique technique in question is what I have called the ‘exo-centric image’, namely an image produced by a camera attached to the body of an actor which, paradoxically, generates the impression of an immobile body in a moving world. Through an analysis of this peculiar technique, I make claims about the illusory nature of camera movement in general. In so doing, this essay concludes that the vocabulary we use to describe camera movement keeps us from seeing some of the more eccentric aspects of the effect we call camera movement. 10.54103/ 2036-461X/16883 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X INTRODUCTION As a recent wave of scholarship has demonstrated, camera movement constitutes a rich, if elusive, topic.1 As David Bordwell put it, ‘camera movement has usually been considered too elusive to be analyzable’.2 Part of the challenge stems from the fact that the vocabulary we typically use to describe camera movement refers to the realities of film production, and not to the ‘phenomenon of camera movement on the screen as it is originally experienced and understood by us as viewers’.3 This fact is best exemplified by edge cases — what Bordwell calls ‘forbidden movements’4 — where expectations about how a camera ought to move are subverted. Trick shots, digitally assisted camera movements and even animation confront us to the challenge of discussing camera movement with any precision. They also reveal the tenuous link between how a camera appears to move on screen and how it actually moved (or not) during production. Consider the opening 17–minute sequence of Alfonso Cuarón’s Gravity (2013), which has been analysed by countless authors for its virtuosic display of camera movement and for the problems it raises as a result of its unique production context.5 While some authors take up Gravity’s daring opening long take to question the use of the term ‘camera’ to discuss this special effects- driven assemblage,6 others address the cognitive7 or proprioceptive8 empathy 37 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X between the movements displayed on screen and those felt by viewers. Instead, I want to focus on the final act of this sequence (00:13:34-00:16:08), when the heretofore unchained camera seemingly becomes fixed to the body of an astronaut spinning out of control in Earth’s orbit. From the beginning of the sequence, the camera has been moving fluidly around the protagonists, Ryan Stone (Sandra Bullock) and Matt Kowalski (George Clooney), two astronauts working on a module of the International Space Station who get caught in a sudden shower of space debris which sends Stone careening into orbit. Around the 13–minute mark, the roving camera seemingly comes to a stop in front of Stone, keeping her locked in the frame even as she continues to spin out of control. In fact, although we know her to be moving rapidly, Stone appears fixed in the frame; it is the earth and the stars instead that we see rotating around her in this part of the sequence. INTRODUCTION While a lot has been written about the narrative functions of — and affective responses to — this particular shot, I want to emphasize the unusual body-camera-space relations on display in this image sequence. By extension, my goal in this essay is also to highlight the tenuous link between the appearance of camera movement and any notion about how (or if) the camera might have moved (or not) during production. Of all the movements showcased in Gravity’s opening sequence, the one I am describing here is an example of a technique I have called the ‘exo-centric’ image.9 Through a unique arrangement of body, camera, and space, this technique creates the illusion of immobilizing on screen the actor who was wearing the camera and moving with it during production. Whether in Gravity or in any of the myriad films that feature exo-centric images, the technique also reveals a deception at the heart of all camera movement, namely that our perception of movement on screen is any indication whatsoever of the actual movements a camera performed during production. Starting from Bordwell’s hypothesis that, ‘there must be perceptual cues which determine a ‘camera- movement effect’ onscreen regardless of whether the camera moved in production or not’ [emphasis in the original],10 this essay seeks to demonstrate the fundamentally illusory nature of camera movement, a fact that is obfuscated by the vocabulary we generally use to describe it. To do so, I approach camera movements from a phenomenological perspective, considering first and foremost the appearance of movement on screen as a signifier of movement. This descriptive approach aims to free us from the preconceptions tied to the vocabulary typically used in film studies to discuss the phenomenon we call ‘camera movement’. While my claims in this essay are meant to reflect on all forms of camera movement, my central example remains the exo-centric technique and the uncanny effect it has of immobilizing heretofore mobile bodies. Beginning by a description of the technique behind exo-centric images, I start by demonstrating the limitations of the language typically used to describe camera movement. I continue with an overview of different ways of describing movement, using frames of references that are either ‘subjective’ or ‘objective’. Namely, I call upon Maurice Merleau-Ponty and James J. INTRODUCTION Gibson to highlight how our perception of 38 Bédard, Points of Anchorage movement is affected by what we focus our attention on, as well as on whether the body is actively moving or passively being moved. Bordwell’s notion of a ‘perceived screen event’ allows me to come back to camera movement with a description of the ‘visual cues’ that enable us to determine whether parts or the whole of an image are moving on screen. Finally, I come back to exo-centric images and, following Jordan Schonig’s lead, analyse it in a way that ‘alters the “normal circumstances” under which the visual effects of the moving camera can be seen’, so as to shed light on what our vocabulary otherwise obfuscates.11 THE EXO-CENTRIC TECHNIQUE Whether it appears in movies, television series, commercials, music videos, or extreme sports videos, the phenomenon I have called the ‘exo-centric image’ stands out for its characteristic way of representing body-space relations: the body appears frozen in the centre of the frame while the world around it is seen moving in its place. This unique effect has been used in films such Requiem for a Dream (Darren Aronofsky, 2000), Mean Streets (Martin Scorsese, 1973) and The Hangover (Todd Phillips, 2009) among countless others. Regardless of the narrative or aesthetic reasons why this effect might have been sought after in these films, I focus on the exo-centric image in this essay because of the reasons for which it succeeds in subverting our expectations as to how cameras appear to move on screen.12 An exo-centric image is produced when a camera is attached to the body of an actor who carries it with them as they move during a scene. Technically speaking, this effect can be broken down into three basic elements: 1. the camera must be connected to the body which it films; 2. the camera must be carried by, but away from, the body (in front or behind); 3. the camera must be facing the body that carries it.13 For example, as the above production still from Requiem for a Dream shows, during her famous hallway scene Jennifer Connelly wore a device called a ‘Snorricam’ which allowed her to move on set with the camera placed about an arm’s length in front of her, looking back at her [fig. 1]. 14 Whether it is placed in front, behind, or anywhere around the actor’s body, the camera keeps them centred in the frame as it moves in unison with them.15 In other words, camera and body maintain their position relative to each other regardless of their displacements through space. This lack of ‘relative movement’ between camera and body is translated on screen by a body that becomes immobilized in the frame despite the movements we know them to have performed during production; the body becomes the camera’s ground, or its point of anchorage. The result is a point of view that, paradoxically, is both physically detached from the body while also being intimately tied to it. THE EXO-CENTRIC TECHNIQUE We see this effect in all forms of the exo-centric image: from early attempts in Kri Kri e il Tango (anonymous, 39 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 1913) and The Last Laugh (Der Letzte Mann, F.W. Murnau, 1924), to more recent and formalized uses in The Wolf of Wall Street (Martin Scorsese, 2013), The Muppets (James Bobin, 2014), The French Dispatch (Wes Anderson, 2021), as well as in animated features such as Frozen II (Chris Buck and Jennifer Lee, 2019), Mitchells vs the Machines (Michael Rianda, 2021), and Turning Red (Domee Shi, 2022). 1913) and The Last Laugh (Der Letzte Mann, F.W. Murnau, 1924), to more recent and formalized uses in The Wolf of Wall Street (Martin Scorsese, 2013), The Muppets (James Bobin, 2014), The French Dispatch (Wes Anderson, 2021), as well as in animated features such as Frozen II (Chris Buck and Jennifer Lee, 2019), Mitchells vs the Machines (Michael Rianda, 2021), and Turning Red (Domee Shi, 2022). By depicting as motionless the body that moved with the camera during filming, the exo-centric image pushes us to recognize the inherent relativity of camera movement. It also highlights the shortcomings of the vocabulary we are taught to describe what we see on screen. Consider the movement the camera performed during the above-mentioned scene from Requiem for a Dream. Although we know full well that the camera moved backward during production as the actress wearing it was walking forward, it would be absurd to describe it as having dollied, tracked, or travelled backward. For one, this would obfuscate the passive nature of the camera’s displacement. Consider Ryan Pierson’s comment on the nature of what constitutes a moving camera or not: If we were to say that every camera ever used on a film was a ‘moving camera’, on the grounds that the Earth underneath it was revolving around the sun, or if we were to say that no camera ever really ‘moves’ because it is always passively part of a dolly or a crane or a human body that’s moving it, then the concept would become useless.16 At the same time, a description of the camera’s movement within profilmic space does not necessarily describe the effect that appears to us on screen. Fig. 1: The so-called ‘Snorricam’ used to shoot Requiem for a Dream (Darren Aronofsky, 2000). Courtesy John Baer (© John Baer 1999) Bédard, Points of Anchorage BODY-SPACE RELATIONS According to David Bordwell, ‘the very notion “camera” already situates us not before the cinema screen, but in a film studio, in production surroundings which include a mechanism called a camera’.17 The same can be said for much of the language we have developed to discuss camera movement, which likewise is grounded in the displacements — whether real or imagined — of the device during production. ‘The category “camera movement”’, Patrick Keating writes, ‘includes several techniques — most notably, pans, tilts, dollies, and cranes’.18 Either these terms describe the tool used to perform a specific kind of movements (cranes are used to move the camera move vertically and dollies are most often used to move the camera in a straight line), or they refer to the particular kind of movement the camera performs (whether it pans left or right, or whether it tilts up or down). Importantly, none of these describes the effects of moving a camera, apart from the terms that metonymically name both the device and the movement for which it is known (e.g., crane, dolly, zoom, etc.). Much like the way we might describe our own movements — or those of other bodies and objects in physical space — the vocabulary we use to describe camera movement assumes that space is fixed and that it is the subject (body or camera) that traverses it.19 That being said, we must recognize that at least two distinct approaches can be used to describe the movements of a given entity in space. Those vary according to the point of reference upon which one anchors one’s descriptions. For the sake of simplicity, I will call these ‘subjective’ and ‘objective’ frames of reference.20 Using what I am calling a ‘subjective’ frame of reference means taking the moving subject itself as the point of anchorage in one’s description of their movements. For example, this could include describing someone as turning to their left or, in our case, saying that a camera pans right or tilts up. These descriptions are ‘subjective’ because they take the body of the subject as anchor; their point of reference is centred in the body. Such descriptions pay no mind to external factors that might affect or relativize how the individual (or the camera) is moving: a person sitting with their back to the direction that their train is moving might say that the vehicle turned (to their) right. THE EXO-CENTRIC TECHNIQUE In the case of the exo-centric image, no profilmic account can satisfactorily describe the impression of an immovable body around which space seems to gravitate. To understand this strange reversal and the implication it has on our appreciation of camera movement, we must first understand the frames of 40 Bédard, Points of Anchorage reference that allow us to make sense of the movement we perceive, on screen as much as in real life. BODY-SPACE RELATIONS Meanwhile, a bystander might describe the same movement as the train having banked left, as seen from their external perspective. This is what I am calling an ‘objective’ frame of reference, taking the individual who is moving — or being moved — as simply one among many other objects within a more expansive field of action. Using this approach is what allows sailors, among other examples, to give spatial cues that relate ‘objectively’ to their vessel (starboard, port, forward, aft, etc.), regardless of their own orientation at any particular moment. The individual that performs the movements remains the subject of the statement, but its status as a point of 41 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X anchorage is revoked in favour of more ‘objective’ points of anchorage. A number of major issues keep me from suggesting this ‘objective’ model might reliably be used to describe the movements of a camera, at least as well as it can be used to describe the movements of objects in the real world. Firstly, the fact remains the vocabulary we have developed to speak of camera movement remains intimately tied to subjective referents. When we write of the camera panning left, tilting up, or tracking forward, we are in fact describing its movements from its own first-person perspective. Secondly, we cannot describe camera movement from an objective perspective precisely because our only access to camera movement — on screen, within a finished film — presents us exclusively with this subjective, first-person experience of movement. Note that this is different from suggesting that camera movement draws us into diegetic space, that it fosters empathy for or identification with the camera, or that it allows us to feel at the position of the camera, as Daniel Morgan has criticized of the notion of point of view.21 On the contrary, my point is simply that, as viewers, we cannot observe a camera’s movement from an external or third- person perspective. BODY-SPACE RELATIONS That is why I am advocating for an approach that is less reliant on the terminology used to describe the displacements of the camera in profilmic space than on ‘the phenomenon of camera movement on the screen as it is originally experienced and understood by us as viewers’.22 In turn, this shift brings us back to the question of the appearance of camera movement, and to the issue of our perception of movement in general. Bédard, Points of Anchorage POINTS OF ANCHORAGE The question of our perception of movement is a large and complex one, having to do not only with the way we perceive our own subjective movements but also the way we come to understand the motion of objects in the world. James J. Gibson’s questions are programmatic in this regard: ‘How do we see the motion of an object? How do we see the stability of the environment? How do we perceive ourselves as moving in a stable environment?’.23 These questions will bear on our appreciation of camera movement, but it is important to consider first how they are dealt with within our habitual modes of perception. Put simply, the answers Gibson gives to these questions have to do with whether parts or the whole of the retina are stimulated at any given time, suggesting objective motion or subjective movement respectively. This interpretation echoes that proposed by Jacques Paillard who, using the ‘efference’ model developed by Von Holst and Mittelstaedt, concludes that ‘the movement of the retinal image resulting from the controlled displacement of the eye would be interpreted perceptually as a movement of the body in relation to a stable universe and not as a mobility of the external universe in relation to the body’.24 In both cases of the movements of the body in its entirety and of those specific to the eye, subjective movement furnishes our perception with sufficient data (visual, but also kinesthetic, vestibular, etc.) to determine whether the movement the eye 42 Bédard, Points of Anchorage Bédard, Points of Anchorage records is due to its own displacement, to those of the body through space, or to those of objects external to the body. While the field of psychophysiology offers illuminating answers to these questions, Maurice Merleau-Ponty’ phenomenology of perception offers an equally stimulating explanation. It will also help us make our way back to the specific issue of movement in the cinema. Merleau-Ponty evokes a similar problem to that raised by Gibson when he describes how easy it is for our perception of movement to be altered by whatever we choose to focus our gaze on. POINTS OF ANCHORAGE Using as an example an individual sailing toward the coast, the philosopher recounts: ‘It is the coast which slips by if we keep our eyes fixed on the rail, and the boat which glides along if we look at the coast’.25 While an objective frame of reference might allow us to give a definitive answer as to what is moving and what is not, subjectively speaking, either one of these things may appear as though it is moving relative to the other. Movement, then, becomes a question of whether one chooses to focus on this or that part of the visual field, and of whether one allows oneself to attend to their perception of said movement. Several other examples of this relativity of movement in the eye of the subject can be found throughout The phenomenology of perception: ‘The cloud floats over the steeple and the river flows under the bridge, if it is the cloud and the river that we are looking at. The steeple falls across the sky and the bridge slides over a static river if we are looking at the steeple or the bridge’.26 Importantly, Merleau-Ponty’s concern for the phenomenological description of appearances enjoins us not to see in these examples an immanent relativity of objects in motion, but rather a purely subjective relativity.27 Indeed, Merleau- Ponty explains that ‘[w]hat makes part of the [visual] field count as an object in motion, and another as the background, is the way in which we establish our relations with them by the act of looking. […] The relation between the moving object and its background passes through our body’.28 Here, Merleau-Ponty’s phenomenology meets Gibson’s ecological approach to perception as both call upon an edge case to highlight the body’s active role in allowing us to make sense of the movement we perceive. The example in question is that of ‘passive or involuntary locomotion’, as is the case when the body is moved in a vehicle, a context in which ‘the kinesthetic component may almost wholly drop out’.29 Deprived of the body’s active contribution, our perception of movement becomes more susceptible to dupery, as Gibson details: It is worth noting that there are special cases of visual stimulation in which it does become equivocal whether the visual scene is moving or whether the observer himself is moving. POINTS OF ANCHORAGE If one sits looking through the window of a stationary railway train at another train on the adjacent track, and if one of the trains begins to move slowly, the impression of moving self with stationary scene may give way to that of stationary self with moving scene, or vice versa.30 For Merleau-Ponty, the interpretation of this illusion depends on the articulation of what, in our field of perception, acts as figure or ground. In the 43 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X case of inattention, the philosopher describes, ‘I can at will see my own train or the train next to it in motion whether on the one hand I do nothing or on the other consider the illusions of motion’.31 If, on the contrary, our consciousness is focused on a particular element, then the nature of the movement we perceive will arise from the relationship between what, at any given point, serves as figure and ground of our perception. As Merleau-Ponty concludes, citing an example by Kurt Koffka, ‘when I am playing at cards in my compartment, I see the neighbouring train move off, even if it is really mine which is starting; when I look at the other train and try to pick out someone, then it is my own train which is set in motion’.32 Koffka’s conclusions about this example are unequivocal: ‘The chief rule for these ambiguous cases is this: that the objects which form the (dynamic) centre of our visual world are at the same time our points of anchorage’.33 Since it is always a question of the relationship between the subject and their environment, the conclusions of Merleau-Ponty, Koffka, and Gibson highlight the role played by points of reference in our perception of movement. These conclusions also prove stimulating for our appreciation of camera movement. Consider a common, if perceptually ambiguous, example: a camera mounted on a moving vehicle, pointed at the characters sitting within it. POINTS OF ANCHORAGE In his analysis of such a shot in Gerry (Gus Van Sant, 2002), Antoine Gaudin asks whether what we are looking at is ‘a camera movement relative to the background of the shot, or a fixed shot of the characters (with a moving setting)?’.34 A similar question might come up when watching Locke (Steven Knight, 2013), where the entirety of the action takes place within a car as Ivan Locke (Tom Hardy) drives from Birmingham to London. As the camera is fixed on the car, pointed at Locke, it would seem absurd to say that it is moving at a constant rate of 100km/h, or thereabouts. While this movement falls into the background of our attention, it is possible to notice when the camera tracks laterally on the hood of the car, or ‘dollies’ in toward Locke. Why is that? In the example of passive locomotion described by Merleau-Ponty, Gibson, and Koffka — but also Bordwell — our perception of what is moving and how depends on whether our attention is directed toward this or that element of the visual field. Unlike the active locomotion of walking — context in which movement provides ‘a dense stream of information about objects’ slants, their edges, their corners, their surfaces, their relations with other objects’35 – the experience of passive locomotion in a train allows us to witness the fundamentally interpretative character of perception, which works by primary anticipation and secondary adaptation.36 If a case of passive locomotion such as riding in a vehicle can give rise to such optical illusions, what of cinema and its equally passive spectatorial position? In the example of passive locomotion described by Merleau-Ponty, Gibson, and Koffka — but also Bordwell — our perception of what is moving and how depends on whether our attention is directed toward this or that element of the visual field. RELATIVE MOVEMENT IN CINEMATIC SPACE(S) Without the clues provided when the body moves of its own free will, perception is quick to fall prey to illusions. This makes the spectator’s passive position all the more interesting for questioning the illusory nature of the ‘perceived screen event’ we call camera movement. ‘Camera movement’, Bordwell writes, ‘presents us with a constricted but effective range of visual cues for subjective movement’.37 With no recourse to an objective frame of reference as to the camera’s actual movements within profilmic space, and without having access to the stimulations their bodies typically afford them in cases of active locomotion, viewers are left to interpret movement solely from the visual cues furnished by the camera’s own subjective movements, be they active, passive or nonexistent. As Gaudin and Schonig have demonstrated following Bordwell’s lead, several additional conditions can affect our understanding of camera movement.38 More specifically, Bordwell reminds us that the ‘the profilmic-event model cannot specify the perceived screen event which we identify as camera movement’ and that, on the contrary, ‘[t]here must be perceptual cues which determine a “camera-movement effect” onscreen regardless of whether the camera moved in production or not (since we recognize camera movement without necessarily making any inferences about production circumstances)’.39 Here, Bordwell shifts the focus from the movements that the camera might have made during production to what we can actually attest to: the appearance of camera movement as we perceive it on screen. Pierson makes a similar argument by calling to our attention to the fact that the camera movement we see in Gravity’s opening shot were actually stitched together from ‘thousands of digitally composited events’.40 The same can be said for animated films in general, as Pierson demonstrates once more, since the impression of camera movement can easily be produced even when no camera was ever used during production. The exo-centric image also illustrates this perfectly, since all traces of the camera’s displacement during filming are eliminated in the final image; a profilmic account cannot adequately describe the onscreen effect. In turn, this shift opens up a discussion regarding the nature of camera movement as an optical illusion. Part of the challenge when viewing camera movement is understanding what elements of the phenomenon seen on screen pertain to the movements of the camera as opposed to the movements of surrounding elements. POINTS OF ANCHORAGE Unlike the active locomotion of walking — context in which movement provides ‘a dense stream of information about objects’ slants, their edges, their corners, their surfaces, their relations with other objects’35 – the experience of passive locomotion in a train allows us to witness the fundamentally interpretative character of perception, which works by primary – the experience of passive locomotion in a train allows us to witness the fundamentally interpretative character of perception, which works by primary anticipation and secondary adaptation.36 If a case of passive locomotion such as riding in a vehicle can give rise to such optical illusions, what of cinema and its equally passive spectatorial position? 44 Bédard, Points of Anchorage Bédard, Points of Anchorage RELATIVE MOVEMENT IN CINEMATIC SPACE(S) In other words, how do we come to determine that it is the camera that moves and not the other objects in the frame?41 As opposed to Gibson’s discussion of subjective movement and objective motion, however, we must also remember that camera movement offers only (second degree) visual cues of subjective movement, leaving up much to interpretation.42 To demonstrate the hermeneutic character of camera movement, consider Rudolf Arnheim’s account of the fundamental distinction between the active 45 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X perception of a body in motion and the stimuli presented through camera movement to the immobile body of the spectator. To set the stage for the effect produced by camera movement, Arnheim chooses first to describe the visual impressions produced by the movements of one’s head: If I turn my eyes or my head, the field of vision is altered. Perhaps a moment ago I was looking at the door; now I am looking at the bookcase; then at the dining-room table, then at the window. This panorama, however, does not pass before my eyes and give the impression that the various objects are moving. Instead I realize that the room is stationary as usual, but that the direction of my gaze is changing, and that that is why I see other parts of the motionless room.43 In accordance with Gibson’s hypotheses, the contributions of the active body are what allow Arnheim to understand that the movement he sees is due to his own ‘subjective movement’ rather than the ‘objective motion’ of external objects. Arnheim insists, however, on the ambiguity that arises when this same movement is executed by a camera and projected on screen: If the camera was rotated while the picture was being shot, the bookcase, table, window, and door will proceed across the screen when the picture is projected; it is they which are moving. For since the camera is not a part of the spectator’s body like his head and his eyes, he cannot tell that it has been turned. He can see the objects on the screen being displaced and at first is led to assume that they are in motion.44 Although counterintuitive at first glance, this conclusion stems from the fact that for Arnheim, the camera’s position is ‘presumed to be fixed. Bédard, Points of Anchorage RELATIVE MOVEMENT IN CINEMATIC SPACE(S) Hence if something moves in the picture this motion is at first seen as a movement of the thing itself and not as the result of a movement of the camera gliding past a stationary object’.45 This ‘relativity of movement in film’, as Arnheim calls it, can even result in movements on screen that completely contradict those performed during production.46 This is also what I am suggesting is demonstrated by exo- centric images. Even if we forego Arnheim’s assumption that the camera is fixed until proven otherwise, the author’s explanations perfectly exemplify Bordwell’s conclusion that: ‘For the camera movement effect to occur, monocular movement parallax must be read from the entire visual field. If only a part or item in the visual field yields that differential angular velocity across time, then camera movement will not be specified — only the movement of that object will be specified’.47 In doing so, Arnheim also confronts us with the hermeneutic character of camera movement. To wit, perceiving the movement of the camera in the image asks of us to decipher purely visual cues, without the use of the kinesthetic stimuli that would usually allow us to perceive movement as either subjectively performed or objectively witnessed. 46 Bédard, Points of Anchorage Bédard, Points of Anchorage Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X EXO-CENTRIC IMAGES AND THE REVERSAL OF BODY-SPACE RELATIONS Arnheim’s inclination to describe a simple panoramic camera movement as depicting the displacement of all objects in space around a stationary camera is an inspiring springboard toward an analysis of the effect produced by exo-centric images. If, like Bordwell and Arnheim before him we choose to focus on the appearance of camera movement on screen rather than on any preconceived notions about if or how a camera moved during production, what ‘camera movement effect’ can we say is produced by the exo-centric technique? Consider the exo-centric image featured in Requiem for a Dream. Although there have been scores of exo-centric images in films and moving image media in the decades since the film’s release, it remains the paradigmatic example of this technique and of its strange reversal of body-camera-space relations. Among the three sequences in Aronofsky’s film that use this technique, the best known one occurs halfway through (01:00:20-01:01:25), when Marion (Jennifer Connelly), leaves the apartment of her psychiatrist after exchanging sexual favours for drug money. The ‘camera movement effect’ used to convey the unsettling feeling of this scene has the side effect of leaving us with a deep uncertainty as to the (camera-)body-space relations presented on screen.48 If we were to describe the movements of the camera ‘objectively’, as it took place on set, we could easily start by noting that it was attached in front of the body of the actress. We would then describe how, equipped with this device, Connelly walked down a lengthy corridor before turning right, calling the elevator, going down to the ground floor and exiting the building, all the while transporting the camera along with her. In this case of passive locomotion — where the camera was simply carried by the actress — should we say that the camera was moving backward in the corridor, that it turned left to get to the elevator, and so on? In the strictest technical sense, these descriptions would indeed be correct with regard to the displacements of the camera within profilmic space. However, several problems would come from this approach. Firstly, such an attempt at describing the camera’s movements ‘objectively’ would obfuscate the fact that the camera did not move autonomously; that it was subject to the movements of the actress. EXO-CENTRIC IMAGES AND THE REVERSAL OF BODY-SPACE RELATIONS Secondly, and as a result of our own limited access to this movement, we must also ask ourselves how this unique camera-body-space relation appears on screen, regardless of the movements we imagine the camera to have performed during production. Limiting ourselves to the visual cues this exo-centric sequence affords us, what can we glean about the origin and nature of the movement depicted on screen? The sequence starts with Marion in the centre of the image, a position from which she does not move during the entire scene. The first of the three shots contained in this one-minute sequence begins when the wall behind Marion starts to move around her, counterclockwise. The vanishing lines that pointed to the left of the screen disappear when the wall fills the frame, then reappear Firstly, such an attempt at describing the camera’s movements ‘objectively’ would obfuscate the fact that the camera did not move autonomously; that it was subject to the movements of the actress. Secondly, and as a result of our own limited access to this movement, we must also ask ourselves how this unique camera-body-space relation appears on screen, regardless of the movements we imagine the camera to have performed during production. Limiting ourselves to the visual cues this exo-centric sequence affords us, what can we glean about the origin and nature of the movement depicted on screen? The sequence starts with Marion in the centre of the image, a position from which she does not move during the entire scene. The first of the three shots contained in this one-minute sequence begins when the wall behind Marion starts to move around her, counterclockwise. The vanishing lines that pointed to the left of the screen disappear when the wall fills the frame, then reappear 47 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X to the right of the frame as the wall continues its rotation totalling 180° [fig. 2]. Once the rotation is complete, the camera faces Marion and the man’s apartment; all three are aligned. As the sequence advances, the man’s apartment recedes into the background while Marion remains motionless in the centre of the frame [fig. 3]. Her immutable position at the centre of the image is crystallized by the rotations of the world around her once she has reached the end of the corridor. Fig. 2: Beginning and end of the 180° rotation. The wall identified in yellow moves around Marion. Screen grab, Requiem for a Dream (Darren Aronofsky, 2000) EXO-CENTRIC IMAGES AND THE REVERSAL OF BODY-SPACE RELATIONS Already, we find that all the visual cues contained in the image convey the impression of a body in a state of (relative) immobility; in the eyes of the camera, Marion has not changed position since the beginning of the sequence. According to Bordwell’s conclusions — indebted to Gibson’s theories and shared by his successors, including Gaudin and Schonig — the appearance of a partial transformation of the environment suggests that only changing elements are in motion. In other words, as Arnheim similarly concluded, ‘if something moves in the picture this motion is at first seen as a movement of the thing itself and not as the result of a movement of the camera gliding past a stationary object’.49 Otherwise, everything that remains fixed in the image is presumed to have been motionless. By extension, and as Bordwell concludes, ‘[f]or the camera movement effect to occur, monocular movement parallax must be read from 48 Bédard, Points of Anchorage the entire visual field’.50 In the case of Requiem for a Dream, these cues suggest the immobility of the character’s body and the movement of all other elements in space around her. This is the very impression that all examples of exo-centric images in narrative cinema evoke. Although the Requiem for a Dream sequence continues with two changes in the camera’s position (it moves behind Marion and then back in front of her), these descriptions are enough to draw some conclusions regarding the shift that occurs when leaving behind the presuppositions that come with the vocabulary of film analysis. Specifically, I have chosen to describe this exo-centric image as depicting a body perfectly fixed in the centre of an otherwise moving space. This interpretation is encouraged by the relative immobility of the body in the eyes of the camera. Echoing Arnheim’s equally eccentric description of a panning camera which gives rise to the impression that space is moving around a stationary camera, my analysis of Requiem for a Dream’s exo-centric image highlights how ambiguous the ‘camera movement effect’ really is. Similarly, borrowing from Jordan Schonig’s interpretation of the gestalt shift that occurs when seeing camera movements in altered viewing conditions, I suggest the approach I have proposed here, ‘doesn’t modify the image itself, but merely Fig. Fig. 3: The vanishing point (identified by yellow lines) moves into depth as Marion remains fixed in the centre of the frame Requiem for a Dream (Darren Aronofsky, 2000) EXO-CENTRIC IMAGES AND THE REVERSAL OF BODY-SPACE RELATIONS 3: The vanishing point (identified by yellow lines) moves into depth as Marion remains fixed in the centre of the frame Requiem for a Dream (Darren Aronofsky, 2000) 49 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X alters the “normal circumstances” under which the visual effects of the moving camera can be seen’.51 CONCLUSION Returning to the example with which we opened this essay, however, leaves us with further questions about the way camera movement might be analysed. The opening shot of Gravity is emblematic of a new production context that is transforming both the camera, and the things it is expected to do in narrative films: virtual production. In virtual production, camera, space, and all elements that compose the film (e.g., sets, lighting, actors, costumes, etc.) are transformed into digital data. The immateriality of the ‘function’ that the camera has become also allows it to cross space without any physical limits (volume, speed, course, etc.).52 As there is no physical film set to speak of in some of these virtual productions, this context raises new questions as to the relevance of an analytical language based in descriptions of the movements of a camera in profilmic space. What is the nature of movement in this virtual cinema? What happens to the division between profilmic and scenographic spaces? How can we deduce the movements of the device in relation to space if they are now, ontologically speaking, one and the same thing? Thankfully, recent scholarship has shown that, now more than ever, camera movement is recognized as a complex phenomenon deserving of a more sustained theoretical engagement. Returning to the example with which we opened this essay, however, leaves us with further questions about the way camera movement might be analysed. The opening shot of Gravity is emblematic of a new production context that is transforming both the camera, and the things it is expected to do in narrative films: virtual production. In virtual production, camera, space, and all elements that compose the film (e.g., sets, lighting, actors, costumes, etc.) are transformed into digital data. The immateriality of the ‘function’ that the camera has become also allows it to cross space without any physical limits (volume, speed, course, etc.).52 As there is no physical film set to speak of in some of these virtual productions, this context raises new questions as to the relevance of an analytical language based in descriptions of the movements of a camera in profilmic space. What is the nature of movement in this virtual cinema? What happens to the division between profilmic and scenographic spaces? How can we deduce the movements of the device in relation to space if they are now, ontologically speaking, one and the same thing? CONCLUSION By modifying a single aspect of our engagement with camera movement — rejecting the habitual, profilmic-focused vocabulary in favour of descriptions based purely on the appearance of visual cues — my goal in this essay has been to shed light on the inherent relativity of camera movement. We know that the camera did in fact move on set while filming Requiem for a Dream, but the absence of any relative movement between the camera and the actress who was carrying reflects another reality. A similar conclusion can be drawn from less unusual techniques for moving the camera. Even the seemingly simple example of a camera fixed onto the front of a moving car — as in the case of Locke described earlier — becomes ambiguous when we start questioning what the frame of reference should be for describing its movements. Ultimately, using a vocabulary anchored to the profilmic context keeps us from engaging more viscerally with the formal effects that camera movements produce on screen regardless — even in spite — of if or how the camera moved during production. By modifying a single aspect of our engagement with camera movement — rejecting the habitual, profilmic-focused vocabulary in favour of descriptions based purely on the appearance of visual cues — my goal in this essay has been to shed light on the inherent relativity of camera movement. We know that the camera did in fact move on set while filming Requiem for a Dream, but the absence of any relative movement between the camera and the actress who was carrying reflects another reality. A similar conclusion can be drawn from less unusual techniques for moving the camera. Even the seemingly simple example of a camera fixed onto the front of a moving car — as in the case of Locke described earlier — becomes ambiguous when we start questioning what the frame of reference should be for describing its movements. Ultimately, using a vocabulary anchored to the profilmic context keeps us from engaging more viscerally with the formal effects that camera movements produce on screen regardless — even in spite — of if or how the camera moved during production. Thankfully, recent scholarship has shown that, now more than ever, camera movement is recognized as a complex phenomenon deserving of a more sustained theoretical engagement. Notes 1 See Scott C. Richmond, Cinema’s Bodily Illusions (Minneapolis: University of Minnesota Press, 2016); Ryan Pierson, Figure and Force in Animation Aesthetics (New York: Oxford University Press, 2019); Daniel Morgan, The Lure of the Image: Epistemic Fantasies of the Moving Camera (Oakland: University of California Press, 2021); Jordan Schonig, The Shape of Motion: Cinema and the Aesthetics of Movement (New York: Oxford University Press, 2021). 1 See Scott C. Richmond, Cinema’s Bodily Illusions (Minneapolis: University of Minnesota Press, 2016); Ryan Pierson, Figure and Force in Animation Aesthetics (New York: Oxford University Press, 2019); Daniel Morgan, The Lure of the Image: Epistemic Fantasies of the Moving Camera (Oakland: University of California Press, 2021); Jordan Schonig, The Shape of Motion: Cinema and the Aesthetics of Movement (New York: Oxford University Press, 2021). y 2 David Bordwell, ‘Camera Movement and Cinematic Space’, Ciné-Tracts, 1 (1977), 19. 3 Vivian Sobchack, ‘Toward Inhabited Space: The Semiotic Structure of Camera Movement in the Cinema’, Semiotica, 41 (1982), 317–335 (317). 3 Vivian Sobchack, ‘Toward Inhabited Space: The Semiotic Structure of Camera Movement in the Cinema’, Semiotica, 41 (1982), 317–335 (317). 3 Vivian Sobchack, ‘Toward Inhabited Space: The Semiotic Structure of Camera Movement in the Cinema’, Semiotica, 41 (1982), 317–335 (317). 5 Adriano D’Aloia, ‘The Character’s Body and the Viewer: Cinematic Empathy and Embodied Simulation in the Film Experience’, in Embodied Cognition and Cinema, ed. by Maarten Coëgnarts and Peter Kravanja (Leuven: Leuven University Press, 2015), 187–199; Kristen Whissel, ‘Parallax Effects: Epistemology, Affect and Digital 3d Cinema’, Journal of Visual Culture, 15 (2016), 233–249; Ryan Pierson, ‘Whole-Screen Metamorphosis and the Imagined Camera (Notes on Perspectival Movement in Animation)’, Animation, 10 (2015), 6–21; Tom Gunning, ‘“Nothing Will Have Taken Place – except Place”: The Unsettling Nature of Camera Movement’, in Screen Space Reconfigured, ed. by Susanne Ø. Sæther and Synne T. Bull (Amsterdam: Amsterdam University Press, 2020), 263–281; Morgan, 17–19. 8 Richmond. 9 Philippe Bédard, ‘L’espace exo-centrique au cinéma’, Écranosphère, 4 (2020), 7–23; Bédard, ‘Going Beyond the Human Perspective: GoPro Cameras and (Non)Anthropocentric Ways of Seeing’, in Versatile Camcorders: Looking at the Gopro Movement, ed. by Winfried Gerling and Florian Krautkrämer (Berlin: Kadmos, 2021), 45–61; Bédard, ‘Un regard hors de soi : Étude des rapports entre corps, caméra et espace dans l’histoire des techniques de prise de vues au cinéma’ (Ph. D. thesis, Université de Montréal, 2021). 10 Bordwell, 21. 11 Schonig, 121. 11 Schonig, 121. CONCLUSION 50 Bédard, Points of Anchorage 17 Bordwell, 20, emphasis in the original. Notes g 12 For an in-depth study of the narrative functions of exo-centric images, see Bédard, ‘Situating the Camera: Third-Person Images and the Question of Point of View in Narrative Cinema’ (Master thesis, Concordia University, 2015). Note that while what I call here ‘exo-centric images’ can be found in almost any type of moving image media — as long as there are a body, a camera, and a space – I have limited my examples and sources to narrative films here simply for the sake of efficiency.ii ii 13 Here too, we can find exceptions to this definition. The camera can be attached to a vehicle, an animal, or any other nonhuman body. For the sake of clarity and because there are more prevalent, I will focus on exo-centric images that feature the human body. 13 Here too, we can find exceptions to this definition. The camera can be attached to a vehicle, an animal, or any other nonhuman body. For the sake of clarity and because there are more prevalent, I will focus on exo-centric images that feature the human body. 14 Like Steadicam, Kodak, GoPro and so many other genericized trademarks, the name Snorricam is often used indiscriminately to describe ‘a camera device used in filmmaking that is rigged to the body of the actor, facing the actor directly, so when they walk, they do not appear to move, but everything around them does’. <https://www.snorricam.com/home> [accessed 23 April 2022]. 14 Like Steadicam, Kodak, GoPro and so many other genericized trademarks, the name Snorricam is often used indiscriminately to describe ‘a camera device used in filmmaking that is rigged to the body of the actor, facing the actor directly, so when they walk, they do not appear to move, but everything around them does’. <https://www.snorricam.com/home> [accessed 23 April 2022]. 15 Most films use the frontal arrangement, as seen in Requiem for Dream, Mean Streets, and The Hangover. Cameras mounted behind the actor in this exo-centric position would produce an over-the-shoulder perspective reminiscent of third-person points of view in video games, as showcased in Seconds (John Frankenheimer, 1966). Finally, still fewer films have used a rotating exo-centric perspective, with only Angst (Gerald Kargl, 1983) coming to mind. However, this orbiting exo-centric point of view is much more common in GoPro videos. See ‘Situating the camera’. 17 Bordwell, 20, emphasis in the original. 17 Bordwell, 20, emphasis in the original. 7 D’Aloia. 8 Richmond. Notes p g 18 Patrick Keating, The Dynamic Frame: Camera Movement in Classical Hollywood (New York: Columbia University Press, 2019). 19 S A t i G di L’ i é t hi E théti t d t i (P i A d C li 18 Patrick Keating, The Dynamic Frame: Camera Movement in Classical Hollywood (New York: Columbia University Press, 2019). 19 See Antoine Gaudin, L’espace cinématographique: Esthétique et dramaturgie (Paris: Armand Colin, 2015), 7–8. 19 See Antoine Gaudin, L’espace cinématographique: Esthétique et dramaturgie (Paris: Armand Colin, 2015), 7–8. 51 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 20 In navigational sciences, these would be called egocentric and exocentric, respectively. For a more sustained discussion of these concepts and their relation to camera movement, see ‘Un regard hors de soi’. 21 Morgan. 23 James J. Gibson, ‘The Visual Perception of Objective Motion and Subjective Movement’, Psychological Review, 101 (1994), 318–323 (318). 23 James J. Gibson, ‘The Visual Perception of Objective Motion and Subjective Movement’, Psychological Review, 101 (1994), 318–323 (318). 24 Jacques Paillard, ‘Les déterminants moteurs de l’organisation de l’espace’, Cahiers de psychologie, 14 (1971), 261–316 (272), emphasis added. My translation of: ‘le mouvement de l’image rétinienne résultant du déplacement commandé de l’œil serait interprété au plan perceptif comme un mouvement du corps par rapport à un univers stable et non comme une mobilité de l’univers extérieur par rapport au corps’. 24 Jacques Paillard, ‘Les déterminants moteurs de l’organisation de l’espace’, Cahiers de psychologie, 14 (1971), 261–316 (272), emphasis added. My translation of: ‘le mouvement de l’image rétinienne résultant du déplacement commandé de l’œil serait interprété au plan perceptif comme un mouvement du corps par rapport à un univers stable et non comme une mobilité de l’univers extérieur par rapport au corps’. 25 Maurice Merleau-Ponty, The Phenomenology of Perception (New York: Routledge, 2010), 324. 26 Ibidem. 27 Indeed, Merleau-Ponty issues a warning that ‘to say that motion is a structural phenomenon is not to say that it is ‘relative’. The very peculiar relationship which constitutes movement does not exist between objects’, ibidem, 323–324. Nevertheless, I insist on calling ‘relative’ this perception of movement which imparts on this object or that the movement an individual perceives, according to whether they are focussing on this or that. 27 Indeed, Merleau-Ponty issues a warning that ‘to say that motion is a structural phenomenon is not to say that it is ‘relative’. Notes The very peculiar relationship which constitutes movement does not exist between objects’, ibidem, 323–324. Nevertheless, I insist on calling ‘relative’ this perception of movement which imparts on this object or that the movement an individual perceives, according to whether they are focussing on this or that. 28 Ibidem, 324, emphasis added. Another instructive example in this regard is Merleau-Ponty’s description of a patient with paresis of the oculo-motor muscles: ‘A subject whose oculo-motor muscles are paralysed sees objects moving to his left whenever he believes that he is turning his eyes towards the left’, ibidem. 55. Convinced that they have moved their eye to the left, the patient’s perception of their surrounding shifts to account for the expected movement; they attribute to the outside world the movement they want to accomplish themself. 29 Gibson, 318. 30 Ibidem, 319. 31 Merleau-Ponty, 326. The illusory subjective movement will be proportionally inverse to that of the visual stimulus caused by the moving train in peripheral vision, so that a neighbouring train that ‘retreats’ (which goes in the opposite direction to that which one is facing) will give one the impression that they are moving forward. 32 Ibidem. 33 Kurt Koffka, ‘Perception: An Introduction to the Gestalt-Theorie’, Psychological Bulletin, 19 (1922), 531– 383 (578). 33 Kurt Koffka, ‘Perception: An Introduction to the Gestalt-Theorie’, Psychological Bulletin, 19 (1922), 531– 383 (578). 34 My translation of: ‘S’agit-il d’un mouvement de caméra par rapport au fond du plan, ou bien d’un plan fixe sur les personnages (avec un décor mouvant) ?’, Gaudin, 129. 34 My translation of: ‘S’agit-il d’un mouvement de caméra par rapport au fond du plan, ou bien d’un plan fixe sur les personnages (avec un décor mouvant) ?’, Gaudin, 129. 36 Art historian Ernst Gombrich quotes J. R. Beloff to the effect that: ‘Perception may be regarded as primarily the modification of an anticipation’, to which Gombrich adds, still about perception: ‘It is always an active process, conditioned by our expectations and adapted to situations’. Ernst H. Gombrich, Art and Illusion: A Study in the Psychology of Pictorial Representation (Princeton: Princeton University Press, 1961), 136. 38 Gaudin, 129; Schonig. 39 Bordwell, 20–21, emphasis in the original. , 41 In a way, these questions are a continuation of those asked by Gibson, namely ‘How do we see the motion of an object? How do we see the stability of the environment? 22 Sobchack, 317. 21 Morgan. 45 Ibidem, 32, emphasis added. 23 James J. Gibson, ‘The Visual Perception of Objective Motion and Subjective Movement’, Psychological Review, 101 (1994), 318–323 (318). 44 Ibidem, 30–31, emphasis added. 24 Jacques Paillard, ‘Les déterminants moteurs de l’organisation de l’espace’, Cahiers de psychologie, 14 (1971), 261–316 (272), emphasis added. My translation of: ‘le mouvement de l’image rétinienne résultant du déplacement commandé de l’œil serait interprété au plan perceptif comme un mouvement du corps par rapport à un univers stable et non comme une mobilité de l’univers extérieur par rapport au corps’. 20 In navigational sciences, these would be called egocentric and exocentric, respectively. For a more sustained discussion of these concepts and their relation to camera movement, see ‘Un regard hors de soi’. 29 Gibson, 318. 39 Bordwell, 20–21, emphasis in the original. 38 Gaudin, 129; Schonig. 37 Bordwell, 21. 30 Ibidem, 319. 51 Schonig, 121. 47 Bordwell, 22. 48 Keating would call these images ‘semi-subjective’, following Jean Mitry’s use of the term. For an in-depth analysis of exo-centric image through the lens of the Mitry’s semi-subjective point of view, see ‘Situating the Camera’. 46 Ibidem. 50 Bordwell, 22. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 49 Arnheim, 32, emphasis added. 46 Ibidem. 47 Bordwell, 22. 48 Keating would call these images ‘semi-subjective’, following Jean Mitry’s use of the term. For an in-d analysis of exo-centric image through the lens of the Mitry’s semi-subjective point of view, see ‘Situ the Camera’. 49 Arnheim, 32, emphasis added. 50 Bordwell, 22. 51 Schonig, 121. 52 See Mike Jones, ‘Vanishing Point: Spatial Composition and the Virtual Camera’, Animation, 2 (2 225–243. Notes How do we perceive ourselves as moving in a stable environment?’, Gibson, 318. 41 In a way, these questions are a continuation of those asked by Gibson, namely ‘How do we see the motion of an object? How do we see the stability of the environment? How do we perceive ourselves as moving in a stable environment?’, Gibson, 318.i 42 Indeed, as Sobchack cleverly articulates, ‘we can see the film’s seeing as the seeing of another who is like myself, but not myself’. Vivian Sobchack, The Address of the Eye: Phenomenology of Film Experience (Princeton: Pinceton University Press, 1992), 136. 42 Indeed, as Sobchack cleverly articulates, ‘we can see the film’s seeing as the seeing of another who is like myself, but not myself’. Vivian Sobchack, The Address of the Eye: Phenomenology of Film Experience (Princeton: Pinceton University Press, 1992), 136. y 43 Rudolf Arnheim, Film as Art (Berkeley, Los Angeles and London: University of California Press, 1957), 30. 43 Rudolf Arnheim, Film as Art (Berkeley, Los Angeles and London: University of California Press, 1957), 30. 44 Ibidem, 30–31, emphasis added. 52 Bédard, Points of Anchorage Bédard, Points of Anchorage Bordwell, 22. 48 Keating would call these images ‘semi-subjective’, following Jean Mitry’s use of the term. For an in-depth analysis of exo-centric image through the lens of the Mitry’s semi-subjective point of view, see ‘Situating the Camera’. 49 Arnheim, 32, emphasis added. 50 Bordwell, 22. Schonig, 121. 52 See Mike Jones, ‘Vanishing Point: Spatial Composition and the Virtual Camera’, Animation, 2 (2007), 225–243. g, 52 See Mike Jones, ‘Vanishing Point: Spatial Composition and the Virtual Camera’, Animation, 2 (2007), 225–243. 53 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X A Gestalt Theory for ‘Disorder’: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art Maria Poulaki, Hellenic Open University The article revisits the concept of entropy in art as discussed by Gestalt psychologist and art theorist Rudolf Arnheim. His discussion of artworks and their reception as complex dynamic fields where the forces of entropy and orderliness counter and complete each other, are brought into dialogue with newer approaches, from the perspective of complexity theory and neuroscience, to the dynamics of perception and to entropic processes in the brain. I will argue that even though Arnheim’s observations can still be valuable for contemporary art criticism they need to be updated as they tend to overstate the tendency for order as well as the visual aspects of reception in the expense of multimodal and embodied aspects. In light of these observations, I will discuss contemporary cases of ‘entropic’ art through the moving image works of Marco Brambilla, their aesthetics as well as the ‘structural themes’ arising and the Gestalt processes involved in their reception. Keywords Rudolf Arnheim Entropy Marco Brambilla Gestalt Dynamical systems Media art DOI Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License sed under a Creative Commons Attribution 4.0 International License 10.54103/ 2036-461X/17087 Entropy as a measure of uncertainty in information theory and as an indication of the tendency for disorder through energy loss according to the second law of thermodynamics could be considered synonymous with dissolution and disintegration of form. In this sense it is antithetical to Gestalt theory’s interest in form and the process of its emergence. This contradiction is the reason we are drawn to prominent art theorist and Gestalt psychologist Rudolf Arnheim’s interest in entropy and its connection to Gestalt principles, as well as its application to art. His contribution is further important as it strengthens the thread that links Gestalt psychology, through the dynamics of perception, with more recent approaches like dynamical systems neuroscience, which is also interested in the concept of entropy and how it plays out in cognitive processes. Following physicist Max Planck,1 Arnheim associates entropy with ‘elementary disorder’ that irreversibly increases and ultimately drives a system and its surroundings towards total degradation. A Gestalt Theory for ‘Disorder’: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art Maria Poulaki, Hellenic Open University On the notion of disorder he also quotes Gestalt psychologist Wolfgang Köhler: ‘The word disorder applies suitably to physical states in which a multiplicity of elements pursue mostly independent paths but, for short times, come into physical connection’.2 So Arnheim applied this notion of entropy-as-disorder to art: ‘A visual parallel can be found in works Entropy as a measure of uncertainty in information theory and as an indication of the tendency for disorder through energy loss according to the second law of thermodynamics could be considered synonymous with dissolution and disintegration of form. In this sense it is antithetical to Gestalt theory’s interest in form and the process of its emergence. This contradiction is the reason we are drawn to prominent art theorist and Gestalt psychologist Rudolf Arnheim’s interest in entropy and its connection to Gestalt principles, as well as its application to art. His contribution is further important as it strengthens the thread that links Gestalt psychology, through the dynamics of perception, with more recent approaches like dynamical systems neuroscience, which is also interested in the concept of entropy and how it plays out in cognitive processes. Following physicist Max Planck,1 Arnheim associates entropy with ‘elementary disorder’ that irreversibly increases and ultimately drives a system and its surroundings towards total degradation. On the notion of disorder he also quotes Gestalt psychologist Wolfgang Köhler: ‘The word disorder applies suitably to physical states in which a multiplicity of elements pursue mostly independent paths but, for short times, come into physical connection’.2 So Arnheim applied this notion of entropy-as-disorder to art: ‘A visual parallel can be found in works 55 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X of art that appear to consist of unrelatable units. The components strain to adapt to one another, fight each other, come apart. The disorderly pattern is perceived as a combination of independent units locked in unreadable conflict’.3 But what would be an entropic artwork for Arnheim? As opposed to the tendency for artistic simplicity, he writes, The other tendency, relying on accidental or deliberately produced disorder, can be traced back to a predilection for compositions of randomly gathered subject matter in Dutch still lifes, untidy scenes of social criticism in the generation of Hogarth, groups of unrelated individuals in French genre scenes of the nineteenth century, and so on. A Gestalt Theory for ‘Disorder’: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art Maria Poulaki, Hellenic Open University In modern painting we note the more or less controlled splashes and sprays of paint, in sculpture a reliance on chance textures, tears or twists of various materials, and found objects.4 His examples here come mainly from painting, although we could also find such in film — notably in Soviet Montage in which conflict played a key role, and multiplicity manifested in space (due to compositional elements maintaining their heterogeneity and certain ‘independence’) as well in time (due to frequent cutting resulting in a high number of shots). Steve Odin refers to Eisenstein’s ‘monism of ensemble’5 at the core of his conception of montage, where shots are juxtaposed to create a total impression, not just accompanying each other but functioning as ‘elements of equal significance’.6 Even though Arnheim writes on entropy as disorder, his main interest is in the way the increase of entropy can build a higher level of order. He refers to order as a process rather than as a set property of a form, being mostly interested in order as emergent from randomness and disorder. No matter how complex a structure, it can show a certain degree of ‘orderliness’. Homogeneity is the simplest way of ordering material (‘the most elementary structural scheme’)7 — and from there various degrees of ordering can emerge. Artistic creation thus becomes a self-regulatory process of ordering — and the same applies to the reception of art. Only from a macro-perspective can order be discerned, as Arnheim argues: ‘only when we look at macrostates rather than at the single elements that comprise them’.8 In this sense, entropy can also be created through repetition, which might be redundant for information theory but useful for art, as it might contribute to the emergence of a structural theme. For example, Arnheim refers to ‘the processions of almost identical human figures on the walls of San Apollinare Nuovo in Ravenna’;9 their grouping makes a new form emerge, that of a multitude of worshipers different than the sum of its parts, which affects the beholder perceptually as well as emotionally. While Gestalt psychology’s concept of ‘Prägnanz’, interpreted (wrongly for Arnheim) as ‘good form’, has been well known and rather influential, Arnheim is critical of confusing the notion of order with Prägnanz. Poulaki, A Gestalt Theory for ‘Disorder’ A Gestalt Theory for ‘Disorder’: From Arnheim’s Ordered Chaos to Brambilla’s Entropic Art Maria Poulaki, Hellenic Open University As he argues, Order can be analyzed with the tools of Gestalt psychology, which, in principle, has ways of determining levels of complexity as well as degrees of orderliness. This does not mean that a high level 56 Poulaki, A Gestalt Theory for ‘Disorder’ of order is the same as a ‘good Gestalt’ — an unfortunate term, which, in some of the early Gestalt writings, burdened a purely descriptive concept with a value judgment and made a definite structural condition look subjective and vague. The term was used to describe the tendency toward regularity, symmetry, simplicity, best named ‘the law of simplicity’ or perhaps ‘the law of dynamic direction,’ as Köhler called it in 1938. Because of the vagueness of the term, ‘good Gestalt’. the law of simplicity was readily confused with ‘praegnanz’, meaning clear-cut structure, or with whatever else may be perceptually and aesthetically enjoyable, interesting, appropriate, or useful. [...] [T]he law of simplicity refers only to orderliness attained by tension reduction.10 As it becomes apparent from this passage, Arnheim’s view of order is dynamic, aligned with a tendency for tension reduction, rather than a finite state — and much less, indeed, a value judgement associated with ‘good’ Gestalt. Artworks are not interesting only when they offer harmonic compositions and well- recognizable shapes, but when they invite perception to respond with ‘acts of recognition’,11 through a dynamic movement that makes forms emerge. Here Arnheim of course stays faithful to the basic tenets of Gestalt theory, and to Köhler’s ‘law of dynamic direction’ expressed in field processes where wholes and parts interact, and where the tendency for tension reduction coexists with ‘a tension-increasing articulation’.12 Despite this, however, Arnheim insists on the necessity of order, not only in art (where he finds it ‘a necessary although not a sufficient condition of aesthetic excellence’),13 but also in life, as ‘order is a prerequisite for survival; therefore the impulse to produce orderly arrangements is inbred by evolution. […] A pervasive striving for order seems to be inherent […] in the human mind — an inclination that applies mostly for good practical reasons’.14 Artistic striving for order is embedded in this wider tendency. Entropy is still fascinating for Arnheim as it brings forward these dynamics of orderliness that counter entropy’s drive towards disorganization, homogenization and shapelessness. ENTROPIC ART Arnheim finds art that engages with chance and contingency and contains units coexisting in tension to be in dialogue with entropy, countering its formless tendencies. He insisted that in order to be meaningful, contingency should be subsumed to a certain structural theme ‘anabolically’ established (referring to anabolism as a molecular process of building complexity through energy storage), ‘which introduces and maintains tension’.15 He was thus rather critical of artistic attempts to create the impression of randomness and entropy without subsuming it to a structural theme. He particularly refers to ‘certain avant garde attempts in film editing [insisting on this point since the 1930s when in his writings on film, he criticized Carl Theodor Dreyer’s La Passion de Jeanne d’Arc (1928) for the same reason],16 or the multiplication or mixing of 57 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X media to combine disparate elements more or less at random. […] [B]ut mere randomness of combination does not suffice to create readable complexity’.17 Such works are not entropic for Arnheim, as they do not involve the anabolic process of spending energy and building tension through the process of organization: A mind released from the demands of organized experience may content itself with the shapelessness of accidental materials, happenings, or sounds. Mere noise involves a minimum of structural tension and therefore calls for a minimum of energy expended by producer and recipient, in spite of creating the illusion that much is going on. In the extreme case, again, it will reach the emptiness of homogeneity.18 He concludes that artistic techniques used to create noise in art, when not handled with competence, lead to chaos, ‘which is very close to saying nothing’.19 A good sense of form on behalf of the artist can bring forth interesting and even beautiful structural themes, but ‘mere randomness of combination does not suffice to create readable complexity’.20 Neither, however, a complex order, even if it corresponds to a level of complexity that the human mind can handle is for Arnheim a sufficient condition to make an artwork valuable. ENTROPIC ART What is mostly important, he remarks, ‘is that this order reflect a genuine, true, profound view of life’.21 We should not interpret Arnheim’s remark as a call to naturalism or representational realism (Arnheim was after all a devoted formalist), but rather as a call to art that, as he writes, ‘makes visible or audible’ a particular form of order of the human condition. Art that becomes a message exactly because of this coupling with the human observer or listener — and not because of its complexity per se, but because of its profound meaning communicating something anew, or offering a new perspective on life: ‘A structural theme deserves to be ordered, to become a message, because of what it says about man and world’.22 Poulaki, A Gestalt Theory for ‘Disorder’ GESTALT THEORY AS COMPLEXITY THEORY? Arnheim’s fascination with entropy and his discussion of it in the context of artistic complexity is insightful and important in that it brings the Gestalt theory of art in dialogue with complex systems theory. A number of points he makes is compatible with approaches that were developed in later decades — even though it could be said that complexity theory was not unknown to the art and humanities in the 1970s, for example in the work by Gene Youngblood on expanded cinema or Buckminster Fuller’s ‘synergetics’.23 Particularly, Arnheim places emphasis on the independence of elements out of which wholes emerge rather than on wholes alone. In this sense, to an extent his analysis in Entropy and Art could be considered to share the perspective of what Ian Bogost calls ‘new complex systems theory’.24 The latter differs from earlier ‘classical’ systems theory in that it privileges bottom-up approaches, placing emphasis on 58 Poulaki, A Gestalt Theory for ‘Disorder’ the way units interact and form aggregates that are different than the sum of their parts, further becoming themselves subsystems of larger organizations in a process of growing complexity. In a later essay from 1990, Arnheim confirms his earlier stance defending with a greater awareness Gestalt theory as a complex systems theory and insisting on a two-way determination of Gestalt wholes, as ‘any description uniquely favoring the role of either the part or the whole will fail to do justice to the richness of field processes’.25 Arnheim’s emphasis on dynamics of orderliness that use energy to create structure out of disorder is compatible with a paradigm change that according to Katherine Hayles took place in systems theory after the 1970s, according to which randomness is not ‘simply [...] the lack of pattern [...] but […] the creative ground from which pattern can emerge’.26 Pattern and randomness are found in a productive dialectic, as systems achieve higher levels of complexity with the ‘infusion of noise’.27 Even though Arnheim might not have embraced chaos as a force of emergence so enthusiastically, he nonetheless regards it as a necessary condition for order and meaning to emerge, thus his perspective is quite progressive in this respect, and reveals how Gestalt theory —on which his thinking is based, more than it is based on physics and thermodynamics — is linked to these later developments. GESTALT THEORY AS COMPLEXITY THEORY? Moreover, Arnheim’s view of making meaning is compatible with ideas emerging around the time of Entropy and Art on autopoietic coupling of system and environment — as proposed by Humberto Maturana and Francisco Varela in 1972 in their theory of autopoiesis.28 In their seminal work on cognitive neuroscience and philosophy, The Embodied Mind (1992), which drew on autopoiesis, Varela, Evan Thompson and Eleanor Rosch point out that the constitution of patterns is fundamental to the way a system ‘couples’ with its environment, and is associated with the way autopoietic organisms self-organize by relating and dealing with external complexity: ‘over time this coupling [of a system with its milieu] selects or enacts from a world of randomness a domain of distinctions [...] that has relevance for the structure of the system. In other words, on the basis of its autonomy the system selects or enacts a domain of significance’.29 The meaning Arnheim looks for in an artwork does not have to do so much, as already pointed out, with its technical competence and formal complexity per se, but with its ability, through its structural theme or pattern, to create a domain of significance, and therefore a world that is meaningful to the beholder, who, in this perspective, would be a system as well, coupling with the work. 59 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X FROM ENTROPY TO ORDER AND BACK TO ENTROPY: THE COMPLEXITY OF SYSTEMS FROM GESTALT TO DYNAMICAL SYSTEMS THEORY Even though, as Wagemans et al. argue, the Berlin School of Gestalt psychology, the teaching of which Arnheim follows in Entropy and Art (particularly that of Köhler), ‘tended to emphasize properties of the system above properties of the system elements’ and to consider form superseding its elements in a ‘one way global to local determination’,30 they recognize that Gestalt theory’s ideas paved the way for later theories of dynamic cognitive organization, such as dynamical systems neuroscience. Arnheim’s reference to the neuronal basis of Gestalts in art’s reception is through Köhler’s electromagnetic field theory of brain functioning. Perceived forms, for example in a visual composition of an artwork in art s reception is through Köhler s electromagnetic field theory of brain functioning. Perceived forms, for example in a visual composition of an artwork — a dynamic field in itself according to Arnheim — corresponds to a neuronal cortical field that is isomorphically dynamic, ‘because only when the forces constituting a process are sufficiently free to interact can a pattern organize itself spontaneously according to the structure prevailing in the whole’.31 Even though it is debatable whether Köhler’s particular theory still has bearing in modern neuroscience (with some claiming it has been disproved while others that it is compatible with newer theories of consciousness),32 field theory takes a holistic and dynamic perspective on neuronal organization; but its tendency to consider these dynamics tending always to a certain equilibrium is less supported when taking a contemporary dynamical systems theory perspective that sees brain dynamics inherently entropic and considers instability the fundamental state of neuronal functioning. Ordered areas, represented by attractors in the brain’s state-space, are never stable, as the mind’s dynamics is ‘metastable’, meaning that its normal condition is to drift, as neuroscientists Emmanuelle Tognoli and Scott Kelso note, between and away from stable regions (represented by ‘attractors’ in the brain’s state-space). 33 The concept of the ‘entropic brain’ that has been recently proposed by Carhart-Harris et al. is based on states of ‘disorganization’ of the brain’s function. Such are states of ‘criticality’, ‘the property of being poised at a “critical” point in a transition zone between order and disorder’.34 Entropy increases when the mind-brain is under the influence of substances (such as hallucinogenics) but also in different conditions and states of uncertainty, which can be reached through different avenues, ranging from dreaming to art. Poulaki, A Gestalt Theory for ‘Disorder’ FROM ENTROPY TO ORDER AND BACK TO ENTROPY: THE COMPLEXITY OF SYSTEMS FROM GESTALT TO DYNAMICAL SYSTEMS THEORY When the brain system’s entropy increases, the multiplicity of potential states rises, and the system acquires a ‘maximum sensitivity to perturbation’,35 which means that it can easily and unpredictably switch to different directions. In this perspective, processes of formation of Gestalts in perception and consciousness are never complete or stable. Carhart-Harris et al. distinguish between the secondary ‘waking’ 60 Poulaki, A Gestalt Theory for ‘Disorder’ consciousness and the ‘primary’ brain states of risen entropy. Secondary consciousness shows an ‘entropy-suppressing function’ that ‘serves to promote realism, foresight, careful reflection and an ability to recognize and overcome wishful and paranoid fantasies. Equally however, it could be seen as exerting a limiting or narrowing influence on consciousness’.36 It is interesting how both Arnheim in his discussion of entropy in the early 1970s as well as the entropic brain theorists (re)turn to Freud: Arnheim in stressing the need for ‘tension reduction’, expressed in suppression of drives as well as in the drive towards order and structure, and Carhart-Harris et al., from the opposite end, in arguing that the entropic brain in its primary states corresponds to the Freudian unconscious, with the rise in criticality releasing tension that the secondary, normal waking consciousness suppresses. A similar attitude, open to the destabilizing forces of entropy as a positive organizing force, can be discerned in both cases, however more emphatically and decisively in the case of Carhart- Harris et al. because of the change of paradigm that took place in cognitive science during the past few decades.37 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART Brambilla worked with Photoshop in his studio to make the collages as still canvases on which, aided by technicians and VFX artists (Crush studio), he overlaid sampled clips from Hollywood movies projected in loops on these canvases. Over four hundred video clips thus composed a huge ‘video mural’, which the elevator passengers could experience as a trip from hell to heaven, as the lift goes up, and from heaven to hell as it goes down.38 Brambilla often adopts in his work the technique of collage, consisting of — in Arnheim’s expression — ‘unrelatable units’ which bring into dynamic interplay the unit with the whole. As he explains in an interview, ‘Collage is the point of departure, juxtaposing imagery then superimposing looping visuals onto one another and setting cuts from various films into each other to create original narratives. They function much like the parabolic style of Hieronymus Bosch who layered fables and proverbs as detailed notes within a big picture’.39 As already noted, the piece Civilization gave birth to a trilogy of large-scale 3D video installations called Megaplex to evoke the homonymous cinema theaters in the US, hinting at Brambilla’s background as a filmmaker and his passion for cinema. The embodied engagement of the beholder in this series of works (in the case of Civilization, ascending — or even descending in the case of the elevator projection — on a journey to heaven) presumably changes the affect of the images as well as the emerging Gestalts and narratives each time a visitor takes a journey. Experiencing Civilization in an elevator differs from experiencing this and the other vivid ‘tableaus’ of the series in a museum or gallery space — which has been the case, as Megaplex was exhibited in various places internationally. Following the regular, steady and mechanical movement of the elevator differs from the less restricted bodily engagement of the gallery visitor, who, unlike the hotel visitor, can experience the work in 3D. The three parts of the trilogy involve camera movement in three different axes: Civilization as already noted moves on the vertical axis, Evolution on the horizontal axis (as it unfolds like a pre-cinematic panorama, scrolling sideways), while Creation is a ‘cosmic pull back’ on the z-axis. NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART Arnheim’s observations on art and entropy can be useful when considering contemporary works representing the ‘tendency for disorder’ through multiplicity and randomness in their composition. One could argue that complex compositions, which resemble the ‘French genre scenes of the 19th century’ Arnheim mentions but have also evolved in their complex and mixed-media environments such as those I will shortly discuss, resist macrostructure, or rather offer dynamic and unstable macrostructures, because from the beholder’s perspective, engaging with different parts of the work might make new macrostructures and patterns emerge. This is not, of course, a characteristic of new media art only. Such dynamics bring to mind works in the tradition of op art, for example Bridget Riley’s Composition with Circles 2 (2001): the more you look at its multiple, almost identical patterns, the more constellations and shapes you can discern, while old ones fade and new ones emerge continuously. The dimension of time is fundamental in the experience of such dynamic artworks, and becomes even more so in the arts of the moving image. It would thus not be entirely accurate to call these dynamic and transitive macrostructures ‘order’. Moreover, even orderliness as a tendency for Gestalt ordering might not be the most essential aspect of the beholder’s experience, as the mind-body is engaged and challenged in different ways in works that surpass the pictorial. Contemporary media artworks such as those of Marco Brambilla make an interesting case to explore such issues. Brambilla produces moving image work that is quite versatile in terms of styles, techniques and media used, ranging 61 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X from large-scale 3D video collages and panoramas (e.g., the Megaplex series: Civilization, Evolution, Creation, 2008-2012) to music videos (for rapper Kanye West’s song ‘Power’ (2010)) or the stage video-projections for Debussy’s opera Pelléas et Mélisande (2018) produced by Opera Vlaanderen and directed by Aviel Cahn. Brambilla’s work Civilization, the first piece of the Megaplex trilogy, is a piece originally commissioned by the NYC Standard Hotel and designed to be installed on the side of its elevator shaft which would thus function as projection surface for the work to be watched from the lift’s interior. The shaft’s long vertical surface became a tableau populated with an excessive multiplicity: hundreds of videos projected and overlaid to compose baroque collages. Poulaki, A Gestalt Theory for ‘Disorder’ NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART These types of movement are conceptually linked to the works (Civilization for example evokes religious themes while Evolution refers to historical and chronological development), building embodied metaphors that make such concepts felt on a precognitive level. 62 Poulaki, A Gestalt Theory for ‘Disorder’ Areas of orderliness are certainly present within this multiplicity, as the swarm of looping videos in each moving mural is placed in a certain way with an intention to form patterns discerned from a vantage point. In all cases, however, there is an effect of ‘excess’ — not only spatial (due to the multiplicity of scenes, the large scale of the installation and the high number of these ‘detailed notes’ contained in the canvases) but also temporal, as what the viewer ‘catches’ each time is dependent on the speed of the images’ (as well as the viewer’s own) movement — which is never enough to properly attend to the work, and makes it hard to remember it in detail. In Megaplex, as described on Brambilla’s website, ‘The hyper-saturated tableaus test the limits of visual overload, looping and interlacing in a way that confounds the temporal parameters of the moving image’. Brambilla’s work as a whole often evokes the sense of visual overload which certainly exceeds the ‘visual’ itself, involving the whole sensorium. This also applies to the music video Brambilla made for artist Kanye West’s song ‘Power’, where influences from Renaissance paintings are here too discernible in the multiplicitous composition and arrangement of elements. The visual, or rather ‘sensory’,40 overload consists of trying to include as much as possible within the limits of a projection surface, experimenting with its form, as well as extending it in time. Brambilla’s interest in excess, as well as in infinity, becomes manifest through the use of multiplicity and superimpositions, looping elements and kaleidoscopic elements, as well as formal and mathematical infinite multiplication, as in his other work under the title Constellation (2015), described as ‘a computer- generated video sculpture’ performing a multiplication based on the recursive series of Fibonacci numbers, creating a fractal shape through a sphere ‘surrounded by a tryptic of projections’, and ‘replicated many times in space’.41 The multiplicity and heterogeneity that characterizes Brambilla’s moving- image work challenges, as already broached, the formation of Prägnanz. In fact, entropy seems more dominant here, as well as the dynamical processes of formal change rather than equilibrium. NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART Brambilla’s works can in this sense be considered contemporary combinations of order and chaos. The relationship between this excessive multiplicity and entropy becomes perhaps even clearer in Brambilla’s recent kinetic sculpture Winklevii: Bigger Than Both of Us (2021). In this digital animated sculpture, the busts of Cameron and Tyler Winklevoss, twin brothers considered among the early adopters and ambassadors of the bitcoin cryptocurrency, are shown back-to-back, rotating, inflating, deforming, multiplying and dissolving. The work, inspired by the paintings of Francis Bacon, uses digital morphing to visually alter the form of the avatars, while it is accompanied by sampled audio pieces of speech, soundbites from interviews with the brothers containing their most used words and phrases representing the terminology of cryptocurrency. These make a soundtrack that, in Brambilla’s words, ‘becomes a mantra, and when you repeat and you loop it and you cycle it, it becomes almost hypnotic’. Thus the aural modality of the work matches the visual one’s tendency for excess and oversaturation. As a result its energy ‘becomes more and more about entropy’, ‘a cyclone of 63 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X information’.42 As arts editor Virginia Valenzuela remarks in her article about the piece for SuperRare magazine, entropy in this sense represents ‘a decline into disorder, a theme which permeates throughout Brambilla’s Winklevii:43 ‘the artwork reaches its manic climax of revolving figures and mantras before crashing back down to the start of its original aspirational anthem, only to build up again in a never ending cycle of rise and fall’.44 Winklevii might be dealing, as the artist intends, with entropy and seemingly resisting the tendencies for ‘self-regulation’, submitting to chaotic and dispersive drives. It would still, however, be of interest to a Gestaltist like Arnheim because of its processes of trans-formation: the initial forms of the brothers might be changing and deforming but they acquire new, albeit monstrous form — before indeed ‘crashing’ and reemerging in a loop. They are therefore subjected to processes of orderliness in a way, even though this term would not be doing merit to the work’s continuously changing and looping nature. NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART Any emerging forms as well as meaning is volatile and unstable, just like the mind that tends to be continuously drifting from established ‘Gestalts’; thus a work not locking to a unifying principle of a structural theme might be more profoundly engaging with the dynamics of the entropic brain. In any case it is important to keep in mind that orderliness does not emerge (only) in the work itself but in the mind of the perceiver, and is related to the emergence of meaning. One could say that some structural theme(s), even unstable and uncertain ones, can always emerge in the perception and interpretation of an artwork, even when the latter seems to actively resist a unifying principle, such as Brambilla’s Winklevii. The meaninglessness of Winklevii reflects that of the crypto-jargon, in an isomorphic kind of way, which is a Gestalt principle after all. The work adopts the mode of communication of its subject, only to inflate and dismember as a result. It is not only vision and hearing that contribute to such emergence of meaning (even if this meaning is meaninglessness). Arnheim’s discussion of formal structure and meaning through an interplay of entropy and order in art includes the audiovisual but leaves out other non-visual and non-audible ways and modalities through which artworks make meaning, even if they are not subjected to visual or sonic ‘orderliness’. For example, his criticism in his 1930s writings on film of Dreyer’s Jeanne d’Arc for its pointless formalism can be understood, in the context of his later writings on entropy, as equivalent to his criticism of other (‘avant-garde’) works that create disorder without subsuming it to the powers of orderliness. However, in his criticism Arnheim seems to ignore the work’s embodied impact upon the viewer. Effects of cinematography and montage are bodily affective (practiced and theorized as such since Eisenstein) without constructing a specific formal ‘structural theme’ — still, meaning can emerge through the body. Dismemberment, fragmentation of body and speech and subjection of the body to many different points of view from invisible lookers, as in the case of Dreyer’s film, might as well constitute a meaningful message communicated by the choices of cinematography and editing, which invite the viewer to share the protagonist’s experience in an embodied way. NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART 64 Poulaki, A Gestalt Theory for ‘Disorder’ When it is the proper image of the body that the work involves, if only to dissolve or dismember it either through editing or digital morphing, certain processes of mirroring are at play (following arguments like those posed by the theory of ‘embodied simulation’),45 as well as what Semir Zeki and Tomohiro Ishizu called a disruption of the ‘inherited concepts’ of face and body (discussed in the reception of Bacon’s paintings) that creates a ‘visual shock’ and an abnormal neuronal reaction.46 But techniques such as montage, collage-like juxtaposition, or flicker, also extensively explored in the 1960s avant-garde, primarily invite a bodily sharing of rhythms of image change, and secondarily of movements of actual seen figures and bodies. Thus the corresponding feeling of body of the beholder should not only be discussed from the aspect of ‘mirorring’ or simulating but also from that of an isomorphism addressing the very processes of image and sound movement and the energies and rhythms that bring the compositional units of the film — in their heterogeneity — into relation and conflict. Phenomenologically such isomorphism might be expressed as altered rhythms of breathing, heartbeat, changes in bodily posture and movements, interoception, etc. Something similar has been proposed by Ellen Esrock through the concept of ‘transomatization’.47 While Arnheim saw the possibilities of multiplicity, heterogeneity, contingency, and redundancy in art (in various forms and examples from Renaissance to modern art) to increase entropy and thus build complexity through processes of ordering and self-organization, he did not escape some reservation towards what he saw as a misuse of such qualities, in ‘avant-garde attempts to mix elements at random’, as already broached. In Brambilla’s digital works Metaform No. 1, No. 2, No. 3 (2021), random objects chosen from a digital database compose dense animated collages. They shine, some slowly move or rotate, in compositions evoking the 16th century ‘cabinets of curiosities’, displays of significant or curious objects that collectors kept and demonstrated in their houses, before museums were established. Each of the Metaform collages suggests a multiplicity that does not assimilate or make any meaning as a whole, apart from seen as a collection visualizing that of the stock library of 3D assets it originates from — each object notably accompanied by its URL. NEW ENTROPIC ART? THE CASE OF MARCO BRAMBILLA’ S MEDIA ART It is the unit (as in Bogost’s ‘unit operations’) that digital technology and culture builds upon, making it not only demonstrable as in these digital Wunderkammers Brambilla replicated, but also exchangeable. In its properly meta-gestaltist title, ‘Metaform’ invites us to reflect on what is art, what is left when entropy renders art and cultural objects into a ‘heap’, i.e. a collection of unrelatable units, and how this noise can again be turned into art that is somehow meaningful. 65 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Notes 26 K h i H l H W B P h (Chi U i i f Chi P 1999) 286 24 See Ian Bogost, Unit Operations: An Approach to Videogame Criticism (Cambridge: The MIT Press, 2006). 25 Arnheim, ‘The Vanishing World and Köhler’s Inkwell’, in The Legacy of Solomon Asch: Essays in Cognition and Social Psychology, ed. by Irvin Rock (New York: Psychology Press, 1990), 271–278 (277). 26 K h i H l H W B P h (Chi U i i f Chi P 1999) 286 24 See Ian Bogost, Unit Operations: An Approach to Videogame Criticism (Cambridge: The MIT Press, 2006). 25 Arnheim, ‘The Vanishing World and Köhler’s Inkwell’, in The Legacy of Solomon Asch: Essays in Cognition and Social Psychology, ed. by Irvin Rock (New York: Psychology Press, 1990), 271–278 (277). 25 Arnheim, ‘The Vanishing World and Köhler’s Inkwell’, in The Legacy of Solomon Asch: Essays i nd Social Psychology, ed. by Irvin Rock (New York: Psychology Press, 1990), 271–278 (277). ne Hayles, How We Became Posthuman (Chicago: University of Chicago Press, 1999), 286. atherine Hayles, How We Became Posthuman (Chicago: University of Chicago Press, 1999), 28 bidem, 25. 28 See Humberto R. Maturana and Francisco J. Varela, Autopoiesis and Cognition: The Realization of the Living (Dordrecht: D. Reidel, 1972). It is worth noting that later Arnheim (in ‘The Vanishing World’ essay, 1990) criticized autopoiesis for what he saw as an exaggerated emphasis on the living organism as an operationally closed system, and insisted on the Gestalt perspective of a higher reciprocity between organism and environment. 28 See Humberto R. Maturana and Francisco J. Varela, Autopoiesis and Cognition: The Realization of the Living (Dordrecht: D. Reidel, 1972). It is worth noting that later Arnheim (in ‘The Vanishing World’ essay, 1990) criticized autopoiesis for what he saw as an exaggerated emphasis on the living organism as an operationally closed system, and insisted on the Gestalt perspective of a higher reciprocity between organism and environment. 29 Francisco J. Varela, Eleanor Rosch and Evan Thompson, The Embodied Mind: Cognitive Science and Human Experience (Cambridge: The MIT Press, 1992), 155–156. 29 Francisco J. Varela, Eleanor Rosch and Evan Thompson, The Embodied Mind: Cognitive Science and Human Experience (Cambridge: The MIT Press, 1992), 155–156. 30 Johan Wagemans, Jacob Feldman, Sergei Gepshtein, Ruth Kimchi, James R. Pomerantz, Peter A. Notes 1See Max Planck, ‘Entropy and Temperature of Radiant Heat’ [Entropie und Temperatur Strahlender Warme], Annalen der Physik, 1.4 (1900), 719–737. 1See Max Planck, ‘Entropy and Temperature of Radiant Heat’ [Entropie und Temperatur Strahlender Warme], Annalen der Physik, 1.4 (1900), 719–737. 2 Rudolf Arnheim, Entropy and Art: An Essay on Order and Disorder (Berkeley: University of California Press, 1971), 13; quoting Wolfgang Köhler from Die physischen Gestalten in Ruhe und im stationären Zustand: eine naturphilosophische Untersuchung (Braunschweig: Vieweg & Sohn, 1920). 2 Rudolf Arnheim, Entropy and Art: An Essay on Order and Disorder (Berkeley: University of California Press, 1971), 13; quoting Wolfgang Köhler from Die physischen Gestalten in Ruhe und im stationären Zustand: eine naturphilosophische Untersuchung (Braunschweig: Vieweg & Sohn, 1920). 3 Arnheim, Entropy and Art, 13. 3 Arnheim, Entropy and Art, 13. 5 Steve Odin, ‘Blossom Scents Take Up the Ringing: Synaesthesia in Japanese and Weste Soundings, 69.3 (1986), 256–281 (274). 7 Arnheim, Entropy and Art, 51. 8 Ibidem, 28. 9 Ibidem, 17. 10 Ibidem, 51–52. 11 Arnheim, Visual Thinking (Berkeley: University of California Press, 1997), 90. 12 See Arnheim, ‘The Two Faces of Gestalt Psychology’, American Psychologist, 41.7 (1986), 820–824. 13 Arnheim Entropy and Art 51 12 See Arnheim, ‘The Two Faces of Gestalt Psychology’, American Psychologist, 41.7 (1986), 820–824. 13 Arnheim, Entropy and Art, 51. 14 Ibidem, 3. 15 Ibidem, 52, emphasis in the original. 16 See Arnheim, Film as Art (Berkeley: University of California Press, 1957), 40–41. 17 Ibidem, 19. 18 Ibidem, 53. 19 Ibidem, 19. 20 Ibidem. 21 Ibidem, 56. 22 Ibidem, 55. 22 Ibidem, 55. 23 See Gene Youngblood, Expanded Cinema (New York: P. Dutton & Co, 1970). 23 See Gene Youngblood, Expanded Cinema (New York: P. Dutton & Co, 1970). 24 See Ian Bogost, Unit Operations: An Approach to Videogame Criticism (Cambridge: The MIT Press, 2006). 25 Arnheim, ‘The Vanishing World and Köhler’s Inkwell’, in The Legacy of Solomon Asch: Essays in Cognition 24 See Ian Bogost, Unit Operations: An Approach to Videogame Criticism (Cambridge: The MIT P 24 See Ian Bogost, Unit Operations: An Approach to Videogame Criticism (Cambridge: The MIT Press, 2006). 25 Arnheim, ‘The Vanishing World and Köhler’s Inkwell’, in The Legacy of Solomon Asch: Essays in Cognition and Social Psychology, ed. by Irvin Rock (New York: Psychology Press, 1990), 271–278 (277). 1See Max Planck, ‘Entropy and Temperature of Radiant Heat’ [Entropie und Temperatur Strahlender Warme], Annalen der Physik, 1.4 (1900), 719–737. 2 Rudolf Arnheim, Entropy and Art: An Essay on Order and Disorder (Berkeley: University of California Press, 1971), 13; quoting Wolfgang Köhler from Die physischen Gestalten in Ruhe und im stationären Zustand: eine naturphilosophische Untersuchung (Braunschweig: Vieweg & Sohn, 1920). 3 A h i E t d A t 13 Notes van der Helm and Cees van Leeuwen, ‘A Century of Gestalt Psychology in Visual Perception II. Conceptual and Theoretical Foundations’, Psychological Bulletin, 138.6 (2012), 1218–1252 (11). 30 Johan Wagemans, Jacob Feldman, Sergei Gepshtein, Ruth Kimchi, James R. Pomerantz, Peter A. van der Helm and Cees van Leeuwen, ‘A Century of Gestalt Psychology in Visual Perception II. Conceptual and Theoretical Foundations’, Psychological Bulletin, 138.6 (2012), 1218–1252 (11). 32 See Gerald C. Cupchik, ‘A Critical Reflection on Arnheim’s Gestalt Theory of Aesthetics’, Psychology of Aesthetics, Creativity, and the Arts, 1.1. (2007), 16; also see B. I. B. Lindahl and Peter Århem, ‘Consciousness and Neural Force Fields’, Journal of Consciousness Studies, 23.7–8 (2016), 228–253. 32 See Gerald C. Cupchik, ‘A Critical Reflection on Arnheim’s Gestalt Theory of Aesthetics’, Psychology of Aesthetics, Creativity, and the Arts, 1.1. (2007), 16; also see B. I. B. Lindahl and Peter Århem, ‘Consciousness and Neural Force Fields’, Journal of Consciousness Studies, 23.7–8 (2016), 228–253. 33 Emmanuelle Tognoli and Scott J. A. Kelso, ‘Enlarging the Scope: Grasping Brain Complexity’, Frontiers in Systems Neuroscience, 8.122 (2014), <doi: 10.3389/fnsys.2014.00122> [accessed 25 April 2022]. 33 Emmanuelle Tognoli and Scott J. A. Kelso, ‘Enlarging the Scope: Grasping Brain Complexity’, Frontiers in Systems Neuroscience, 8.122 (2014), <doi: 10.3389/fnsys.2014.00122> [accessed 25 April 2022]. 34 Robin L. Carhart-Harris, Robert Leech, Peter J. Hellyer, Murray Shanahan, Amanda Feilding, Enzo Tagliazucchi, Dante R. Chialvo and David Nutt, ‘The Entropic Brain: A Theory of Conscious States Informed by Neuroimaging Research With Psychedelic Drugs’, Frontiers in Human Neuroscience, 8.20 (2014), 66 Poulaki, A Gestalt Theory for ‘Disorder’ Poulaki, A Gestalt Theory for ‘Disorder’ <doi:10.3389/fnhum.2014.00020> [accessed 25 April 2022]. <doi:10.3389/fnhum.2014.00020> [accessed 25 April 2022]. 35 Ibidem. 36 Ibidem. 37 An indication of this much more favorable stance towards entropy is given, perhaps, by recent research arguing that increased brain entropy has been associated with increased intelligence; see Glenn N. Saxe, Daniel Calderone and Leah J. Morales, ‘Brain Entropy and Human Intelligence: A Resting-state fMRI Study’, PLoS ONE, 13.2 (2018), e0191582. 38 Marco Brambilla and Sean Cochrane, ‘Q&A with Marco Brambilla & Civilization – Notes from Crush Senior Artist Sean Cochrane’, Glossy, <http://glossyinc.com/misc/civilization.html> [accessed 10 January 2022]. 38 Marco Brambilla and Sean Cochrane, ‘Q&A with Marco Brambilla & Civilization – Notes from Crush Senior Artist Sean Cochrane’, Glossy, <http://glossyinc.com/misc/civilization.html> [accessed 10 January 2022]. 38 Marco Brambilla and Sean Cochrane, ‘Q&A with Marco Brambilla & Civilization – Notes from Crush Senior Artist Sean Cochrane’, Glossy, <http://glossyinc.com/misc/civilization.html> [accessed 10 January 2022]. 39 Brambilla, ‘Marco Brambilla: Astral Projections’, Elephant, 3 February 2018, <https://elephant.art/ marco-brambilla-astral-projections> [accessed 12 January 2022]. 40 Ibid 39 Brambilla, ‘Marco Brambilla: Astral Projections’, Elephant, 3 February 2018, <https://elephant.art/ marco-brambilla-astral-projections> [accessed 12 January 2022]. 40 Ibidem Ibidem. 41 As described on Brambilla’s website, <https://www.marcobrambilla.com/work> [accessed 12 January 2022] 41 As described on Brambilla’s website, <https://www.marcobrambilla.com/work> [accessed 12 January 2022]. 42 Brambilla, excerpt from interview in article by Virginia Valenzuela ‘Very Excited, Pumped, Excellent: Marco Brambilla and the Winklevii’, SuperRare, 24 August 2021, <https://editorial.superrare.com/2021/08/24/ very-excited-pumped-excellent-marco-brambilla-and-the-winklevii/> [accessed 13 January 2022]. 42 Brambilla, excerpt from interview in article by Virginia Valenzuela ‘Very Excited, Pumped, Excellent: Marco Brambilla and the Winklevii’, SuperRare, 24 August 2021, <https://editorial.superrare.com/2021/08/24/ / Brambilla and the Winklevii , SuperRare, 24 August 2021, <https://editorial.superrare.com/2021/08/24/ very-excited-pumped-excellent-marco-brambilla-and-the-winklevii/> [accessed 13 January 2022]. 43 Valenzuela. very-excited-pumped-excellent-marco-brambilla-and-the-winklevii/> [accessed 13 January 2022]. 43 Valenzuela. 44 SuperRare, ‘Winklevii: Bigger Than Both of Us’, <https://superrare.com/artwork-v2/winklevii:-bigger- than-both-of-us-27285> [accessed 12 January 2022]. 44 SuperRare, ‘Winklevii: Bigger Than Both of Us’, <https://superrare.com/artwork-v2/winklevii:-bigger- than-both-of-us-27285> [accessed 12 January 2022]. y Gallese and Michele Guerra, The Empathic Screen (Oxford: Oxford University Press, 2020). , p ( y , ) 46 Semir Zeki and Tomohiro Ishizu, ‘The “Visual Shock” of Francis Bacon: An Essay in Neuroesthetics’, Frontiers in Human Neuroscience, 7.850 (2013), <doi: 10.3389/fnhum.2013.00850> [accessed 25 April 2022]. 47 Esrock J. Ellen, ‘Body Forth in Narrative’, in Narrative Complexity: Cognition, Embodiment, Evolution, ed. by Marina Grishakova and Maria Poulaki (Lincoln: Nebraska University Press, 2019), 270–290. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Keywords Gestalt Animation theory Design theory Jacques Rancière Film criticism DOI 10.54103/ 2036-461X/17107 This article explores the affinities between animation practice and experiments in perception by Gestalt psychologists. By drawing out a Gestalt style of seeing — a sensitivity to the visual forces that scaffold an image — we can better describe movements, figures, and spaces in animation. Although these affinities make Gestalt appropriate for discussing animation, they do not necessarily imply that animated films merely illustrate or independently verify Gestalt laws of perception. Rather, they suggest two branches of cultural practice sharing what philosopher of science Ian Hacking calls a ‘style of reasoning’: a regularized procedure whose consistent results form a basis for knowledge in a given culture. This article argues that Gestalt and animation are co-participants in the ‘culture of design’: a project of shaping sensory arrangements in order to shape populations, which began in the nineteenth century and has gained force through the present day. It is this culture of design, which includes the exploration of cinema as an art of graphic arrangement, that has become all-but-ubiquitous in the twenty-first century and has led to the ubiquity of animation. This article explores the affinities between animation practice and experiments in perception by Gestalt psychologists. By drawing out a Gestalt style of seeing Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License sed under a Creative Commons Attribution 4.0 International License DOI 10.54103/ 2036-461X/17107 Some scholars have begun using the term ‘Gestalt’ when they refer to certain effects related to animation. <doi:10.3389/fnhum.2014.00020> [accessed 25 April 2022]. 46 Semir Zeki and Tomohiro Ishizu, ‘The “Visual Shock” of Francis Bacon: An Essay in Neuroesthetics’, Frontiers in Human Neuroscience, 7.850 (2013), <doi: 10.3389/fnhum.2013.00850> [accessed 25 April 2022]. Semir Zeki and Tomohiro Ishizu, The Visual Shock of Francis Bacon: An Essay in Neuroesthetics , Frontiers in Human Neuroscience, 7.850 (2013), <doi: 10.3389/fnhum.2013.00850> [accessed 25 April 2022]. 47 Esrock J. Ellen, ‘Body Forth in Narrative’, in Narrative Complexity: Cognition, Embodiment, Evolution, ed. by Marina Grishakova and Maria Poulaki (Lincoln: Nebraska University Press, 2019), 270–290. 47 Esrock J. Ellen, ‘Body Forth in Narrative’, in Narrative Complexity: Cognition, Embodiment, Evolution, ed. by Marina Grishakova and Maria Poulaki (Lincoln: Nebraska University Press, 2019), 270–290. 67 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Hannah Frank describes an inky patch on a card in Robert Breer’s Blazes (1961) as bearing a certain ‘Gestalt’, one that suggests ‘a bird flapping its wings’.1 Elsewhere she calls Breer’s film Fuji (1974) an ‘experiment in Gestalt’, wherein we are invited to see that all it takes for us to recognize Mount Fuji is a triangle, or an upside-down V with a certain obtuse slope.2 Andrew Johnston describes an early experiment in electrical image reproduction (a predecessor of the CRT technology in television sets) which brought an image into resolution ‘through a pointillist Gestalt’.3 Jordan Schonig describes the effect of a compression glitch in Chairlift’s music video for ‘Evident Utensil’, wherein an abstract collage of colour begins to move like a man’s face, as a ‘perceptual effect where we seize a recognizable form from the temporal Gestalt of its movement’.4 As these authors use it, the term is not being used in a very technical sense; aside from Schonig, none of these authors cite Gestalt psychologists in their work. Yet it’s significant that these authors choose the word ‘Gestalt’, rather than a cognate like ‘shape’ or ‘form’. Each author is describing something like 69 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X a shape, but not something that is fixed or assured. In each example, there is a sense that the percept in question is fragile, contingent. It might have been perceived otherwise. The inky patch might not make a bird; a triangle or an upside-down V might not make Mt. Fuji; an abstract collage might not make a face. Each of these percepts might have remained a chaotic visual soup, bearing no configuration at all. A kind of work is required, on the part of the perceiver, to complete the impression in question. Each example yields something that feels like it ‘holds together’ before our eyes, and not because we are directly seeing conditions in the physical world. These conditions are created out of whole cloth, or heavily technologically mediated, such that their visual coherence is an open question. The ‘togetherness’ of these examples is not a given. It must be earned, by being arranged before our eyes just so. Each of these arrangements forms one half of a kind of perceptual agreement, an agreement that a viewer will complete by grasping the arrangements as being organized just so. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary It is precisely these kinds of perceptions — perceptions that might be grasped otherwise, and which therefore seem to tell us something about how we grasp things in the world through our senses — that Gestalt psychology was constructed to explain. In this article, I argue that Gestalt psychologists and animators found many of the same perceptual effects, implying a similar picture of human nature as primarily tasked with organizing the world and organizing ourselves in concordance with it. Because Gestalt and animation have been so historically preoccupied with how sensory arrangements must be ‘earned’, Gestalt turns out to offer an excellent framework for describing animation — especially a period and class of animation practice that explicitly toyed with the limits of perception.5 However, I will hold back from claiming that animators ended up ‘proving’ the theoretical claims of Gestalt psychology. (They certainly did not prove any of Gestalt’s more controversial theories, such as brain isomorphism). Rather, I wish to draw out some historical implications of the overlap between Gestalt and animation as styles of manipulating or knowing the world. Ultimately, I want to suggest, Gestalt and animation were silent partners in what we might call the ‘culture of design’: a dual obsession with shaping arrangements of the senses and shaping arrangements of populations, an obsession that begins in the nineteenth century and continues to this day. It is the ubiquity of design, and not merely the ubiquity of digital imagery per se, that has allowed animation to saturate moving image culture so thoroughly today. How then is animation generally conceived, and how does Gestalt offer a helpful alternative? By and large, animation has been written about in its distinctness from live-action film — in the fact that its movements, figures, and spaces are not recorded in real time but created from scratch. Writers on film such as Siegfried Kracauer, Erwin Panofsky, and Lewis Jacobs celebrated cartoons (especially Disney) for achieving sights and sounds that seemed effortless when drawn, but which would have been awkward or even impossible to achieve through direct photography.6 Cartoons were often conceived as bearing their own kind of medium-specificity — a specificity that lie within the technical possibilities of 70 Pierson, Gestalt, Animation, and the Culture of Design moving pictures, yet stood apart from ‘cinema’ proper. This opposition between synthesized movement and recorded movement has persisted to this day. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Notably, it helped set the terms for many of the debates around the status of digital film.7 From noting that animation is different live-action, it is but a small step in logic to argue that animation should be as different as possible from live- action. This hidden value judgment lay in many critics’ assessments of Disney’s early feature films, when the unruly physics that had once dominated cartoon worlds gave way to more rigid principles of movement and suggestions of three-dimensional space.8 That value judgment also underlies the most well- known feature of animation: plasmaticness. First coined by Sergei Eisenstein, plasmaticness is the sense of freedom from worldly constraints that we sometimes feel when we watch animated figures stretch themselves or change their shape.9 For Eisenstein, this was an imaginary freedom Americans felt from the tedium of industrial production. Though the concept was originally intended only to describe Disney cartoons of the 1930s, plasmaticness has been so commonly cited, inside and outside of animation studies, that it has nearly been hypostatized into a timeless essence of frame-by-frame filmmaking more generally.10 There is an obvious problem here. If we assume that a film is more animated the less less resembles live-action, we risk ignoring all the ways that animators acknowledge or embrace the secular world. Examples of animators doing exactly this are numerous. Before the digital era, almost all animation had to be photographed; this meant that matters of camera and lighting were essential to animation aesthetics.11 In addition, animators often studied photographed human and animal motion, and their studies resonated with — and in some cases were directly inspired by — scientific studies of motion.12 Moreover, since World War I animators have made liberal use of the rotoscope, a tool for tracing recorded movements; the Fleischer studio’s 1920s hero Koko the Clown was traced from reference footage of Max Fleischer in a clown suit, and the Out of the Inkwell shorts that featured Koko were celebrated in part because those traced movements looked more ‘animated’ than customary cartoon movements.13 Finally, animation techniques have long been a part of scientific study itself, diagramming all manner of unseen processes.14 Animation’s powers of visualization and reduction formed a cornerstone of animation practice during and after World War II, most famously by the United Productions of America studio.15 Animators don’t just study the world around them, though. They study perception, albeit often indirectly. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary This is where Gestalt becomes helpful. Recall that I said about the opening examples that their holding-together, their manifoldness as unified perceptions, must be ‘earned’. This is a logical consequence of how animation, as movement and space constructed frame by frame, works. In a live-action film, a filmmaker can create the impression of a character walking across a room simply by filming an actor walking across a room. As viewers, we would explain our impression of the event by describing 71 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X the event itself; if the walk had some idiosyncrasy, we would attribute that idiosyncrasy to the actor. In animation, none of these things is a given. The manner of the walk, the size and shape of the character, even the dimensions of the room — any of these can change at any time. Hume’s problem of induction looms large over animation technique. Thinking within a framework of plasmaticness, we might be inclined to celebrate this openness as a liberation from the impositions of earthly physics; but Hume conceived induction as a problem precisely because it leaves us unprotected from vertiginous, chaotic meaninglessness. In animation practice, we get a perceptual analogue of this problem: how does an animator make anything look like anything at all? When physical forces do not hold in an aesthetic world — when gravity, friction, inertia, and the properties of chemical compounds will not hold together a walking figure or the room it wants to walk across — how does an animator create forces that will hold? The answer, arrived at by animators and Gestalt psychologists alike is, by exploiting the forces within the sensory field itself: the intuitive impressions of attraction, repulsion, and coordination among units of perception that seem to spontaneously arise from within a picture. Like a square that emerges from an array of dots — . . . . . . . . — movements, figures, and spaces can emerge from relations of spacing and timing within and across frames. Some animated films do this more self-consciously than others. Take a film by Norman McLaren, Blinkity Blank (1952). In this film, McLaren scratches figures into the emulsion of a film strip, often leaving frames completely black. Pierson, Gestalt, Animation, and the Culture of Design Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary The early part of the film is, in part, a kind of test to see how frames can be arranged so that, even though we see black frames, we still see movement. McLaren uses blank frames in a number of ways. Sometimes he alternates blank frames with figured frames, which slows down the movement into a fragile kind of stutter. Sometimes he places a few blank frames in a row after a quick movement, as if the figure has sped up beyond our threshold of vision. Sometimes he ‘cuts’ to black after a burst of action, only to have a figure wander back into the blank frame; it is as if the cut to black turned into an empty space, right before our eyes. In all these cases, the black frames are plainly visible. And yet, we are amazed that movement still holds across those gaps — that McLaren can even make a gap add to the impression of movement. Collectively, these optical tricks demonstrate that a blank frame is not just a blank frame. How we see it will depend on how the frames around it are arranged. Any given blank frame will look more like the figured frames surrounding it than like other blank frames. This demonstration is remarkably similar to a set of experiments in motion that Gestalt psychologist Max Wertheimer published in 1912. Wertheimer used a tachistoscope to show test subjects three phases of a movement: a vertical 72 Pierson, Gestalt, Animation, and the Culture of Design Pierson, Gestalt, Animation, and the Culture of Design strip, a blank space, and a horizontal strip. By playing with the interval at which the middle phase — the blank space — was presented, Wertheimer got subjects to see a variety of impressions. Most famously, subjects saw phi: a ‘movement that did not appear to belong to either strip but hovered faintly between the two’. Presented with a differently-timed interval, subjects would see one strip moving and the other strip standing still; this was described as ‘dancing’. With yet another interval, a subject would see two phi motions simultaneously, one on the left and one on the right.16 As in McLaren’s film, a blank interval is not just a blank interval. Manipulating that interval will alter the impression of motion. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary We can see more remarkable similarities in another pair of examples: McLaren’s Animated Motion instructional series (1976–1978) and Gestalt psychologist Albert Michotte’s experiments in the impression of causality. In the third Animated Motion video, McLaren uses two circles to demonstrate that when an animator manipulates the number of frames it takes for one thing to hit another. Depending on what the other thing does upon impact, the animator can suggest different kinds of movement: a punch, a gentle push, a cautious touch. Underneath each movement is a set of tick-marks that show the frame- by-frame positions of each circle, demonstrating that what appears seamless and spontaneous is a series of precisely-measured distances. Albert Michotte experimented with impressions of moving squares hitting each other. Using a ‘paper disc’ method, Michotte manipulated the timing of each square’s movement and found that different timings yielded qualitatively different impressions. If one square approached slowly and the other square shot off quickly upon impact, the impression of causality was especially strong (despite this motion behaving contrary to the laws of physics). If there was a short time lag between the moment of impact and the second square skittering off, it looked as if some mysterious force had been triggered inside the second square.17 In both films, McLaren is intentionally isolating simple movement effects (for experimental and pedagogical purposes, respectively); most animated films contain figures, movements, and spaces that are considerably more complex. Yet the similarities between McLaren’s presentations of movement and Gestalt experiments seem to indicate something deeper, precisely because they are so stripped down. One thing we might want to say here is that animators have independently verified Gestalt laws of perception. This is more or less what Rudolf Arnheim asserts. He describes an experiment by Fritz Heider and Marianne Simmel, wherein subjects viewed a short film of two triangles and a circle interacting in various ways. Subjects described the larger triangle as ‘aggressive’ and ‘belligerent’, solely from its movements. Arnheim notes similarly expressive movements by geometric figures ‘in the more elaborate “abstract” films of Oskar Fischinger, Norman McLaren, Walt Disney, and others’, taking them to demonstrate the Gestalt theory of expression.18 I believe a more fruitful path of inquiry, however, might lie in taking Gestalt seriously for animation criticism and history. Arnheim writes of Gestalt as a 73 Cinéma & Cie vol. 22 no. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary 38 2022 · ISSN 2036-461X ‘style…of science’, likening it to art.19 Philosopher of science Ian Hacking has picked up on something like this when he theorizes, following A.C. Crombie, ‘styles of reasoning’. Broadly speaking, a style of reasoning is a set of procedures that creates its own standards for correctness.20 Like styles of art, styles of reasoning can coexist within a given historical period, and they can accumulate over time. (Statistical analysis, for example, is a style of reasoning). Speaking to the area of criticism: one of the biggest challenges in animation scholarship is simply describing what we see and hear. Our inherited vocabulary of formal film analysis was forged in the 1960s and 1970s to describe live-action films. It offers little help when we want to capture what is most striking about a piece of animation, save for some CGI films that are constructed to resemble live-action feature films. (And unlike live-action, animation has precious little of a tradition of criticism for us to rely on). We are often left with what animation theorist Suzanne Buchan calls ‘the inarticulate “mmm…” that is often the response to what we see on screen’.21 This is one of the reasons plasmaticness has proven to be such an appealing concept: it’s easy to apply. It saves us the trouble of having to closely describe how things are moving. Paraphrasing or summarizing what we see, noting the fantastical elements, we glide past the initial ‘mmm…’ and go straight to interpretation.22 Gestalt gives us a way to push into the initial ‘mmm…’ and come out the other side with a firmer grasp of perceptual subtleties. In the experiments described above, we can discern something like a style of Gestalt experimentation — and, by extension, a Gestalt style of looking at animation. Designing Gestalt experiments, such as the phi experiments, required a special sensitivity to the organizational features and thresholds of perception, tweaking an apparatus or a presentation such that one impression would become something else. Gathering the results for these experiments involved soliciting spontaneous, open-ended responses from subjects. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Wertheimer, Michotte, and Heider and Simmel collected lively self-reports of what viewers saw — they reported ‘dancing’, or ‘a sort of two-stroke’, or, ‘It is as if A in touching B induced an electric current which set B going’ — and these reports make for some of the most convincing evidence of the effects the authors are arguing for. Putting these factors together, we can note that a Gestalt style of seeing involves being sensitive to the ways that picture and sound organize themselves before us, describing those ways as closely as we can, however counterintuitive the descriptions might be. We are prompted to pay greater attention to the qualities of visible movements, figures, and spaces themselves. By using our intuitions about organization as our primary means of description and asking questions like, how does this figure, movement, or space fit together?, what forces seem to scaffold it? how am I seeing it as one configuration and not another?, we can perform criticism with more precision, staying with the surfaces of what we see and hear without falling back on simply paraphrasing what happens. In this manner, Gestalt becomes less like a science than like formalist art criticism or ordinary language philosophy. I stress that this way of looking at animation need not be limited to looking for I stress that this way of looking at animation need not be limited to looking for 74 Pierson, Gestalt, Animation, and the Culture of Design Pierson, Gestalt, Animation, and the Culture of Design Gestalt principles in animated figures (similarity, continuity, closure, Prägnanz, and so on).23 If we look more generally for figures and forces in what we see, we gain new insights into animation techniques throughout history. Take the line, one of the most basic units of two-dimensional animation. Traditionally, the moving or transforming line has been conceptualized as the formal analogue of plasmaticness. Vivian Sobchack, for one, argues that when the line moves, it effectively rebels against its own form. Whatever a line may represent at any given time, the line itself is always visible as a mark on a surface, irreducible to that representation. That moving, irreducible mark always threatens to overtake the figure, asserting its own power as a sort of inbetween-ness of lively being: Thus the animated line never ‘flattens’ itself out into something geometrically ‘straightforward’ – nor does it ever become pure figure. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Recursive, it insists on the mobility of its becoming, on its unfixing of and separation from itself, on its capacity to simultaneously both posit and negate itself.24 Taking as her privileged example Raimond Krumme’s 2000s commercials for Hilton hotels, in which a single line metamorphoses into various scenes of travel, Sobchack hints that the power of this single transformative line is ‘perhaps, the DNA of animation’.25 I don’t want to directly argue with the claim that some originary hint of this transformative power is present anytime we see a moving line, but because the claim is so totalizing, it leaves us unable to describe any other functions a line might have. A theoretically posited ‘essence’ makes it more difficult to perform specific criticism. Instead of assuming that this power of the line is found everywhere, we may do better to ask: what makes us want to attribute this power to the line in cases like these? Why does the line seem to be moving or transforming itself? Why doesn’t it look like it’s being transformed by something else? Here, the ‘pure’ line against a blank space seems to be enacting a mysterious power to change itself not because that is a natural property of the moving line but because there are no other visual forces competing with it. A solid line, with nothing around it to make us see it any other way (such as a repeating pattern of the same line), appears to hold itself together. It appears as an abstract version of what Gestalt psychologist Fritz Heider calls a ‘thing’: a manifold whose parts attract each other more strongly than they attract outside forces or entities.26 Things are, by and large, solid and stable: persons, trees, rocks. When we see a manifold whose parts do not strongly attract each other, we grasp it as what Heider calls a ‘medium’: a loose arrangement of parts. In everyday life, fluids and gases are mediums. In pictorial terms, if we see a tangled layer of intersecting lines (such as the whorls of a Jackson Pollock painting), rather than a spare line against a blank background, we will be inclined to grasp that tangle as a medium. A thing is that which we press against; a medium is that which we press through. Because a thing holds itself together, it tends to move as a single, whole 75 Cinéma & Cie vol. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary 22 no. 38 2022 · ISSN 2036-461X entity. Push a rock and the entire rock will move. Because a medium does not hold itself together, its parts will move in different ways, and at different rates of speed. Push a volume of water and it will re-form around your hand, making temporary whirlpools or folds, eventually resettling itself. It is clear that we do not see a naked line in empty space as a medium. When it moves, it moves as one. When it transforms, it does not appear to be transforming according to an outside force, as happens with the volume of water. Its principle of transformation is active: it seems to be changing its own shape. What matters for this description is that the line is holding shape as it is changing shape. We can observe that the line seems to have transformative powers not because of properties within the line itself, but because the line is being depicted as a unified thing that holds itself together whose force of alteration comes from within itself. We can usefully contrast this kind of line with another kind of line: the outlines of Disney characters after the mid-1930s. Over the course of the 1930s, animators at Disney thought of outlines less as fixed boundaries of bodies than as flexible skins that contained a principle of movement within them. Disney animation instructor Donald Graham appropriately refers to this change as going from ‘animating forms’ to ‘animating forces’.27 When an animator is animating by forces, the ‘essence’ of a character is not in its shape but in a kind of linear scaffolding held within the character, a flexible vector of movement. The role of the outline is to register the forces of this movement. Thus, the outline of the duckling in The Ugly Duckling (Burt Gillette, 1939) is extremely flexible, but we are hardly inclined to attribute powers of transformation to it, because its changes appear to be under the sway of an internal motive principle, unseen but palpable. We can even note that the camera itself — not the physical apparatus on the animation stand that photographs drawings, but the internally-coherent view of a diegetic animated world — functions as a kind of figure in two-dimensional animation. Certain visual conditions, such as parallax, must hold in order for a camera movement to manifest itself. Pierson, Gestalt, Animation, and the Culture of Design Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary The things onscreen must move in a special synchronization with each other. When this happens, we intuit something inside the space taking views of it. Animators like Caroline Leaf and Kathy Rose have played with these conditions, creating strange and nonsensical camera movements. These movements cannot be described in live-action terms; they offer a feeling of movement through space without offering a coherent space. With an eye toward the visual forces of configuration, we can also see other animation techniques in new ways, such as sound synchronization and rotoscoping. What is implied by this overlap between Gestalt experimentation and animation technique? We can use Gestalt to describe things closely, making animation more amenable to formal analysis; but descriptions are rarely, if ever, epistemically neutral. They entail certain philosophical and political commitments. As philosopher of science Thomas Kuhn has demonstrated, even our descriptions of something as simple as the swing of a pendulum will 76 Pierson, Gestalt, Animation, and the Culture of Design Pierson, Gestalt, Animation, and the Culture of Design imply some overall picture of the world around that pendulum. An adherent of modern physics will see it primarily as a revolution around a center, which is being interrupted by the force of gravity. An Aristotelian would see it as a fall toward the earth, interrupted by the arm of the pendulum.28 What might taking Gestalt seriously commit us to? What happens when we see a labile line in blank space as a thing with the power to remain itself through change, or when we see a labile line around a cartoon duck as a skin being reshaped according to a vector of forces inside it? Even if we stop short of taking the Gestalt style of seeing all the way to its proponents’ most extreme theoretical conclusions, by its very nature of seeking out perceptual arrangements the style will incline us toward a view of perception as arrangement. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Somewhat like the way phenomenological film criticism tends to take the camera as a model of phenomenology’s own picture of being-in-the-world, attending to Gestalt forces leads us to reflect on ourselves as arrangements of forces that organize themselves in relation to the world and each other.29 One upshot here is that instead of modeling political engagement as an opposition between dominant structures of power and acts of resistance to those structures — a kind of binary thinking that may lead us to resist the idea of ‘structure’ altogether, potentially slipping into what feminist author Jo Freedman has called the ‘tyranny of structurelessness’30 — we may think about structure itself as an ally and a weapon. Instead of, ‘how do we resist?’ our primary question becomes, ‘how might we organize?’. This is precisely the way many animators of the middle twentieth century thought about their medium. Animators of this period took inspiration from graphic design — a field that itself took inspiration from Gestalt psychology.31 György Kepes’s seminal design textbook Language of Vision argued that the graphic arts provided a kind of sensory education that could unite the public. By encouraging citizens to see themselves in terms of relationships with others, Kepes argued, designers provided a defense against the threats of fascism and haphazard technologism; not coincidentally, Kepes openly acknowledges an intellectual debt to the Gestalt psychologists.32 (To this day, Gestalt laws are routinely included in graphic design textbooks). While the high modernists of the postwar era were growing disenchanted with collectivist politics and turning to esoteric aesthetic forms, postwar animators working outside the American studio system held a commitment to organizing with a public.33 In 1975, the International Association of Animated Film issued a manifesto that read, in part: We must prove that apart from being an art media of its own, a useful tool in entertainment and in advertising, animation could also contribute to the understanding of basic human and social problems. […] In fact, given a chance, animation can contribute to serve humanity on a far broader level than it has done in the past.34 This is not to say that mid-century animators made especially radical films or held to an especially radical politics; by and large, they did not. By extension, engaging with Gestalt psychology will not automatically produce a radically 77 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X new kind of world. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary But the affinity between animation practice and Gestalt by means of design should give us pause. Dealing with that affinity can help us see historical conditions to which we already find ourselves committed. What might those historical conditions be? More pointedly, why did Gestalt principles seem like an appropriate tool for graphic design? I want to suggest here that Gestalt offered clear applications for a project that designers were already engaged in by the time Kepes was writing: the project of arranging the senses in order to arrange populations. As philosopher Jacques Rancière argues, design plays a major role in what he calls the ‘distribution of the sensible’.35 Politics for Rancière always involves the construction of a world that is both sensuously direct and held in common. Aesthetics intervenes in politics by pressing at the scaffolding of that sensorial construction. With the growth of mass production in the nineteenth century, the designer gained an enormous amount of power over this construction (for the simple fact that anything that is mass produced, by definition, must be designed).36 Some of the first major critiques of industrialization, in fact, came around concerns of design — namely, from the British Arts and Crafts movement. For art critic John Ruskin and designer William Morris, the effects of industry were visible not only in labor conditions but in the homes and everyday objects of the citizenry. The Arts and Crafts movement argued for social change through, in part, making the built environment more beautiful.37 By the early twentieth century, designers were routinely recognized as crucial political actors. For the major design schools of this time (the Deutscher Werkbund, the Constructivists, the Bauhaus, and so on), envisioning a set of products or surfaces was inseparable from envisioning an entire society.38 For someone like Kepes, then, Gestalt was appealing because it wove design into human nature itself: even in perception, we are all organizing our environments to find the most balanced relations with it. This is why I didn’t want to argue that Gestalt gets at some timeless ‘truth’ of human nature that animators merely stumbled upon: paradoxically, Gestalt, as an attempt at a scientific psychology, makes arranging — and, by implication, rearranging — the primary task of the human. To think with Gestalt in a historically robust manner is to acknowledge that we live in a culture of design. Pierson, Gestalt, Animation, and the Culture of Design Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary This has been the case for the overdeveloped West since the late nineteenth century, but the importance of this fact has become more and more important, as professional design has encroached into more and more areas of life: with software, with web and app design, and with the rise of ‘design thinking’.39 Film scholarship has so far been limited by its tendency to think about animation as a certain kind of film — and, by extension, to think about the animator as a certain kind of filmmaker. We can see, however, than an equally fruitful path of inquiry opens up when we think about the animator as a certain kind of designer — a designer of movements. Several consequences follow from this. 78 Pierson, Gestalt, Animation, and the Culture of Design First, the field of animation practice stretches beyond the realm of ‘cinema’ in a way that cannot be ignored. Animation takes its place not only in the history of narratively-driven works, such as theatrical cartoons, feature films, and television programs, but in the histories of advertisements, propaganda, scientific visualizations, station identifications, video games, apps, and more. Second, the ubiquity of animation in media culture, which it has become cliché to note, takes on a different tenor. Rather than the ‘return of the repressed’ narrative often told of animation, wherein visuals produced by hand are initially pushed to the margins of cinema’s dominant ‘machine vision’ (i.e., photography), only to come back and become the dominant mode of filmmaking, we can view animation techniques in conversation with the broader expansion of design into everyday life.40 (This has manifested itself in cinema outside of animation techniques as well: note the rise of the sound designer in the 1970s, and the close relationship between film and fashion.41) Finally, we can view animation’s tendency to play with the organization of our senses as bearing at least as much significance as its representational content. As an art that arranges our sensory impressions, animation, whether its practitioners know it or not, bears some of design’s cultural function of arranging populations. Moreover, animation has the ability to arrange these sensory impressions self-consciously. Recall the ‘Gestalts’ that I began with: an impression of a bird or Mt. Gestalt, Animation, and the Culture of Design Ryan Pierson, University of Calgary Fuji or a human face whose arrangement appears fragile, which appears to need something from us in order to be seen properly, which makes salient the fact and the task of organization. As a mode of thinking that mainly concerns itself with the organizational fit between the human and the world, Gestalt is basically an ethos of design. As such, it makes the task of design apparent in ways that other modes of thinking, such as psychoanalysis and phenomenology, do not. It does not in itself promise resistance or utopia.42 But it does hold out the possibility of alternative ways of being and forces us to be specific about what those ways of being might be. Which returns us to the question: how are we to organize? 79 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Notes 6 See Kracauer quoted in Frank, 154–155; Erwin Panofsky, ‘Style and Medium in the Motion Picture’, in The Visual Turn: Classical Film Theory and Art History, ed. Angela Della Vacche (New Brunswick: Rutgers University Press, 2003), 69–64 (75 and 84); and Lewis Jacobs, The Rise of the American Film (New York: Teachers College Press, 1968 [1939]), 496–505. 7 For examples see Lev Manovich, ‘What Is Digital Cinema?’ reprinted in Shane Denson and Julia Leyda, eds., Post-Cinema: Theorizing 21st-Century Film (Falmer: REFRAME Books, 2016), 20–50, and D. N. Rodowick, The Virtual Life of Film (Cambridge: Harvard University Press, 2007), and Dudley Andrew, What Cinema Is!: Bazin’s Quest and Its Charge (Malden: Wiley-Blackwell, 2010). 7 For examples see Lev Manovich, ‘What Is Digital Cinema?’ reprinted in Shane Denson and Julia Leyda, eds., Post-Cinema: Theorizing 21st-Century Film (Falmer: REFRAME Books, 2016), 20–50, and D. N. Rodowick, The Virtual Life of Film (Cambridge: Harvard University Press, 2007), and Dudley Andrew, What Cinema Is!: Bazin’s Quest and Its Charge (Malden: Wiley-Blackwell, 2010). 8 Gregory A. Waller, ‘Mickey, Walt, and Film Criticism from Steamboat Willie to Bambi, in The American Animated Cartoon: A Critical Anthology, ed. by Danny Peary and Gerald Peary (New York: E.P. Dutton, 1980), 49–57. 8 Gregory A. Waller, ‘Mickey, Walt, and Film Criticism from Steamboat Willie to Bambi, in The American Animated Cartoon: A Critical Anthology, ed. by Danny Peary and Gerald Peary (New York: E.P. Dutton, 1980), 49–57. 9 Sergey M. Eisenstein, Disney, trans. by Dustin Condren and ed. by Oksana Bulgakowa and Dietmar Hochmuth (San Francisco: Potemkin Press, 2012), 15. 9 Sergey M. Eisenstein, Disney, trans. by Dustin Condren and ed. by Oksana Bulgakowa and Dietmar Hochmuth (San Francisco: Potemkin Press, 2012), 15. 10 The most thorough exploration of plasmaticness remains Scott Bukatman, The Poetics of Slumberland: Animated Spirits and the Animating Spirit (Berkeley: University of California Press, 2012). I am not arguing against the concept itself: only its hypostatization. 10 The most thorough exploration of plasmaticness remains Scott Bukatman, The Poetics of Slumberland: Animated Spirits and the Animating Spirit (Berkeley: University of California Press, 2012). I am not arguing against the concept itself: only its hypostatization. 12 Alla Gadassik, ‘Assembling Movement: Scientific Motion Analysis and Studio Animation Practice’, Discourse, 37.3 (2015), 269–297. 13 Donald Crafton, Before Mickey: The Animated Film, 1898-1928 (Cambridge: The MIT Press, 1982), 158. 22 This, of course, isn’t to disparage the act of interpretation. Subtle and powerful interpretive claims have been made using plasmaticness — particularly by Nic Sammond, who notes that the freedom of movement in cartoon figures has a history in blackface minstrelsy. See Nic Sammond, Birth of an Industry: Blackface Minstrelsy and the Rise of American Animation (Durham: Duke University Press, 2015). My point here is that 21 Suzanne Buchan, ‘The Animated Spectator: Watching the Quay Brothers’ “Worlds”’, in Animated ‘Worlds’, ed. by Suzanne Buchan (Eastleigh: John Libbey Publishing, 2006), 36. 20 Ian Hacking, ‘“Style” for Historians and Philosophers’, in Id., Historical Ontology (Cambridge: Harvard University Press, 2002), 178–199. Notes 1 Hannah Frank, Frame by Frame: A Materialist Aesthetics of Animated Cartoons, ed. by Daniel Morgan (Oakland: University of California Press), 22. 1 Hannah Frank, Frame by Frame: A Materialist Aesthetics of Animated Cartoons, ed. by Daniel Morgan (Oakland: University of California Press), 22. , 3 Andrew Johnston, Pulses of Abstraction: Episodes from a History of Animation (Minneapolis: University of Minnesota Press, 2021), 158. 3 Andrew Johnston, Pulses of Abstraction: Episodes from a History of Animation (Minneapolis: University of Minnesota Press, 2021), 158. 3 Andrew Johnston, Pulses of Abstraction: Episodes from a History of Animation (Minneapolis: University of Minnesota Press, 2021), 158. 4 Jordan Schonig, The Shape of Motion: Cinema and the Aesthetics of Movement (New York: Oxford University Press, 2021), 166–167. 4 Jordan Schonig, The Shape of Motion: Cinema and the Aesthetics of Movement (New York: Oxford University Press, 2021), 166–167. 5 Of course, each of the arts relies on some kind of organizational principles to achieve coherence; but the kind of ‘holding-together’ I’m referring to is a more basic perceptual matter than the sense of wholeness often implied by an aesthetic judgment (such as the complementarity of colours in a painting, or the sense of ‘rhythm’ that E.M. Forster notes as a unifying force of a novel). 5 Of course, each of the arts relies on some kind of organizational principles to achieve coherence; but the kind of ‘holding-together’ I’m referring to is a more basic perceptual matter than the sense of wholeness often implied by an aesthetic judgment (such as the complementarity of colours in a painting, or the sense of ‘rhythm’ that E.M. Forster notes as a unifying force of a novel). 6 See Kracauer quoted in Frank, 154–155; Erwin Panofsky, ‘Style and Medium in the Motion Picture’, in The Visual Turn: Classical Film Theory and Art History, ed. Angela Della Vacche (New Brunswick: Rutgers University Press, 2003), 69–64 (75 and 84); and Lewis Jacobs, The Rise of the American Film (New York: Teachers College Press, 1968 [1939]), 496–505. 6 See Kracauer quoted in Frank, 154–155; Erwin Panofsky, ‘Style and Medium in the Motion Picture’, in The Visual Turn: Classical Film Theory and Art History, ed. Angela Della Vacche (New Brunswick: Rutgers University Press, 2003), 69–64 (75 and 84); and Lewis Jacobs, The Rise of the American Film (New York: Teachers College Press, 1968 [1939]), 496–505. 19 Arnheim, ‘Gestalt and Art’, The Journal of Aesthetics and Art Criticism, 2.8 (1943), 71. 17 Albert Michotte, The Perception of Causality, trans. by T.R. Miles and Elaine Miles (New York: Methuen, 1963), 121. Notes 14 Scott Curtis, ‘Animated Images in a Media History of Science’, Journal of Cinema and Media Studies, 61.1 (2021), 147–152. 15 Ryan Pierson, ‘Postwar Animation and Modernist Criticism: The Case of Annette Michelson’, Journal of Cinema and Media Studies, forthcoming. On UPA see Dan Bashara, Cartoon Vision: UPA Animation and Postwar Aesthetics (Oakland: University of California Press, 2019). 16 Max Wertheimer, ‘Experimental Studies on Seeing Motion’, trans. by Michael Wertheimer and K.W. Watkins, in Id., On Perceived Motion and Figural Organization, ed. by Lothar Spillman (Cambridge: The MIT Press, 2012), 1–92. 17 Albert Michotte, The Perception of Causality, trans. by T.R. Miles and Elaine Miles (New York: Methuen, 1963), 121. udolf Arnheim, Art and Visual Perception (Berkeley: University of California Press, 1974), 403 19 Arnheim, ‘Gestalt and Art’, The Journal of Aesthetics and Art Criticism, 2.8 (1943), 71. 20 Ian Hacking, ‘“Style” for Historians and Philosophers’, in Id., Historical Ontology (Cambridge: Harvard University Press, 2002), 178–199. 21 Suzanne Buchan, ‘The Animated Spectator: Watching the Quay Brothers’ “Worlds”’, in Animated ‘Worlds’, ed. by Suzanne Buchan (Eastleigh: John Libbey Publishing, 2006), 36. 22 This, of course, isn’t to disparage the act of interpretation. Subtle and powerful interpretive claims have been made using plasmaticness — particularly by Nic Sammond, who notes that the freedom of movement in cartoon figures has a history in blackface minstrelsy. See Nic Sammond, Birth of an Industry: Blackface Minstrelsy and the Rise of American Animation (Durham: Duke University Press, 2015). My point here is that 80 Pierson, Gestalt, Animation, and the Culture of Design Pierson, Gestalt, Animation, and the Culture of Design limitations in our powers of description will in turn limit the scope of our interpretations. ur powers of description will in turn limit the scope of our interpretations. 23 Max Wertheimer, ‘Investigations on Gestalt Principles’, trans. by Michael Wertheimer and K. W. Watkins, in Wertheimer, 127–182. 24 Vivian Sobchack, ‘The Line and the Animorph, or “Travel Is More Than Just A to B”’, Animation, 3.3 (2008), 251–265 (258). Ibidem, 261. 26 Fritz Heider, ‘Thing and Medium’, in Id., On Perception, Event Structure, and Psychological Environment (Ann Arbor: International Universities Press, 1959), 1–34. Notes 27 Gadassik 286 28 Thomas Kuhn, The Structure of Scientific Revolutions (Chicago: University of Chicago Press, 2012), 111–134.i 29 On the importance of the camera (especially the moving camera) for phenomenological film criticism, see Schonig, ‘Seeing Aspects of the Moving Camera: On the Twofoldness of the Mobile Frame’, Synoptique, 5.2 (2017), 57–63. 30 Jo Freedman, ‘The Tyranny of Structurelessness’, Berkeley Journal of Sociology, 17 (1972– 64. 32 György Kepes, Language of Vision (Chicago: Paul Theobald & Co., 1944). 33 On high modernism’s disenchantment with public engagement, see Marci Kwon, Vernacular Modernism: Joseph Cornell and the Art of Populism, Ph.D. dissertation (New York University, 2016), 17–18, and Robert Genter, Late Modernism: Art, Culture, and Politics in Cold War America (Philadelphia: University of Pennsylvania Press, 2011), 24. Annette Michelson follow this narrative when she theorizes American avant- garde filmmaking in ‘Film and the Radical Aspiration’, Film Culture, 42 (1966), 34–42 and 46. 33 On high modernism’s disenchantment with public engagement, see Marci Kwon, Vernacular Modernism: Joseph Cornell and the Art of Populism, Ph.D. dissertation (New York University, 2016), 17–18, and Robert Genter, Late Modernism: Art, Culture, and Politics in Cold War America (Philadelphia: University of Pennsylvania Press, 2011), 24. Annette Michelson follow this narrative when she theorizes American avant- garde filmmaking in ‘Film and the Radical Aspiration’, Film Culture, 42 (1966), 34–42 and 46. p g , , ( ), 35 Jacques Rancière, The Politics of Aesthetics: The Distribution of the Sensible, trans. by Gabriel Rockhill (New York: Continuum, 2004), 12–13. 35 Jacques Rancière, The Politics of Aesthetics: The Distribution of the Sensible, trans. by Gabriel Rockhill (New York: Continuum, 2004), 12–13. 36 Matthew Holt, ‘Baudrillard and the Bauhaus: The Political Economy of Design’, Design Issues, 32.3 (2016), 55–66 (57–58). 36 Matthew Holt, ‘Baudrillard and the Bauhaus: The Political Economy of Design’, Design Issues, 32.3 (2016), 55–66 (57–58). 37 Rancière, Aisthesis: Scenes from the Aesthetic Regime of Art, trans. by Zakir Paul (London: Verso, 2013) 133–153. 37 Rancière, Aisthesis: Scenes from the Aesthetic Regime of Art, trans. by Zakir Paul (London: Verso, 2013) 133 153 37 Rancière, Aisthesis: Scenes from the Aesthetic Regime of Art, trans. by Zakir Paul (London: Verso, 2013) 133–153. e, Aisthesis: Scenes from the Aesthetic Regime of Art, trans. by Zakir Paul (London: Verso, 201 38 On these respective movements see Frederic J. ( y , ) 42 Relatedly, I want to emphasize that there is nothing inherently progressive about design in itself, and I believe Rancière overstates its liberatory potential. See, for example, Arden Stern and Sami Siegelbaum, ‘Special Issue: Design and Neoliberalism’, Design and Culture, 11.3 (2019), 265–277. Paul Fraisse’s Psychology of Rhythm: A Case for Filmology? Massimo Locatelli, Università Cattolica del Sacro Cuore, Milan This essay aims to discuss the topic of rhythm as presented by the classic psychologist and filmologist Paul Fraisse (1911-1994) in his founding studies, to understand its scope, and to consider the possible inheritance to be spent in contemporary research on film. I will first outline Fraisse’s contribution to the psychology of rhythm, a model grounded both on the value of Gestalt organisation and on related dimensions of sensory-motor activation. Secondly, I will investigate contemporary thinking in this field, showing on the one hand how Fraisse’s contribution still helps the psychology of music in defining rhythmic listening experiences, and on the other how the French scholar’s multilayered notion of time perception finds legitimation in neuroscientific research on timing. Finally, I will delve into film theory. In particular, I will put forward the assumption that the sense of rhythm, due to its values of Gestalt organisation, plays a fundamental role in narrative and event-based viewing, enhancing it; yet, due to the dimensions of sensory-motor activation, sound rhythms, in particular, can induce both bodily and neural entrainment and constitute in film an auditive analogon of those embodied and enactive visual processes recognised by the most recent neurofilmological approaches. Keywords Paul Fraisse Rhythm Gesltat Filmology Sound studies DOI 10.54103/ 2036-461X/17202 This work is licensed under a Creative Commons Attribution 4.0 International License Creative Commons Attribution 4.0 International License censed under a Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International Licen Rhythm is a Gestalt, but it is much more: a preferential mode of uniting perception and action, thze source of social manifestations and the basis of arts of succession and movement. Paul Fraisse, Is Rhythm a Gestalt? Notes Schwartz, The Werkbund: Design Theory and Mass Culture before the First World War (New Haven: Yale University Press, 1996); Victor Margolin, The Struggle for Utopia: Rodchenko, Lissitsky, and Moholy-Nagy, 1917-1946 (Chicago: University of Chicago Press, 1997), and T’ai Smith, Bauhaus Weaving Theory: From Feminine Craft to Mode of Design (Minneapolis: University of Minnesota Press, 2014). 38 On these respective movements see Frederic J. Schwartz, The Werkbund: Design Theory and Mass Culture before the First World War (New Haven: Yale University Press, 1996); Victor Margolin, The Struggle for Utopia: Rodchenko, Lissitsky, and Moholy-Nagy, 1917-1946 (Chicago: University of Chicago Press, 1997), and T’ai Smith, Bauhaus Weaving Theory: From Feminine Craft to Mode of Design (Minneapolis: University of Minnesota Press, 2014). 39 On the spread of design thinking see Annemarie Rose Ennis Dorland, ‘Doing Design Thinking: An Ethnography of the Digital Graphic Design Studio’ (Ph. D. dissertation, University of Calgary, 2018), 33–43. 39 On the spread of design thinking see Annemarie Rose Ennis Dorland, ‘Doing Design Thinking: An Ethnography of the Digital Graphic Design Studio’ (Ph. D. dissertation, University of Calgary, 2018), 33–43. 40 Manovich offers the most popular version of the return-of-the-repressed narrative. Manovich offers the most popular version of the return of the repressed narrative. 41 On sound design see William Whittington, Sound Design and Science Fiction (Austin: University of Texas Press, 2007); on film and fashion see, for example, Lucy Fischer, Designing Women: Cinema, Art Deco, and the Female Form (New York: Columbia University Press, 2003). 41 On sound design see William Whittington, Sound Design and Science Fiction (Austin: University of Texas Press, 2007); on film and fashion see, for example, Lucy Fischer, Designing Women: Cinema, Art Deco, and the Female Form (New York: Columbia University Press, 2003). y 42 Relatedly, I want to emphasize that there is nothing inherently progressive about design in itself, and I believe Rancière overstates its liberatory potential. See, for example, Arden Stern and Sami Siegelbaum, ‘Special Issue: Design and Neoliberalism’, Design and Culture, 11.3 (2019), 265–277. 81 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Keywords Paul Fraisse Rhythm Gesltat Filmology Sound studies DOI 10.54103/ 2036-461X/17202 PAUL FRAISSE Paul Fraisse (1911-1994), a French psychologist, who was a pupil of Albert Michotte van den Berck in Louvain and a collaborator with leading exponents of the French school of psychology of perception, made his mark on research into the experience of time as few other scholars of his age did, significantly contributing to founding the studies which after him came to be known as chronopsychology. In The Psychology of Time (1957) he demonstrated the existence of a multiplicity of temporal structures in the life of the individual, and shifted the attention of then nascent chronobiology from endogenous mechanisms to external conditioning, 83 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X making explicit the dimension of adaptation to periodicities connected both ‘with the main biological activities of the organism, namely motility, rest and food’ and with induced rhythms, ‘which bear no relationship with organic alterations’, hence induced in the laboratory or, hypothetically, attributable to social activity.1 Among social activities, Fraisse always attributed an important role in his studies to creative practices, such as music, supremely considered the art of time. Moreover, he also followed his master Michotte in the years of his filmological adventure, when between the end of the Second World War and the early 1960s he tried to establish a first methodological study with categories of the experimental psychology of the new media, primarily cinema.2 Together with Germaine de Montmollin, another of Michotte’s students, Fraisse conducted research into the understanding and memorisation of films for the Sorbonne filmology laboratories. PAUL FRAISSE As its starting point the research took the famous studies by Frederic Bartlett on the understanding of narrative and, as in this case, it strongly emphasised the active role of the subject in the cognitive process.3 Fraisse did not continue his film studies, but these early researches were useful to focus attention on the themes of the perception of time.4 In The Psychology of Time he succeeded in defining some essential traits that are still the basis of research today: the twofold nature of the experience of time, based on both immediacy and duration, in fact on multiple durations (as we shall see in §3), and the organised nature of this experience directed towards synchrony and succession, such that the tick-tock of the clock would never become a tock-tick nor a waltz change its tempo.5 The unity of the perceived succession between two minimum intervals, our clock or dance pattern, then forms what Fraisse calls ‘the psychological or perceived present’.6 The study of the experience of time would from then on focus on the measurement on the one hand of the subjective and intersubjective perception of duration, as a factor that qualifies every temporal experience,7 and on the other of the dimensions relating to rhythm and repetition, as its structuring element.8 Indeed, in keeping with his chronopsychological studies, Fraisse recognised rhythm as being above all a perceptual process with its basis in Gestalt reorganisation. The perception of time, in relation to rhythmic phenomena, recognises pregnant forms, which tend to be perceived first or produce a greater perceptual impact. ‘The reorganization takes the following two forms: differentiation, which sharpens the differences, and assimilation, which tends to diminish or suppress minor differences. Rhythmic reproductions are constituted by intervals having two values: short durations and long durations, which have a ratio of approximately 2/1; within each category, intervals are equal.’9 The short duration acts as a structural basis of rhythmic understanding, close to the limit of immediacy and perceptible in the form of the ‘collection’ of stimuli; the long duration is constituted in relation to the short duration. It is the place of duration and the constitution of multiple durations.10 The repetition of the formal scheme in a succession of homogeneous groups separated by pauses, tending to last for long durations, is fundamental in the recognition of rhythms. PAUL FRAISSE Cadences lower than 20 hundredths of a second and higher than 180 hundredths of a second, on the other hand, require conscious intervention and memory recall to maintain synchronisation.15 Finally, he describes each rhythmic fraction included in the motor rhythm band as a place of spontaneous grouping, which allows its overall perceptual organisation on the basis of precise logics of intensity (accent) and succession (pause). The idea is that the accent elicits the repetition also of complex isochronous groups (the rhythmic experience) and that the rhythmic movement, or its outline, is synchronised with the accent.16 We can distinguish three steps in Fraisse’s research that establish the rhythmic experience as eminently linked to body motor sequences. Firstly, in his laboratory experiments, Fraisse recognized a spontaneous motor rhythm, which was set within the temporal arc of the perceived present, i.e. on average within the band between 15/20 and 150/200 hundredths of a second, with a preference for intervals close to 60 hundredths of a second.13 A sort of basic rhythm, that of rocking or tapping the foot to the rhythm of music, that enables the subject to physically enter the rhythmic experiential field.14 Next, he demonstrates that spontaneous rhythmisation is the result of a sensory-motor response or ‘motor induction’ at regular intervals that activates a precise system of anticipation of bodily movement with respect to the rhythmic beat. Cadences lower than 20 hundredths of a second and higher than 180 hundredths of a second, on the other hand, require conscious intervention and memory recall to maintain synchronisation.15 Finally, he describes each rhythmic fraction included in the motor rhythm band as a place of spontaneous grouping, which allows its overall perceptual organisation on the basis of precise logics of intensity (accent) and succession (pause). The idea is that the accent elicits the repetition also of complex isochronous groups (the rhythmic experience) and that the rhythmic movement, or its outline, is synchronised with the accent.16 PAUL FRAISSE The pause, as Fraisse notes, is conceptually different 84 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm from the interval, even when it consists of an identical time span. It is a linkage, at the limit ‘analogous to the role of frame […] in the case of spatial figures’.11 While the structure in intervals of the single measure is related to the relationship between background and figure, the grouping and rhythm, albeit operating on the same Gestalt bases, through the pause-repetition mechanism, transfer the formal dynamism of the single unit to series of complex units that may require sensory-motor involvement by the subject. Psychological historians such as Fernández and Travieso have strongly emphasised this latter component in Fraisse’s thinking about rhythm, namely the motor basis of rhythmic experience.12 from the interval, even when it consists of an identical time span. It is a linkage, at the limit ‘analogous to the role of frame […] in the case of spatial figures’.11 While the structure in intervals of the single measure is related to the relationship between background and figure, the grouping and rhythm, albeit operating on the same Gestalt bases, through the pause-repetition mechanism, transfer the formal dynamism of the single unit to series of complex units that may require sensory-motor involvement by the subject. Psychological historians such as Fernández and Travieso have strongly emphasised this latter component in Fraisse’s thinking about rhythm, namely the motor basis of rhythmic experience.12 We can distinguish three steps in Fraisse’s research that establish the rhythmic experience as eminently linked to body motor sequences. Firstly, in his laboratory experiments, Fraisse recognized a spontaneous motor rhythm, which was set within the temporal arc of the perceived present, i.e. on average within the band between 15/20 and 150/200 hundredths of a second, with a preference for intervals close to 60 hundredths of a second.13 A sort of basic rhythm, that of rocking or tapping the foot to the rhythm of music, that enables the subject to physically enter the rhythmic experiential field.14 Next, he demonstrates that spontaneous rhythmisation is the result of a sensory-motor response or ‘motor induction’ at regular intervals that activates a precise system of anticipation of bodily movement with respect to the rhythmic beat. THE PSYCHOLOGY OF RHYTHM The correspondence or circularity between a perceptual mechanism that generally responds to Gestalt laws of perception and a sensory-motor activation system that intervenes on the level of basic rhythmic synchronisation seems to lead us back to the contemporary debate. The psychological field today implicitly or explicitly refers to cognitive theories of time, generally based on the notion of an internal clock at the neurophysiological level and on attentional and decisional mechanisms at the psychological level.17 But even researchers who share this paradigm address the problem of the relationship with the pre-cognitive dimension of experience, supplementing their research through a comparison with theoretical approaches of a markedly phenomenological nature, in which time on a psychological level is a function of the neurophysiological response or arousal.18 Understandably, the latter have a strong impact on studies in the cultural field, where case studies raise issues that are difficult to relate 85 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X to the rigid protocols of laboratory research. A constructivist vision, based on the recognition of qualitatively different processes, such as that of Paul Fraisse, could certainly assist the dialogue. Three key concepts taken from the psychology of music should be reconsidered after neuroscientific verification. Entrainment. The synchronisation of the sensory-motor response with the sound stimulus, especially in the presence of complexity or variations of the stimulus, is an emblematic case of this. Today we know that synchronisation, which we now refer to with the term entrainment, can occur at both a sensory and a neural level. THE PSYCHOLOGY OF RHYTHM Jones and Large, while working within an attentional model, conjecture, in the Dynamic Attending Theory, that the experience of listening to rhythmic stimuli develops a continuous and cyclical temporal experience, a transition from state to state and an internal resonance that dynamises the attentional effort.19 More recently, Chen, Penhune and Zatorre shored up the idea that different motor areas underlie the perception of rhythms, while rhythm fosters functional connectivity between auditory and motor networks; in these fMRI-based studies, musical rhythm processing has been shown to activate (also) neural mirroring, in analogy to visual perception.20 In the psychology of music, an attempt has recently been made to redefine the notion of entrainment by relating it to the associated experience of the topical and exciting moment of listening, and measuring it in terms of peak experience.21 The peak can also be reached through other paths, for instance melodic ones, but a fundamental key seems to be that of rhythmic beat timing, and the system of rhythmic accents identified by Paul Fraisse comes across as a good example of the construction of peak experiences. Anticipation. The most recent studies have also confirmed Fraisse’s intuition of the anticipatory character of the sensory-motor response to the rhythmic stimulus. David Huron, in his influential ITPRA model, strongly insists on the centrality of the expectation-prediction system as a cue to understanding the music experience.22 Working in an evolutionary perspective, Huron stresses that rhythm not only concerns low-order relationships (short rhythmic motives) but also higher-order relationships (meters), whereas ‘the basis for temporal perception is not periodicity — but predictability’, as it is an accurate prediction of the biological goal of expectation.23 A fundamental part of contemporary research rests on this premise. More specifically, the actual coupling between the auditory system and the dopamine system (referred to as the reward system) has been theorised, and in particular the two-phase structure of this coupling. In laboratory tests, a peak of pleasure felt while listening to a much-loved piece of music was connected to the activation of one sub-portion of the striatum. But a first anticipatory phase occurred a few seconds before in a different sub-region of the striatum.24 This occurrence indicates that the pleasure of the musical experience is to all intents and purposes built, at the neurophysiological level, on the interplay between expectation and resolution, an interplay that rhythm and repetition can expand and reiterate endlessly. Attention. Locatelli, Paul Fraisse’s Psychology of Rhythm THE PSYCHOLOGY OF RHYTHM The complexity of higher-order structures or musical meters has been widely debated. For Justin London, metrical experience ‘involves our 86 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm initial perception as well as subsequent anticipation of a series of beats that we abstract from the rhythm surface of the music as it unfolds in time’.25 London interestingly proposes a model, the Many Meters Hypothesis, which considers meter as a kind of “learnable” entrainment, a synchronization of our attention and response to the rhythms of the musical environment which we are exposed to. London openly admits his debt to Paul Fraisse. Thus, physical involvement can develop within metrically complex compositions and a universe of different possible interpretations, styles, and arrangements.26 From a purely neurophysiological point of view, it has also proved possible to describe a double circuit that integrates a typically bottom-up path, that of listening to music, with a typically top-down path, that of memorisation and recall.27 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X THE NEUROSCIENCE OF TIMING 38 2022 · ISSN 2036-461X milliseconds/seconds, many activities in our daily lives are related to the scale of seconds/minutes, which psychologists often refer to in terms of interval timing, and which recurs in the most explicitly aware activities involving the cognitive system.33 Studies of this kind, again, tell us of the central role played by memorisation and learning processes in the musical experience.34 The sense of individual time is therefore constructed starting from the synchronisation of different time frames, which include times of long-duration (such as the circadian rhythms regulating the alternation between sleeping and waking), all of which, in the light of recent analyses of neuroimaging, work on a semi-autonomous basis.35 In any case, in a logic of layering of the neural activation systems of the rhythmic and temporal sense, the perception of temporally organised stimuli is reinterpreted in terms of a binary functioning in different neural regions: on the one side an accumulation mechanism, which can correspond to the traditional internal clock, rhythmically organises the entry of stimuli (though they may not be rhythmic in origin), and therefore it can activate attentional and/or memorisation mechanisms. On the other side a comparison mechanism re-processes the data of the internal clock in the light of analogue or parallel systems and of the mnestic activity, and allows for an estimation of different complex intervals.36 If this is the case, despite the methodological issues that exploring the level of interval timing raises, perhaps it is not out of place to take as a first working hypothesis that the idea of enjoying audio-visual media (listening to music or watching a movie) is an essential training ground for a psychology of rhythm and apply it to complex segments and repetitions with a duration of seconds and minutes: meters and hyper-meters on the one hand, shots, scenes, sequences, musical insertions on the other. Music and film share the power to accumulate modules organised by rhythm but based on intervals and durations organised in the order of seconds and minutes (the order of our internal clocks engaged in interval timing), and to refine our power of comparison (of rhythmic-temporal dimensions). Starting from these strongly embodied images and sounds we shall nonetheless finally restore a cognitive dimension of the sense of rhythm, especially where the perception of durations and times implies evaluations. THE NEUROSCIENCE OF TIMING At this point, we need to delve into the core component of our rhythmical experience, assuming with contemporary research that the notion of timing describes our ability to live in time through the perception of rhythms and intervals. At this point the topic stops being purely a musicological one. Today we define the rhythmic perception that arises from the constant interaction between the auditory and motor systems of our mind, directed towards the recognition and anticipation of temporal organisations of experience, as beat-based timing.28 The beat is understood as the value of the psychological response to the rhythmic sequence and therefore epitomises in itself the organising value of the clicking of the metronome and that of a sensory- motor activation mechanism.29 In beat timing, succession and repetition seem to be a training ground for many important challenges that arise from it. These challenges above all concern the temporal structuring of perception itself, first, and clearly describe some characteristics of our involvement, namely a) the predictivity of experience. We already know that one of the ‘miracles’ of human cognition of time is the ability to anticipate rhythms (which enables us to dance, for example);30 b) its duration: the cyclical nature of the rhythm makes it possible to prolong the sensory-motor response and timing;31 and c) its permanence: we are capable of maintaining the sense of the rhythm that we have experienced for many cycles after the stimulus has ceased.32 The pattern of repetition, as we have seen, is not attributable only to the simplest rhythms, but also to complex meters and events stimulating predictive or attentional behaviours. We need to introduce a second timing level, called interval timing, in which one analyses the system of recognition of complex rhythmic temporal experiences and assesses their duration. As concerns this dimension, to date we have far fewer laboratory observations. We can still only consider this issue on the level of hypotheses. However, in recent years, scientific research has widely explored different scales for assessing the passing of time (or in general the experience of time). In particular, while musical rhythm, like the rhythm of language, is related to a scale of 87 Cinéma & Cie vol. 22 no. THE NEUROSCIENCE OF TIMING Evaluating and deciding, hence reasoning on a temporal basis, is a process that in turn acts on the neurophysiological mechanisms of timing, in keeping with a logic that could be defined as re-entry: the decision-making system ultimately intervenes on the internal clocks — whatever their form or scale — and modifies their computation.37 FOR A FILMOLOGY OF RHYTHM The basic assumption of this essay actually concerns the usefulness of reference to Fraisse’s work in contemporary research on film and the moving image. Film culture has always dealt with the problem of rhythm, both as a 88 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm question of style, and as a tool to get the audience involved. Cultural historians have shown how there is a qualifying link between the sense of modernity embodied by cinema and its avant-gardes in the first half of the 20th century, and the visual rhythms with which directors and editors experimented on multiple levels.38 The ‘founding fathers’ of film theory all gave ample space to reflection on rhythm, influencing subsequent debates: Béla Balázs’ film aesthetics, on which generations of directors were trained, prescribes a relationship of a musical type (harmony, counterpoint) between the ‘rhythm of the shots’, that is the degree of dynamism of the image, and the ‘rhythm of the montage’.39 In the sound era, by the mid-1930s, film rhythm was widely understood as the product of editing sound and image and of synchronizing multiple layers or strata of temporal development in an effort to gain control over the image.40 Film semiotics was later able to systematise the many possible regimes of inner- and inter-textual relationships apt to govern narrative film rhythm.41 Sergei Eisenstein has proposed the broadest and most refined reflection on cinematic rhythm, integrating the different rhythmic codes into a unitary concept of vertical editing. Vertical editing aims to involve the viewer ‘mechanically’ in a sensuous experience. The Russian director found his first inspiration in the primordial and tribal rhythm of the drum, and then broadened his observation to more complex rhythms of multiple cultural expressions. The most evident break with respect to Balázs and the classic style of montage can be found in the principle of the ‘opposite movement’ (otkaznoe dviženie) or mutual contrast between the elements, which enhances the impact of film rhythm by emphasizing its own dialectical process.42 The Eisensteinian perspective influenced later reflection on the potential of film for rhythmic involvement. We can distinguish between broad categories of intervention: one of a meta-psychological nature, one of phenomenological inspiration, and one in the cognitivist area. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X FOR A FILMOLOGY OF RHYTHM In this case, a multi-layered notion of rhythm, as inherited from Paul Fraisse, could offer a point of balance between models for understanding film experience which, by analogy with what happens in the more general framework of the psychological and neuroscientific debate, underline the cognitive challenge of film comprehension and narrative,49 and models that have strongly reassessed the pre-cognitive and embodied dimensions of the act of viewing.50 The Event segmentation theory postulates that continuity editing matches our brain’s use of discrete representation to predict the immediate course of events, and to create an internal, interconnected representation in memory.51 David Bordwell, though, from a clear cognitivist point of view, has insisted on the affective intensification effects of modern editing rhythm in terms of cutting pace,52 while Torben Grodal has brought back the effectiveness of the filmic experience to the body rhythms (‘fluctuations’) of the viewer.53 On this basis, Karen Pearlman has suggested reconsidering the work of the editor as a real technique of the body, assigning to rhythm the task of raising tension and release.54 This reflects also Walter Murch’s influential considerations on the ‘natural’ cut, based both on the actor’s movement and the viewer’s blinking of the eyes.55 In this respect, research into mirror neurons has had a strong impact and enabled authors like Gallese and Guerra to strongly anchor film viewing to a Simulation theory: mirror neuron activation pushes us to an embodied simulation of the character’s action and camera movements.56 The work of Eugeni, Balzaretti, Cavaletti, and D’Aloia demonstrates how in film viewing ‘the body schemata of movement and action possess an intrinsic temporal dimension’, and that this temporal dimension is expressed ‘both in terms of speed and duration’.57 The Italian authors questioned the influence of different editing rhythms (neutral, classical, intensified) on time perception. All these contributions solicit a ‘somatic intelligence’ of rhythm in film and the moving image,58 allowing us to extend its semiotics from a logic of control and sense strata to a logic of dynamic resonance and ‘production of affects’.59 Overall, though, they prevalently come across as being focused on visual factors, probably the most immediate and the easiest to test. An additional strand of research we need to consider is therefore research on film music. FOR A FILMOLOGY OF RHYTHM The first, which probably arises from Christian Metz’s founding observations on the fetish of technology,43 is best defined in Raymond Bellour’s most recent work.44 For Bellour, film has a corporeal quality that produces the constant illusion of a sensory, almost hypnotic ‘agreement’ with the spectator’s body. Bellour relies on Daniel Stern’s concept of the ‘present moment’, which is in turn indebted to French phenomenology and Paul Fraisse’s ‘psychological present’. In Stern a timing correspondence stems from the immediateness of any relationship, starting from the first face-to-face contacts of the child.45 For Bellour, film gives us the illusion of a similar immediate, bodily correspondence. Rhythm defines here the ‘transmodal character’ of this correspondence, ensuring the coordination of sound and image.46 The conclusions of the authors of the second line of thinking are indeed not far off: for example Vivian Sobchack also stresses the embodied character of rhythmically expressed ‘temporal reciprocity’.47 And already according to Jean Mitry the phenomenological perspective accounts for the subjective and sensorial value of rhythmic experience: rhythm is an intentional structure, which opens up to the construction of meaning through a perceptual experience. Notably, Mitry states that this experience must be interpreted with psychological tools 89 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X like those enunciated by Paul Fraisse, from the subjective value of rhythmic perception to the binary Gestalt principle of assimilation and differentiation (see earlier in §1).48 The psychological implications of the experience of rhythm have also been considered in the third area of study. FOR A FILMOLOGY OF RHYTHM On the one hand, related studies place great weight on the aspect of emotional activation of the original musical soundtrack.60 On the other hand, general reflections on affective intensification have been also related to dimensions such as those of the spatialisation of sound61 and the amplitude of usable frequencies following the introduction of noise reduction. The richness of timbre and sounds and noises of the contemporary film soundtrack, and, last but not least, the volume of sound in the cinema seem to enhance the emotional involvement of the audience.62 Conversely, there are few specific and dedicated in-depth laboratory studies Conversely, there are few specific and dedicated in-depth laboratory studies 90 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm on the auditive dimensions of film rhythm, or on multimodal and cross-modal rhythm processing, an exception being the recent work by Swenberg and Carlgren. Here, once again, it has been proved that ‘the relationship of the visual beat to the musical beat affects human viewers’ visual perception of the edits’, by enhancing both continuity and discontinuity effects.63 Carol Vernallis also aptly described the particular ‘responsiveness’ of video music editing to musical rhythms.64 This allows me to take a step further. Paul Fraisse had already studied the relationships between visual stimuli and sound stimuli, and his consolidated starting point lay in the basic differences between the two sensory systems: e.g., the response to the sound stimulus is faster than the visual one, the sound stimulus seems longer than a visual one of equal duration.65 These observations are substantiated by contemporary psychological and neuroscientific studies, which confirm the greater accuracy of the timing in auditory stimuli than in visual ones. Sound appears to be a more immediate instrument, but also a more precise mechanism.66 The signals of the visual organisation require a longer time to re-process and also a longer time to learn in childhood.67 We could refer to an auditive efficiency, in respect to visual perception. The French scholar further reported a fundamental qualitative difference between visual rhythm perception and sound rhythm perception. FOR A FILMOLOGY OF RHYTHM Listening allows a direct connection and synchronisation between the bodily response and rhythmic stimuli (entrainment), while vision allows only formal recognition of repetitions and symmetries that tend not to lead to a synchronised bodily response.68 Jean Mitry confirmed this view, affirming that film bases its rhythm on “discontinuous forms”, and therefore cannot achieve bodily synchronization through visual means only, if not on very particular occasions. An example of this would be the master sequence of the Teutonic Knights in Alexander Nevsky (Sergei Eisenstein, 1938).69 Eisenstein himself, criticising the complex scene of the religious dances in Storm over Asia (Vsevolod Pudovkin, 1928), noticed that in this case the visual editing just foregrounded an abstract cadence of spatial repetitions and combinations (metric montage, in his terms). He explicitly asked here for a musical track, in order to gain real rhythmical effectiveness 70 We can then put forward a second working hypothesis regarding the role of sound rhythms in film and the moving image. Rhythmic aptitude in listening, due to the automatisms of sensory-motor activation and synchronisation that constitute it, seems to be functional to film experience in the same way as the embodied and enactive visual processes recognised by the most recent neurofilmological theories are. That is, not only as simple emotional reinforcement, as in the case of a melodic commentary, but as a bodily scheme of action and movement, which allows an embodied and enactive response to the stimulus. The differences between the visual and the auditive-induced involvement need, indeed, to be stressed: in the first case, neural mirroring and simulation involve a personal relationship with a represented subject or action, while rhythmic entrainment does not require it. Furthermore, we might think that this response is not simply simulated but shared, with the body of the 91 91 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X text and the spectator community’s bodies. An impulse of extreme power and intensity. The limitation of the hypothesis lies in the lack of laboratory evidence, but the studies mentioned previously in support of the Dynamic attending theory (§2) seem to confirm at least its legitimacy. For now, we have confirmation of phenomena of both sensory and neural entrainment. CONCLUSIONS We have enough material to draw a conclusion, in a first attempt to delineate a Rhythmic Involvement Theory for audiovisual Media Experience (RITMEx). According to Fraisses’ founding definitions, and later debates on the psychology of rhythm, the neuroscience of timing, and the theory of film and the moving image, RITMEx can state at least that: 1. Rhythmic involvement in audiovisual media experiences is grounded both on a value of Gestalt organisation and on related dimensions of sensory-motor activation (§1) and is generally to be interpreted as a training ground for our multilayered timing skills (§3). 2. Rhythmic involvement can be traced back to two different ways of experiencing audiovisual media: on the one hand, a dynamic mode, closely but not necessarily related to style-centered filmmaking, music videos, and social media contents, where rhythm itself ensures entrainment and embodied anticipation (§2). On the other, a narrative or event-related one, closely but not necessarily related to continuity editing, that exploits the enhancing effects of rhythmic stimuli in order to reinforce attentional focusing and low and high- order cognitive prediction skills (§2 and 4). 3. Sound rhythms are more effective than visual ones in beat timing and short interval timing; they can induce both bodily and neural entrainment and consequently a dynamic mode of experience. They are a grounding element in a ‘somatic’ relationship to the body of the film or any moving image viewed. They can nonetheless also have enhancing, empowering, or even contrapunctual value in narrative or event-based experiences (§4). 92 Locatelli, Paul Fraisse’s Psychology of Rhythm 20 Joyce L. Chen, Virginia B. Penhune and Robert J. Zatorre, ‘Moving on Time: Brain Network for Auditory- Notes 1 Paul Fraisse, Psychologie du temps (Paris: Presses Universitaires de France, 1957), eng. ed. Psychology of Time (New York: Harper and Row, 1963), 28.i 2 On the filmological enterprise in post-war Paris and its relationship with psychology, see at least Cinémas, 19.2-3 (special issue La filmologie, de nouveau, ed. by François Albera and Martin Lefebvre, 2009).i 3 Paul Fraisse and Germaine de Montmollin, ‘Sur la mémoire des films’, Revue Internationale de Filmologie, 9 (1952), 37–69 (69). See also Frederic Charles Bartlett, Remembering: A Study in Experimental and Social Psychology (Cambridge: Cambridge University Press, 1932), 64–84. 3 Paul Fraisse and Germaine de Montmollin, ‘Sur la mémoire des films’, Revue Internationale de Filmologie, 9 (1952), 37–69 (69). See also Frederic Charles Bartlett, Remembering: A Study in Experimental and Social Psychology (Cambridge: Cambridge University Press, 1932), 64–84. 4 In the course of the above-mentioned study, Fraisse collected data on the judgment of duration that enabled him to subsequently demonstrate that the judgment of duration of single actions which one is called on to make directly tends to underestimate the time taken. This contrasts with the judgment of duration of complex activities, not clearly addressed and of which in particular one is a spectator: ‘For a spectator the interest, although real enough, does not give the task unity. The act of perception has its end in itself and not in an objective to be reached or a task to be carried out.’ Fraisse, Psychology of Time, 228. 5 Fraisse, Psychology of Time, 98. ‘Humans tend to perceive an isochronous stream of identical sounds as having alternating strong and weak notes — the “tick tock” phenomenon’. J. Daniel Cameron and Jessica A. Grahn, ‘Perception of Rhythm’, in The Cambridge Companion to Rhythm, ed. by Russel Hartenberger and Ryan McClelland (Cambridge: Cambridge University Press 2020), 20–38 (21). 5 Fraisse, Psychology of Time, 98. Humans tend to perceive an isochronous stream of identical sounds as having alternating strong and weak notes — the “tick tock” phenomenon’. J. Daniel Cameron and Jessica A. Grahn, ‘Perception of Rhythm’, in The Cambridge Companion to Rhythm, ed. by Russel Hartenberger and Ryan McClelland (Cambridge: Cambridge University Press 2020), 20–38 (21). 6 Fraisse, Psychology of Time, 84–85. 6 Fraisse, Psychology of Time, 84–85. 7 Fraisse Estimation and Perception of Time Annual Review of Psychology 35 (1984) 1–36 6 Fraisse, Psychology of Time, 84–85. 6 Fraisse, Psychology of Time, 84–85. Notes 7 Fraisse, Estimation and Perception of Time, Annual Review of Psychology, 35 (1984), 1–36 8 Fraisse brought together all of his studies on rhythm in a monograph published in France in 1974, Psychologie du rythme, which was soon translated into Italian, of which only an abridged version is available in English, collected by Diana Deutsch in the first edition of her influential Psychology of Music: Paul Fraisse, ‘Rhythm and Tempo’, in The Psychology of Music, ed. by Diana Deutsch (New York: Academic Press, 1982), 149–180. We will refer here to the Italian edition: Fraisse, Psycologie du rythme (Paris: Presses Universitaires de France, 1974), it. ed. Psicologia del ritmo (Roma: Armando, 1979). g 9 Fraisse, ‘Is Rhythm a Gestalt?’, in Gestalttheorie in der modernen Psychologie, ed. by Suitbert Ertel, Lilly Kemmler and Michael Stadler (Darmstadt: Steinkopff, 1975), 227–232 (231). g 9 Fraisse, ‘Is Rhythm a Gestalt?’, in Gestalttheorie in der modernen Psychologie, ed. by Suitbert Ertel, Lilly Kemmler and Michael Stadler (Darmstadt: Steinkopff, 1975), 227–232 (231). 9 Fraisse, ‘Is Rhythm a Gestalt?’, in Gestalttheorie in der modernen Psychologie, ed. by Suitbert Ertel, Lilly Kemmler and Michael Stadler (Darmstadt: Steinkopff, 1975), 227–232 (231). 10 Fraisse, Psicologia del ritmo, 82. Note that the distinction between a short and a long time is still the basis of one of the fundamental tasks in psychological research into time, termed temporal bisection: John Wearden, The Psychology of Time Perception (London: Palgrave Macmillan, 2016), 71–83. 11 ‘ rnández and David Travieso, ‘Paul Fraisse y la psicología del ritmo’, Revista de Historia de la 2-3 (2006), 31–43. 12 Marcos Fernández and David Travieso, ‘Paul Fraisse y la psicología del ritmo’, Revista de Historia de la Psicología, 27.2-3 (2006), 31–43. 12 Marcos Fernández and David Travieso, ‘Paul Fraisse y la psicología del ritmo’, Revista de Historia de la Psicología, 27.2-3 (2006), 31–43. 13 Fraisse, Psicologia del ritmo, 52. 15 Fraisse particularly stresses that they involve systematic errors in response, ibidem, 50–58 16 Fraisse, Psicologia del ritmo, 67–68. ‘These productions would have a perceptual regulation (grouping and structuration) but the basis would be motoric’, Fraisse, ‘Is Rhythm a Gestalt?’, 232. 16 Fraisse, Psicologia del ritmo, 67–68. ‘These productions would have a perceptual regulation (grouping and structuration) but the basis would be motoric’, Fraisse, ‘Is Rhythm a Gestalt?’, 232. 16 Fraisse, Psicologia del ritmo, 67–68. 20 Joyce L. Chen, Virginia B. Penhune and Robert J. Zatorre, ‘Moving on Time: Brain Networ . Chen, Virginia B. Penhune and Robert J. Zatorre, ‘Moving on Time: Brain Network for Auditor 13 Fraisse, Psicologia del ritmo, 52. Notes 27 Yue Ding, Yang Zhang, Wenjing Zhou, Zhipei Ling, Juan Huang, Bo Hong and Xiaoqin Wang, ‘Neural Correlates of Music Listening and Recall in the Human Brain’, Journal of Neuroscience, 39.41 (2019), 8112– 8123. 28 ‘This beat-based form of sensory-motor timing allows humans to flexibly extract a regular temporal structure from a range of rhythms, from simple isochronous sequences, in which all the intervals are identical, to more-complex meters like those of waltzes, marches, and sambas.’ Virginia B. Penhune and Robert J. Zatorre, ‘Rhythm and Time in the Premotor Cortex’, PLoS Biology, 17.6 (2019), 1–6 (1). See also, for a general description and bibliographic overview, Cameron and Grahn, ‘Perception of Rhythm.’ 28 ‘This beat-based form of sensory-motor timing allows humans to flexibly extract a regular temporal structure from a range of rhythms, from simple isochronous sequences, in which all the intervals are identical, to more-complex meters like those of waltzes, marches, and sambas.’ Virginia B. Penhune and Robert J. Zatorre, ‘Rhythm and Time in the Premotor Cortex’, PLoS Biology, 17.6 (2019), 1–6 (1). See also, for a general description and bibliographic overview, Cameron and Grahn, ‘Perception of Rhythm.’ 29 Cameron and Grahn, ‘The Neuroscience of Rhythm’, in The Oxford Handbook of Music Psychology, ed. by Suan Hallam, Ian Cross and Michael Thaut (Oxford and New York: Oxford University Press, 2016), 357–368. 29 Cameron and Grahn, ‘The Neuroscience of Rhythm’, in The Oxford Handbook of Music Psychology, ed. by Suan Hallam, Ian Cross and Michael Thaut (Oxford and New York: Oxford University Press, 2016), 357–368. 30 F l i th l b t th ti i t i d t d hil li t i t h th ti i t Cameron and Grahn, The Neuroscience of Rhythm , in The Oxford Handbook of Music Psychology, ed. by Suan Hallam, Ian Cross and Michael Thaut (Oxford and New York: Oxford University Press, 2016), 357–368. 30 For example, in the laboratory, the participants required to drum while listening to a rhythm anticipate the finger stroke by approx. 50 ms: Li-Ann Leow and Jessica A. Grahn, ‘Neural Mechanisms of Rhythm Perception: Present Findings and Future Directions’, Advances in Experimental Medicine and Biology, 829 (2014), 325–338 (327). Notes motor Synchronization is Modulated by Rhythm Complexity and Musical Training’, Journal of Cognitive Neuroscience, 20 (2008), 226–239, and ‘Listening to Musical Rhythms Recruits Motor Regions of the Brain’, Cerebral Cortex, 18.12 (2008), 2844–2854. 21 Alf Gabrielsson, John Whaley and John Sloboda, ‘Peak Experiences in Music’, in The Oxford Handbook of Music Psychology, ed. by Suan Hallam, Ian Cross and Michael Thaut (Oxford and New York: Oxford University Press, 2016), 745–758. 22 ‘A mnemonic for the sequence of five expectation-related responses: Imagination response, Tension response, Prediction response, Reaction response, Appraisal response.’ David Huron, Sweet Anticipation: Music and the Psychology of Expectation (Cambridge and London: The MIT Press, 2006), 7–18 and 416. 23 Ibidem 199 24 The striatum is a subcortical nucleus of the telencephalon, involved in many cognitive functions, including the motoric and reward systems. Valorie N. Salimpoor, Mitchel Benovoy, Kevin Larcher, Alain Dagher and Robert J. Zatorre, ‘Anatomically Distinct Dopamine Release During Anticipation and Experience of Peak Emotion to Music’, Nature Neuroscience, 14.2 (2011), 257–262. 24 The striatum is a subcortical nucleus of the telencephalon, involved in many cognitive functions, including the motoric and reward systems. Valorie N. Salimpoor, Mitchel Benovoy, Kevin Larcher, Alain Dagher and Robert J. Zatorre, ‘Anatomically Distinct Dopamine Release During Anticipation and Experience of Peak Emotion to Music’, Nature Neuroscience, 14.2 (2011), 257–262. 24 The striatum is a subcortical nucleus of the telencephalon, involved in many cognitive functions, including the motoric and reward systems. Valorie N. Salimpoor, Mitchel Benovoy, Kevin Larcher, Alain Dagher and Robert J. Zatorre, ‘Anatomically Distinct Dopamine Release During Anticipation and Experience of Peak Emotion to Music’, Nature Neuroscience, 14.2 (2011), 257–262. 25 Justin London, Hearing in Time: Psychological Aspects of Musical Meters (Oxford and New York: Oxford University Press, 2004), 4. 25 Justin London, Hearing in Time: Psychological Aspects of Musical Meters (Oxford and New York: Oxford University Press, 2004), 4. 25 Justin London, Hearing in Time: Psychological Aspects of Musical Meters (Oxford and New York: Oxford University Press, 2004), 4. ner’s metric competence resides in her or his knowledge of a very large number of contex trical timing patterns’, ibidem, 152–153. 27 Yue Ding, Yang Zhang, Wenjing Zhou, Zhipei Ling, Juan Huang, Bo Hong and Xiaoqin Wang, ‘Neural Correlates of Music Listening and Recall in the Human Brain’, Journal of Neuroscience, 39.41 (2019), 8112– 8123. 37 Hedderik van Rijn, Bon-Mi Gu and Warren H. Meck, ‘Dedicated Clock/Timing-circuit Theories of Interval Timing and Timed Performance’, in Neurobiology of Interval Timing, ed. by Merchant and de Lafuente, 75–99 Notes On the neurophysiological level, studies based on electroencephalographic research have also shown how, when listening to isochronous rhythms, the oscillations of the beta wave range appear to indicate ‘anticipation of regular, expected tones’, because they end after the sound, but reappear before the next sound. See Cameron and Grahn, ‘The Neuroscience of Rhythm’, 362. y 31 ‘If a sound is predictably repeated, induced responses may be observed around the time of the expected sound, and may occur even if the sound is omitted’, ibidem, 363. 32 See again Penhune and Zatorre, 2019. 33 ‘Time estimation refers to processing in the range of seconds and minutes and is generally seen as the conscious perception of time.’ Michael D. Mauk and Dean V. Buonomano, ‘The Neural Basis of Temporal Processing’, Annual Review of Neuroscience, 27 (2004), 307–340 (309). 33 ‘Time estimation refers to processing in the range of seconds and minutes and is generally seen as the conscious perception of time.’ Michael D. Mauk and Dean V. Buonomano, ‘The Neural Basis of Temporal Processing’, Annual Review of Neuroscience, 27 (2004), 307–340 (309). 34 Benjamin P. Gold, Marcus T. Pearce, Ernest Mas-Herrero and Alain Dagher, Robert J. Zatorre, ‘Predictability and Uncertainty in the Pleasure of Music: A Reward for Learning?’, Journal of Neuroscience, 39.47 (2019), 9397–9409. 34 Benjamin P. Gold, Marcus T. Pearce, Ernest Mas-Herrero and Alain Dagher, Robert J. Zatorre, ‘Predictability and Uncertainty in the Pleasure of Music: A Reward for Learning?’, Journal of Neuroscience, 39.47 (2019), 9397–9409. 35 Note that, rather than referring to dedicated circuits, the most recent proposals are theorising that on the neurophysiological level timing is an intrinsic property of every neural circuit. Cf. Hugo Merchant and Victor de Lafuente, ‘Introduction to the Neurobiology of Interval Timing’, in Neurobiology of Interval Timing, ed. by Hugo Merchant and Victor de Lafuente (New York: Springer, 2016), 1–13 (10). 35 Note that, rather than referring to dedicated circuits, the most recent proposals are theorising that on the neurophysiological level timing is an intrinsic property of every neural circuit. Cf. Hugo Merchant and Victor de Lafuente, ‘Introduction to the Neurobiology of Interval Timing’, in Neurobiology of Interval Timing, ed. by Hugo Merchant and Victor de Lafuente (New York: Springer, 2016), 1–13 (10). 35 Note that, rather than referring to dedicated circuits, the most recent proposals are theorising that on the neurophysiological level timing is an intrinsic property of every neural circuit. Cf. Notes ‘These productions would have a perceptual regulation nd structuration) but the basis would be motoric’, Fraisse, ‘Is Rhythm a Gestalt?’, 232. Fraisse, Psicologia del ritmo, 67 68. These productions would have a perceptual regulati nd structuration) but the basis would be motoric’, Fraisse, ‘Is Rhythm a Gestalt?’, 232. 17 In general, I refer to John Wearden’s compendium, and to the page he devotes to the relation between attention and timing, 88–102. 17 In general, I refer to John Wearden’s compendium, and to the page he devotes to the relation between attention and timing, 88–102. 17 In general, I refer to John Wearden’s compendium, and to the page he devotes to the relation between attention and timing, 88–102. 18 The works of Droit-Volet stress the importance of sensory-motor states in the perception of time, e.g. Sandrine Gil and Sylvie Droit-Volet, ‘Emotional Time Distortions: The Fundamental Role of Arousal’, Cognition and Emotion, 26.5 (2012), 847–862, and in general the discussion in Wearden, 105–115. 19 Mary Riess Jones, ‘Time, Our Lost Dimension: Toward a New Theory of Perception, Attention, and Memory’, Psychological Review, 83.5 (1976), 323–355; Edward W. Large and Mary Riess Jones, ‘The Dynamics of Attending: How People Track Time-varying Events’, Psychological Review, 106.1 (1999), 119–159. For a critical discussion of Dynamic Attending Theory and an updating of its neuroscientific foundations: Anna- Katharina R. Bauer, Manuela Jaeger, Jeremy D. Thorne, Alexandra Bendixen and Stefan Debener, ‘The Auditory Dynamic Attending Theory Revisited: A Closer Look at the Pitch Comparison Task’, Brain Research, 1626 (2015), 198–210. 19 Mary Riess Jones, ‘Time, Our Lost Dimension: Toward a New Theory of Perception, Attention, and Memory’, Psychological Review, 83.5 (1976), 323–355; Edward W. Large and Mary Riess Jones, ‘The Dynamics of Attending: How People Track Time-varying Events’, Psychological Review, 106.1 (1999), 119–159. For a critical discussion of Dynamic Attending Theory and an updating of its neuroscientific foundations: Anna- Katharina R. Bauer, Manuela Jaeger, Jeremy D. Thorne, Alexandra Bendixen and Stefan Debener, ‘The Auditory Dynamic Attending Theory Revisited: A Closer Look at the Pitch Comparison Task’, Brain Research, 1626 (2015), 198–210. 93 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X motor Synchronization is Modulated by Rhythm Complexity and Musical Training’, Journal of Cognitive Neuroscience, 20 (2008), 226–239, and ‘Listening to Musical Rhythms Recruits Motor Regions of the Brain’, Cerebral Cortex, 18.12 (2008), 2844–2854. 56 Vittorio Gallese and Michele Guerra, The Empathic Screen Cinema and Neuroscience (Oxford and New York: Oxford University Press, 2019). For a criticism of the Simulation theory of film: Malcolm Turvey, ‘Mirror Neurons and Film Studies: A Cautionary Tale from a Serious Pessimist’, Projections: The Journal for Movies and Mind, 3 (2020), 21–46. 46 Bellour, 162–163. Notes a in its physical state’, Metz, ‘Le signifiant imaginaire’, Communications, 23 (1975), 3–55 (52). 43 ‘Cinema in its physical state’, Metz, ‘Le signifiant imaginaire’, Communications, 23 (1975), 3–55 (52). 44 é é é ( O 2009) i nd Bellour, Le corps du cinéma. Hypnoses, émotions, animalités (Paris: P.O.L., 2009). y p yp 45 Daniel N. Stern, The Present Moment in Psychotherapy and Everyday Life (New York and London: Norton&C., 2004), 32–33. 45 Daniel N. Stern, The Present Moment in Psychotherapy and Everyday Life (New York and London: Norton&C., 2004), 32–33. 47 Vivian Sobchack, Carnal Thoughts: Embodiment and Moving Image Culture (Berkeley and Los Angeles: University of California Press, 2004), 116. É 48 Mitry, Esthétique et psychologie du cinéma, vol. 2 (Paris: Éditions universitaires, 1965), 166–171. y, q p y g , ( , ), 49 See at least David Bordwell, Narration in the Fiction Film (Madison: University of Wisconsin Press, 1985), and more recent studies of the neuroscientific foundation such as James E. Cutting and Ayse Candan, ‘Movies, Evolution, and Mind: From Fragmentation to Continuity’, The Evolutionary Review, 4.3 (2013), 25– 35, Tim J. Smith, ‘Attentional Theory of Cinematic Continuity’, Projections: The Journal for Movies and Mind, 1 (2012), 1–27. 50 For an overview, see Psychocinematics: Exploring Cognition at the Movies, ed. by Arthur P. Shimamura (Oxford and New York: Oxford University Press, 2013), Neurofilmology: Audiovisual Studies and the Challenge of Neuroscience, ed. by Adriano D’Aloia and Ruggero Eugeni, Cinéma & Cie, 22-23 (2014), and Dan Shaw, ‘Mirror Neurons and Simulation Theory: A Neurophysiological Foundation for Cinematic Empathy’, in Current Controversies in Philosophy of Film, ed. by Katherine Thomson-Jones (London and New York: Routledge, 2016), 148–162. 51 Jeffrey M. Zacks and Khena M. Swallow, ‘Event Segmentation’, Current Directions in Psychological Science, 14.2 (2007), 80–84. 52 Bordwell, The Way Hollywood Tells It: Story and Style in Modern Movies (Berkeley: University of California Press, 2006). 52 Bordwell, The Way Hollywood Tells It: Story and Style in Modern Movies (Berkeley: University of California Press, 2006). 53 Torben Grodal, Moving Pictures: A New Theory of Film Genres, Feelings and Cognition (Oxford: Clarendon Press, 1997), 42. 54 Karen Pearlman, Cutting Rhythms: Shaping the Film Edit (Burlington and Oxford: Focalpress, 2009). 55 Walter Murch, In the Blink of an Eye: A Perspective on Film Editing (Los Angeles: Silman-James Press, 1995), 69. 48 Mitry, Esthétique et psychologie du cinéma, vol. 2 (Paris: Éditions universitaires, 1965), 166–171. 49 See at least David Bordwell, Narration in the Fiction Film (Madison: University of Wisconsin Press, 1985), and more recent studies of the neuroscientific foundation such as James E. Cutting and Ayse Candan, ‘Movies, Evolution, and Mind: From Fragmentation to Continuity’, The Evolutionary Review, 4.3 (2013), 25– 35, Tim J. Smith, ‘Attentional Theory of Cinematic Continuity’, Projections: The Journal for Movies and Mind, 1 (2012), 1–27. Notes Hugo Merchant and Victor de Lafuente, ‘Introduction to the Neurobiology of Interval Timing’, in Neurobiology of Interval Timing, ed. by Hugo Merchant and Victor de Lafuente (New York: Springer, 2016), 1–13 (10). 36 ‘Accumulator and comparator functioning of the internal clock are mediated by distinct as well as partially overlapping neural regions’, Elaine B. Wencil, H. Branch Coslett, Geoffrey K. Aguirre and Anjan Chatterjee, ‘Carving the Clock at Its Component Joints: Neural Bases for Interval Timing’, Journal of Neurophysiology, 104.1 (2010), 160–168 (160). 37 Hedderik van Rijn, Bon-Mi Gu and Warren H. Meck, ‘Dedicated Clock/Timing-circuit Theories of Interval Timing and Timed Performance’, in Neurobiology of Interval Timing, ed. by Merchant and de Lafuente, 75–99 37 Hedderik van Rijn, Bon-Mi Gu and Warren H. Meck, ‘Dedicated Clock/Timing-circuit Theories of Interval Timing and Timed Performance’, in Neurobiology of Interval Timing, ed. by Merchant and de Lafuente, 75–99 37 Hedderik van Rijn, Bon-Mi Gu and Warren H. Meck, ‘Dedicated Clock/Timing-circuit Theories of Interval Timing and Timed Performance’, in Neurobiology of Interval Timing, ed. by Merchant and de Lafuente, 75–99 94 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm (76). ( ) 38 Laurent Guido, L’Age du rythme. Cinéma, musicalité et culture du corps dans les années 1900-1930 (Lausanne: Payot, 2007), Michael Cowan, Technology’s Pulse: Essays on Rhythm in German Modernism (London: School of Advanced Study, 2011). y 39 Béla Balázs, Der sichtbare Mensch oder die Kultur des Films (Frankfurt a.M., Suhrkamp 2001), 50 (my translation). 40 Cf. Lisa Jacobs, Film Rhythm After Sound: Technology, Music, and Performance (Berkeley and Los Angeles: University of California Press, 2015). 41 Cf. at least the grounding project of a grande syntagmatique, Christian Metz, Film Language: A Semiotics of the Cinema (Oxford and New York: Oxford University Press, 1974). More recently, Eugeni proposed to articulate film rhythms in three components: segmentation of the visual and sound continuum, evaluation of the reciprocal lengths of the segments, and detection of visual and sound accentuation, Ruggero Eugeni, Semiotica dei media. Le forme dell’esperienza (Roma: Carocci, 2010), 86ff. 42 Sergei M. Eisenstein, Il metodo (2002), vol. 1 (Venice: Marsilio, 2018), 183 and 203. A keen scholar of Eisenstein’s work, Jean Mitry, has included the entire classic debate on montage in a broader reflection on rhythmic styles, Jean Mitry, Esthétique et psychologie du cinéma, vol. 1 (Paris: Éditions universitaires, 1963). 54 Karen Pearlman, Cutting Rhythms: Shaping the Film Edit (Burlington and Oxford: Focalpress, 2009). 55 Walter Murch, In the Blink of an Eye: A Perspective on Film Editing (Los Angeles: Silman-James Press, 1995), 69. 57 Ruggero Eugeni, Stefania Balzarotti, Federica Cavaletti, Adriano D’Aloia, ‘It Doesn’t Seem_It, But It Is. A Neurofilmological Approach to the Subjective Experience of Moving-Image Time’, in The Extended Theory of Cognitive Creativity: Interdisciplinary Approaches to Performativity, ed. by Antonino Pennisi and Alessandra Falzone (Cham: Springer, 2020), 243–267 (257). 58 Pearlman, Cutting Rhythms, xvii. 70 Eisenstein, ‘The Fourth Dimension in Cinema (1929)’, in Selected Works, vol. 1 (London: British Film Institute, 1988), 186ff. 69 Mitry, Esthétique et psychologie du cinéma, vol. 2, 170–171. 67 Pierre S. Zélanti and Sylvie Droit-Volet, ‘Auditory and Visual Differences in Time Perception? An Investigation from a Developmental Perspective with Neuropsychological Tests’, Journal of Experimental Child Psychology, 112.3 (2012), 296–311. 68 Fraisse, Psicologia del ritmo, 56–57. Notes 56 Vittorio Gallese and Michele Guerra, The Empathic Screen Cinema and Neuroscience (Oxford and New York: Oxford University Press, 2019). For a criticism of the Simulation theory of film: Malcolm Turvey, ‘Mirror Neurons and Film Studies: A Cautionary Tale from a Serious Pessimist’, Projections: The Journal for Movies and Mind, 3 (2020), 21–46. 57 Ruggero Eugeni, Stefania Balzarotti, Federica Cavaletti, Adriano D’Aloia, ‘It Doesn’t Seem_It, But It Is. A Neurofilmological Approach to the Subjective Experience of Moving-Image Time’, in The Extended Theory of Cognitive Creativity: Interdisciplinary Approaches to Performativity, ed. by Antonino Pennisi and Alessandra Falzone (Cham: Springer, 2020), 243–267 (257). 95 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 59 Cf. Roger Odin, ‘Du spectateur ﬁctionalisant au nouveau spectateur: approche semio-pragmatique’, Iris, 8 (1988), 121–139 (128), and Warren Buckland, The Cognitive Semiotics of Film (Cambridge: Cambridge University Press, 2009) 106. 59 Cf. Roger Odin, ‘Du spectateur ﬁctionalisant au nouveau spectateur: approche semio-pragmatique’, Iris, 8 (1988), 121–139 (128), and Warren Buckland, The Cognitive Semiotics of Film (Cambridge: Cambridge University Press, 2009) 106. y 60 See Annabelle Cohen, ‘Film Music from the Perspective of Cognitive Science’, in The Oxford Handbook of Film Music Studies, ed. by David Neumeyer (Oxford and New York: Oxford University Press, 2014), 96– 130, and Lars Kuchinke, Hermann Kappellhoff and Stefan Koelsch, ‘Emotion and Music in Narrative Films: A Neuroscientific Perspective’, in The Psychology of Music in Multimedia, ed. by Siu-Lan Tan, Annabel J. Cohen, Scott D. Lipscomb and Roger A. Kendall (Oxford and New York: Oxford University Press, 2013), 118–138. 61 Mark Kerins, Beyond Dolby (Stereo): Cinema in the Digital Sound Age (Bloomington: Indiana University Press, 2011).i 62 Jeff Smith, ‘The Sound of Intensified Continuity’, in The Oxford Handbook of New Audiovisual Aesthetics, ed. by John Richardson, Claudia Gorbman and Carol Vernallis (Oxford and New York: Oxford University Press, 2013), 6581–6630. 63 Simon Carlgren, ‘On-Beat/Off-Beat: Visual Responses to Audio-Visual Asynchrony in Projections: The Journal for Movies and Mind, 1 (2021), 28–54 (28). 63 Simon Carlgren, ‘On-Beat/Off-Beat: Visual Responses to Audio-Visual Asynchrony in Music Videos’, Projections: The Journal for Movies and Mind, 1 (2021), 28–54 (28). 64 Carol Vernallis, ‘The Kindest Cut: Functions and Meanings of Music Video Editing’, Screen, 42.1 (2001), 21 48 (21) Projections: The Journal for Movies and Mind, 1 (2021), 28–54 (28). The Aha, Ha! Moment: A Gestalt Perspective on Audiovisual Humour Emilio Audissino, Linnéuniversitetet Emilio Audissino, Linnéuniversitetet In my previous work about film music, I had adopted Gestalt as a theoretical framework to explain the functions and effects of music in film, from a perspective that did not stem from musicology but from film studies. I developed what I call ‘micro/macro configurations’ analysis. In films, music contributes to the overall form with its specific Gestalt (the configuration of the musical structures), and such musical Gestalt meets the Gestalt of some other cinematic device/s. Besides music, any device (light design, colour schemes, dialogue, acting, camerawork, cutting…) has a specific micro-configuration that can fuse with those of the other devices, and it can be analysed in terms of micro/ macro-configuration. The product of the fusion of these micro-configurations is a macro- configuration in which the devices create an audiovisual whole that is ‘something else than the sum of its parts’. In this article I apply this Gestalt-inspired analytical approach to audiovisual humour, more specifically to ‘audiovisual puns’, ‘sight gags’, and ‘perceptual pranks’. The bulk of the examples come from the cinema of the Zucker- Abrahams-Zucker trio, whose comedy is largely based on a clash of incongruous micro- configurations, on perceptual accumulation that creates results similar to multistable figures, and even on comical optical illusions. Closing the article is a proposal that links Gestalt to the Release Theories of humour, explaining the laughter engendered by humour as a ‘Aha, Ha! moment’. Keywords Gestalt and film Neoformalism Zucker-Abrahams-Zucker Textual film analysis DOIi This work is licensed under a Creative Commons Attribution 4.0 International License censed under a Creative Commons Attribution 4.0 International License 10.54103/ 2036-461X/16912 This work is licensed under a Creative Commons Attribution 4.0 International License In this article I start by surveying the contribution that Gestalt theory can give to film analysis; then I present my Gestalt-inspired approach of ‘micro- configuration/macro-configuration analysis’; finally, I offer a Gestalt perspective on audiovisual humour. Given the film-studies oriented nature of this discussion, I shall not consider ‘found humour’ — situations we can find humorous in everyday life — but ‘invented humour’, which is the one we find in film comedies.1 I employ the Law of Prägnanz (‘good configuration’), the Theory of Amodal Completion (which stems from the Law of Good Continuation), and the Theory of Problem Solving (the ‘insight’ or, perhaps more famously, the ‘Aha! Moment’), which all derive from Gestalt Theory. Keywords Gestalt and film Neoformalism Zucker-Abrahams-Zucker Textual film analysis DOI /i Notes 64 Carol Vernallis, ‘The Kindest Cut: Functions and Meanings of Music Video Editing’, Screen, 42.1 (2001), 21 48 (21) 64 Carol Vernallis, ‘The Kindest Cut: Functions and Meanings of Music Video Editing’, Screen, 4 –48 (21). 65 A classic test of cross-modal interference in temporal and rhythmic perception to which Fraisse refers concerns the superimposition of a sound stimulus on an interval between visual stimuli in the so-called ‘Kappa effect’, an illusory perceptual time dilation. Fraisse, ‘Estimation and Perception of Time’, resp. 6 and 14. 66 See, including a general bibliography, Frank R. Schab and Robert G. Crowder, ‘Accuracy of Temporal Coding: Auditory-visual Comparisons’, Memory & Cognition, 17.4 (1989), 384–397. 67 Pierre S. Zélanti and Sylvie Droit-Volet, ‘Auditory and Visual Differences in Time Perception? An Investigation from a Developmental Perspective with Neuropsychological Tests’, Journal of Experimental Child Psychology, 112.3 (2012), 296–311. 69 Mitry, Esthétique et psychologie du cinéma, vol. 2, 170–171. 96 Locatelli, Paul Fraisse’s Psychology of Rhythm Locatelli, Paul Fraisse’s Psychology of Rhythm Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X The Aha, Ha! Moment: A Gestalt Perspective on Audiovisual Humour Emilio Audissino, Linnéuniversitetet I principally refer to the works of Wolfgang Köhler and Gaetano Kanizsa, instead of Rudolf Arnheim, whose theories are already more renowned in film and media studies.2 It is important to point out that this article is inspired by the conceptual framework of Gestalt Theory but it has no pretence to pass itself as a work pertinent to the ‘hard sciences’: its disciplinary 97 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X area is Film Studies, and its concern is the analysis of humour in films. Hence Gestalt is employed in looser terms than it would be in a Psychology journal.3 GESTALT AND FILM ANALYSIS In 1979, the Italian Gestalt Psychologist Gaetano Kanizsa wrote: ‘today Gestalt theory does not have much credit as an explanatory theory’.4 In Film Studies too, Cognitivism has proven more popular in the last decades, at least as an alternative to the post-structuralist and culturalist approaches.5 Yet, the interest for Gestalt Theory has seen some resurgence lately, perhaps as a consequence of the discovery in the 1990s of the mirror neurons, whose action seems to resemble the ‘Psychophysical Isomorphism’ postulated by Gestaltists in the 1910s.6 A renewed interest has consequently emerged in film studies too, especially in the area of sound and music, for example in the work of K. J. Donnelly and Danijela Kulezic-Wilson.7 Gestalt can provide a more holistic view of the audiovisual experience than the more modular view of Cognitivism.8 Often, the computer-like processing of perceptual data appears too central in Cognitivism, with cognition being given a predominant importance. Gaetano Kanizsa, in his works on visual perception, argued for a neater separation between the perception process and the cognition process, calling the two ‘primary process’ and ‘secondary process’, respectively. Primary process, though not exactly immediate — because ‘the organisation is not contained in the stimuli (even if the latter contains the conditions), but is added by the organism’ — is arguably more independent of cognition than Cognitivism would posit.9 In watching a film, for example, the perception of movement and the figure/ground separation would be a matter of primary processes — and not much cognitive processing is required — while the mental reconstruction of the ‘fabula’ from the ‘syuzhet’ would be a matter of secondary processes.10 The primary process, a ‘preconscious process’, is precisely what the Gestalt theorists have concentrated on, and it responds to organisational rules that were categorised into the Gestalt laws.11 An experiment that supports the separation of the two processes is the ‘Ames Room Illusion’: even when one is made aware of the ‘trick’ behind it, the illusion is still perceived, a sign that cognition has little or no effect on that specific perceptual experience.12 Gestalt-oriented film studies have similarly advocated for a neater separation of the two processes. K. J. Donnelly explains: Stimulus recognition takes place before cognition. Unconscious affect always creates emotion, whereas conscious cognition does not necessarily do so. GESTALT AND FILM ANALYSIS […] Cognitive Psychology’s notion of perception is that there is a small amount of stimulus and the ‘work’ all takes place as a cognitive process in the brain. This so-called mental model affirms that stimulus requires the considerable brain input of ‘enriching’ through hypothesis-testing. […] [T]here are distinct aspects of the aesthetic process (for film especially) that are poorly accounted for by such an approach.13 98 Audissino, The Aha, Ha! Moment Within film studies, my own work has also been influenced by Gestalt, in particular in my research on the functions and effects of music in films.14 From this research I developed, from Gestalt Theory, what I called ‘micro/ macro configurations’ analysis.15 In film, music contributes to the overall form with its specific ‘Gestalt’ (the configuration of the musical melody, harmony, timbre, etc.… is such that music is perceived as threatening, for example), and such musical ‘Gestalt’ meets the ‘Gestalt’ of some other cinematic device/s (say, a close-up of a smiling face).16 From the encounter of these micro-configurations (threatening music x smiling face) a macro-configuration is produced in which the two devices fuse to create an audiovisual whole (a ‘creepy’ person). The ‘creepiness’ configuration is neither in the image (the face is smiling) nor in the music (the music is threatening), but it appears as the product of the fusion of the two elements.17 This fusion responds to the Law of Prägnanz: in the apparently incongruous pairing of these two divergent elements, our mind searches for some sense, some stability, some meaningful relation between the two, until an interpretation emerges — that person appears creepy because behind the positive smiling facade some negative intent is hidden — and the percept is thus stabilised. If the perception of the smiling face and the threatening music can be a matter of ‘primary processes’, the interpretation of this apparently incongruous pairing seems to be calling for some higher-level processing, a secondary process more cognitive than perceptual. Yet, in keeping with Gestalt Theory, the secondary process of film interpretation can also be theorised with Gestalt’s theory of problem solving. In ordinary film comprehension, we usually apply ‘reproductive thinking’, that is ‘the application of tried-and-true paths to a solution. GESTALT AND FILM ANALYSIS The thinker reproduces a series of steps that are known to yield a workable answer’.18 With interpretation, we are faced with a problem that requires an act of ‘productive thinking’ on our part, which is ‘characterized by shifts in perspective which allow the problem solver to consider new, sometimes transformational, approaches’.19 If film interpretation is conceptualised as a problem-solving activity, and if a problem is considered like an unstable configuration, then the Gestalt theory of problem solving can be profitably used. According to this theory, a solution is found when the configuration of the problematic object is made stable. To achieve this, the ‘relations’ between the elements of the problem at hand have to be examined, we have to gain an ‘insight’: ‘we may now discover other relations in the material which make the difficulty disappear. In some instances, we are at first unable to see any relations in the material which are relevant to our task. When this happens, we have to inspect the given situation until, eventually, it does exhibit relations from which a solution can be derived’20 Gestalt tackles problem solving not only as a secondary process, and not so much as a cognitive effort of hypothesis-testing, but as a perceptual effort of relation-seeking — ‘seeing relations’, ‘insight’… The problem has to be observed from different angles until the right one is found from which we can see a relation between the elements that can reveal a solution. This is the ‘Aha! Moment’, the 99 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X moment in which the solution to a problem presents itself to the mind: all the pieces fall into the right place all of a sudden because we have found the right angle of observation. Back to our example of the creepy person macro-configuration, we are confronted with a juxtaposition of two micro configurations (threatening music and smiling face) that, taken singularly and compared, are not isomorphic at all: reproductive thinking would expect ‘happy’ music to go with a smiling face. This is the interpretive problem: why the juxtaposition of these two incongruous elements? When we apply productive thinking and find a relation between the elements, a stability of the percept is reached: the two micro-configurations (smiling face, threatening music) reconfigure one another and a macro- configuration of ‘creepy person’ finally emerges. GESTALT AND FILM ANALYSIS We perceive an incongruity between the two micro-configurations, the incongruity is made noticeable as a problem, which alerts us that interpretation (problem solving through productive thinking) is needed. When we eventually have an insight into the relation between the apparently incongruous micro-configurations — music is not incongruous: music is telling me that something threatening is hidden behind that smiling face — then the incongruity itself is removed and a macro- configuration is stabilised: arguably, a creep is about to perpetrate something disturbing. GESTALT AND AUDIOVISUAL HUMOUR When we experience an instance of humour, we are presented with a problem i When we experience an instance of humour, we are presented with a problem — an incongruous situation — and we are required to find a solution. In terms of problem solving, there is a resemblance here between what happens with comprehension through reproductive thinking (the approach we apply in regular situations) and the need to apply productive thinking to something unusual. A Gestalt-based explanation had already been given in 1932 of ‘the relevant mental processes of the humorous experience’, an explanation that gave perception a stronger role: the meaning of a set of elements depends on the specific configuration of said elements; when a sudden change in configuration is experienced — as happens in the incongruous punchline of jokes — the result is a sudden change of meaning.25 More recent contributions directly linked the pleasure that is experienced in solving a problem to the one experienced when understanding a joke: both entail a ‘revelation experience’ and ‘pleasures of the mind’ that are characterised by ‘surprise, violation of expectations and […] a feeling of mastery or virtuosity’.26 While most of these studies focus on verbal jokes or the visual humour of cartoons, I shall direct my attention to the audiovisual humour of films. Steve Neale and Frank Krutnik singled out some categories of comedic devices in film and television: comic events (humorous actions that are built into larger narratives, for example all the ludicrous troubles triggered by the rejuvenating potion in Monkey Business (Howard Hawks, 1952); gags (isolated humorous moments in the ‘field of visual, physical action’); jokes and wisecracks (isolated humorous events and actions that ‘imply a control of language’), and, within the latter, visual puns (‘one of the forms taken by the comic interplay between language and action’).27 Here, I leave aside the comic events and the jokes and wisecracks and concentrate on the gag and the visual pun. The gag is also known as ‘sight gag’, which in films often ‘derives from exploiting the magical properties of cinema, a comedy of metaphysical release that celebrates the possibility of substituting the laws of physics with the laws of the imagination.’28 A classic example of such ‘comedy of metaphysical release’ is the delivery man in Hellzapoppin’ (H.C. Potter, 1941) who is recurrently seen trying to deliver a potted plant to one Mrs. GESTALT AND AUDIOVISUAL HUMOUR I have used the word ‘incongruity’ to describe micro-configurations that do not seem to fit together. And ‘Incongruity Theory’ is precisely the principal orientation currently employed to explain why we experience ‘comic amusement’, the emotional state produced by ‘humour’ in all its forms.21 According to Incongruity Theory, ‘human experience works with learned patterns. […] The core meaning of ‘incongruity’ in standard incongruity theories is that some thing or event we perceive or think about violates our normal mental patterns and normal expectations.22 Faced with such incongruities, our mind at first turns to a state of alert, because any deviation from normalcy might entail a potential danger; but soon after, when the incongruity is assessed as a jocular and unthreatening one, we experience comic amusement for the humorous contestation of normalcy. It is cognitive psychology that has been largely employed to explain the mechanics of humour. Verbal comedy, in particular, has a substantial scholarly literature rooted in Linguistics or Cognitivism, largely falling within the Incongruity Theory — for example, Delia Chiaro’s Pragmatics and Descriptive Linguistics or Victor Raskin’s Script Theory.23 Such explanations — according to which humour is produced through a play with our mental scripts, schemata, and through a set-up that leads us to formulate false inferences that are then subverted by the punchline — are in line with Cognitivism’s focus on the secondary process, and indeed Incongruity Theory is the most popular amongst cognitivists.24 Yet, in the processing of visual and audiovisual humour there is 100 Audissino, The Aha, Ha! Moment arguably a stronger agency of the primary processes than in the more cognition- driven effort of processing verbal humour, which is based on symbolic language that needs stronger cognitive elaboration. Visual humour can be registered, perceived, with a higher level of immediacy than verbal humour: if a written joke in Swedish is read by people who are not able to enact a secondary processing of the language, the humour is not registered at all; on the contrary, the cartoon of some pompous self-important man spectacularly slipping on a banana peel has the potential to elicit comic amusement trans-culturally, because it is registered through primary processing. Hence, Gestalt can perhaps offer some complementing perspectives on audiovisual humour, specifically applying Gestalt’s problem-solving theory. GESTALT AND AUDIOVISUAL HUMOUR Jones in the most absurd situations — always unsuccessfully. Moreover, every time he appears, the plant has grown in size, and in the later attempts it has reached the dimension of a tree. The 101 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X recurring gag has the core of its humour in the sight of the paradoxical rate of growth of the plant and of the delivery man’s increasing exasperation and fatigue for carrying around the item. The visual pun consists in a humorous play with double-meanings and the ambiguities of language, like the verbal pun, but it is realised in the visual domain.29 An oft-cited example is in Horse Feathers (Norman Z. McLeod, 1932) where Groucho Marx, president of a university, inspects an official document before signing it. He stops, alarmed, ‘Wait a minute there’s no seal here. Where’s the seal?’. And Harpo, promptly, brings in a sea seal instead of the expected piece of stationery. Though Maltin’s coinage of ‘visual pun’ for this example is justified by the fact that this is a pun whose punchline takes place visually, I think that audiovisual pun is a fitter qualification. The pun works by means of one visual and one aural element, and it works only because the two are fused together. The visual micro-configuration (Harpo bringing in a sea seal), though certainly bizarre, is not per se humorous. The aural micro-configuration is not humorous at all (a character asks for a stamp). It is the fusion of the two that creates a humorous macro-configuration. Whereas the traditional pun is a wordplay in which the comic effect is produced by a double meaning within the same sensory modality — ‘All calendar’s days are numbered’ — in audiovisual puns the verbal part typically functions as a first leg of the joke (set-up) while the second leg (pay-off) is offered visually. Film comedy typically employs the visual and aural modalities in combination to produce humour. Even in sight gags, sound constitutes an important micro- configuration: consider the various gags about music shifting from the non- diegetic to the diegetic level — what Biancorosso calls ‘epistemological jokes’.30 Viewers are tricked into believing that music is non-diegetic (coming from outside the narrative world) but then they suddenly realise it is in fact diegetic (it comes from some source within the story-world). A classic example is from Mel Brooks’s Blazing Saddles (1974). Audissino, The Aha, Ha! Moment GESTALT AND AUDIOVISUAL HUMOUR We see the sheriff riding his horse in the prairie to the sound of Count Basie’s ‘April in Paris’ — a choice that is per se already incongruous with the western-film setting, but motivated by the incongruous designer’s saddle sported by the sheriff. Yet, as the camera pans to follow the horse ride, we discover that Count Basie and his orchestra are actually there, playing the music from a stage incongruously placed in the middle of the prairie. The music we assessed as non-diegetic accompaniment (following the patterns of reproductive thinking as per our film-viewers’ experience) is actually a diegetic performance.31 Both modalities are actively involved in the production of the gag’s macro-configuration: the aural micro-configuration (Basie’s song) and the visual micro-configuration (the sheriff meeting the orchestra in the prairie). If this had been a silent film, we would have had the surprise of finding an orchestra in the middle of nowhere, but the non-diegetic/diegetic humorous trickery is made possible precisely by the audiovisual fusion. Like this, a sizable number of sight gags involve an aural component, and they are based on some trickery of our perception, not only of our mental schemata and scripts: first we perceive the music as non-diegetic, and then the perception is suddenly shifted 102 Audissino, The Aha, Ha! Moment to another angle — diegetic. Observed from this perception-oriented angle, audiovisual humour of this kind is similar to multistable images, in which ‘sensory information is ambiguous and consistent with two or more mutually exclusive interpretations’.32 The Duck/ Rabbit, the Old Lady/Young Maid, or the Rubin Vase are famous examples: ‘such figures provide the experience of looking at a constant external stimulus whose perceptual appearance changes from one viewing to the next, or indeed from one moment to the next in continuous viewing’.33 In such figures, two co- existing sets of stimuli are juxtaposed, liable to be arranged into two or more different macro-configurations; we try to stabilise one macro-configuration, but that macro-configuration would not really stabilise, some tension remains, something unusual is perceived in the image. Then, we eventually ‘solve the problem’ by noticing, from another angle, that there is another relation between the micro-configuration sets of stimuli. The reaction when we are presented with a multistable figure and we realise the trick it plays on our perception, can be one of smile and comic amusement. GESTALT AND AUDIOVISUAL HUMOUR 38 2022 · ISSN 2036-461X because the effect is both unexpected on the basis of everyday experience and in conflict with the perspective information on depth given by the figure’ and one can also notice ‘the surprise of the observers and their saying, as they did in the case of the fisherman and of the fencer, that there must be some mistake in the picture’.37 Audiovisual gags like Count Basie in the prairie function somewhat like multistable figures: we first see something and then suddenly our perception is flipped to something else, and when we realise the trickery at the base of the image, we experience amusement.38 Apart from the amusement for the sudden switch of perspective, multistable figure-like humour can also derive from the perception of the presence at the same time of two contrasting configurations Audiovisual gags like Count Basie in the prairie function somewhat like multistable figures: we first see something and then suddenly our perception is flipped to something else, and when we realise the trickery at the base of the image, we experience amusement.38 Apart from the amusement for the sudden switch of perspective, multistable figure-like humour can also derive from the perception of the presence at the same time of two contrasting configurations — like in the Old Lady/Young Maid image. For instance, the humour might lie in the contrast between one serious micro-configuration and one ridiculous micro- configuration: in the same macro-configuration we have two different sides, as in a multistable image. The oeuvre of David Zucker, Jim Abrahams, and Jerry Zucker is exemplarily replete with gags based on this multistable ‘audiovisual disjunction’.39 — like in the Old Lady/Young Maid image. For instance, the humour might lie in the contrast between one serious micro-configuration and one ridiculous micro- configuration: in the same macro-configuration we have two different sides, as in a multistable image. The oeuvre of David Zucker, Jim Abrahams, and Jerry Zucker is exemplarily replete with gags based on this multistable ‘audiovisual disjunction’.39 — like in the Old Lady/Young Maid image. For instance, the humour might lie in the contrast between one serious micro-configuration and one ridiculous micro- configuration: in the same macro-configuration we have two different sides, as in a multistable image. The oeuvre of David Zucker, Jim Abrahams, and Jerry Zucker is exemplarily replete with gags based on this multistable ‘audiovisual disjunction’.39 One gag in ZAZ’s TV show Police Squad! GESTALT AND AUDIOVISUAL HUMOUR An example of such effect produced by this multistability — at least on me — is a particular version of the Rubin Vase.34 It is not a drawing but a photorealistic rendering of a vase with the British royal family crest on it. At first, I see the vase, and that seems the way the macro-configuration is stabilised. But there is something weird about this vase: it is asymmetrical and odd-shaped, it does not conform to the Law of Good Form — nor to basic standards of good pottery. By inspecting the vase for solutions to the oddity, the perception suddenly flips to another side: now the profiles of Queen Elisabeth and Prince Philip can be seen, which explains the odd contours of the vase. A new macro-configuration is reached in which what I perceive is not a slovenly shaped vase but a humorous homage to the Royal couple. Kanizsa studied other types of optical illusions, the so-called ‘impossible figures’, like Penrose’s triangle or fork.35 He too detected reactions of comic amusement when people were confronted with such odd images. Kanizsa, also a painter, discussed some of his works in which he created configurations that can be obtained in the bidimensional world of the canvas but that would be impossible in the tridimensional world of real life — as happens in Escher’s lithographies. In Kanizsa’s paintings, the absurd effects are produced by confusing our perception about the distinction between figure and ground through the flatness of bidimentionality, the juxtaposition of bright and dark areas, and a play between thick and thin elements, cues which all tend to be configured automatically, driven by the primary-process Gestalt laws. The result is that elements of the characters in the foreground seem to absurdly fuse or intersect with the objects or characters in the background: ‘The fishing rod is thinner than the sail, so it is “forced” to pass behind […], the fencer’s sword pierces the referee several meters away, the umbrella is threaded through the girl’s hair, and the man and the woman are strangely entangled in the fence. All these figures seem absurd’.36 These absurd paintings suggest ‘a humorous effect to the observer’; the first impression is one ‘of oddity and absurdity, 103 Cinéma & Cie vol. 22 no. GESTALT AND AUDIOVISUAL HUMOUR […] One need only recall the fundamental fact of figure- ground segmentation in the construction of the phenomenal world, in which the articulation always implies the completion (precisely amodal) of the continuous background existing behind the figure.40 Audiovisual puns play with our tendency to anticipate the continuation of an event, to fill the gaps according to the normal expectations that reproductive thinking suggests us. We perform an amodal completion of elements that we suppose are going to come next, even if evidence of this has not been ostensibly presented yet by any modality. In the case of the Marx Brothers’ seal audiovisual pun, the normal amodal completion suggested by reproductive thinking — how we mentally see the event progressing — is the anticipation of an office seal to appear soon; instead, something else appears, which is not in line with the prediction.41 In Police Squad! again we have a number of such instances. For example, in one episode (S01xE04), Hocken and Drebin pay a visit to a night-club to question the resident starlet. They found her in the dressing room, with the scant costume still on. As they talk, she asks, ‘Do you mind if I change’ and then moves behind a dressing screen. Presently, she comes out as a totally different actress, a complete change, not a tall red-head but a petite blonde. Despite the absurd twist, the conversation continues undisturbed, as per the style of Zucker, Abrahams, and Zucker [fig. 1]. A less evident audiovisual puns is in ZAZ’s Top Secrets! (1984): at a performance of Tchaikovsky’s ballet The Nutcracker (1892) we see the male dancers sporting exceptionally prominent crotch bumps in their leotards; as they stand in line, the female dancers leap in the air and continue dancing by stepping on the male anatomical protrusions — a veritable ‘nutcracker’ ballet, for those who recognise the title of the musical piece. There are cases of sight gags that are specifically based on tricks played on our perception. These ‘perceptual pranks’ are abundant in the cinema of Jacques Tati and of Zucker, Abrahams, and Zucker.42 In both, the comedic style is typically based on a play with the viewer’s perception — not only through off- centred, background, or multi-layered gags — but also through veritable optical illusions. GESTALT AND AUDIOVISUAL HUMOUR (S01xE05, 1982) sees Lieutenant Frank Drebin — in an undercover mission — paying a visit to a mob chieftain in his office. As customary after the James Bond films, the stereotypical villain is at his desk, cuddling a white cat on his lap. As the dialogue proceeds on serious tones, the mobster puts the cat away by sticking it inside one of the desk-drawers, and then opens another drawer to grab a gun, but we can peek a white toy poodle inside this other drawer. As these animal gags run, the tone of the dialogue is threatening and confrontational, and the music too accompanies it in a serious mood. Moments later, the mobster opens a file cabinet to put in a document while he assigns to Drebin — who pretends to be wanting to join the crime organisation — a murder as an initiation test. The music punctuates the words ‘I want you to kill him’ with a dramatic figure of the trombones, but as the mobster opens the file cabinet, doves fly out of it, continuing the series of stored-animals gags. If we hear only, the scene seems to belong to the drama genre; if we watch only, it clearly belongs to the comedy genre. The humorous macro-configuration is produced by the multistable tension of the two contrasting micro-configurations. If we consider audiovisual puns, these are not only interpretable in terms of multistability — the flipping from one meaning to another — but they have also a component of trickery played upon our ‘amodal completion’ processes, that is one of the two modalities through which we interpolate non-visible parts to create a good form and stable configuration in our percepts. In Kanizsa’s words: we have phenomena of totalization, of completion, of integration, of ‘filling in the gaps’ — that is, of making present what is absent. The interpolation in the primary process can be modal or amodal. Examples of modal completion are […] the formation of anomalous contours and surfaces. In all these cases the filled-in parts have the characteristics of visual modality [...]. Much more numerous are the 104 Audissino, The Aha, Ha! Moment cases of amodal completion. By ‘amodal presence’ we mean that type of perceptual existence […] that is not verified by any sensory modality. GESTALT AND AUDIOVISUAL HUMOUR A Tati example from Play Time (1967) is the doorman who bends down to open the floor latches of a large glass door, and in doing so the ornate handles of the door gets superimposed to his head and look like a massive pair of antlers. Tati’s films are an examples of ‘parametric cinema’ which ‘exploits the very limits of the viewer’s capacity’.43 Also, ‘the strongly parametric film, in departing from the classical system, must create a degree of perceptual uncertainty’.44 While in classical cinema we can rely on reproductive thinking, with parametric cinema we have to resort to productive thinking to solve the perceptual challenges, like when we are faced with humour. Comedies in the parametric style are thus most interesting from a Gestalt perspective. Within the ‘nonsensical accumulation’ typical of ZAZ’s works, we find numerous 105 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X perceptual pranks at the expense of the viewers.45 A particularly rich repository is Top Secret!. A striking instance of an articulate and technically complex perceptual prank is the Swedish bookstore scene, which I have analysed elsewhere.46 Another is the departing train station: the protagonist is sitting in a train that has just stopped at a station. We hear the whistle signal, the engine warms up, and we see, from inside the wagon, that the train is leaving the station… until we realise that it is actually the station that is leaving the train. The false movement is confirmed when the narration cuts to the exterior of the wagon and we see the station, bizarrely mounted on a wheeled platform, departing from the stationary train — to add more absurdism, a late-coming passenger chases the travelling station. O h i T S ! d i h i b l i h fl f h Fig. 1: ‘Do you mind if I change?’, from Police Squad! (David Zucker, Jim Abrahams, Jerry Zucker, 1982), Episode 4 perceptual pranks at the expense of the viewers.45 A particularly rich repository is Top Secret!. A striking instance of an articulate and technically complex perceptual prank is the Swedish bookstore scene, which I have analysed elsewhere.46 Another is the departing train station: the protagonist is sitting in a train that has just stopped at a station. Fig. 1: ‘Do you mind if I change?’, from Police Squad! (David Zucker, Jim Abrahams, Jerry Zucker, 1982), Episode 4 Audissino, The Aha, Ha! Moment Fig. 2: Trespassing Cows, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 3: Big Phone, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) GESTALT AND AUDIOVISUAL HUMOUR Moment picks it up, and as he walks our perception changes: we realise that the phone is actually oversized [fig. 3]. Fig. 4: Boots, from Secret! (Dav Jim Abraha Zucker, 198 picks it up, and as he walks our perception changes: we realise that the phone is actually oversized [fig. 3]. Perceptual amodal completion is at the base of other gags. The East-Germany commander is seen reading a book in his office, his boots lying on the desk. We are distracted by the book’s title: ‘Hermann Goering’s Workout Book’, and we are led to think that this is the sight gag here. When the attendant enters the office, the commander stands up and his boots keep staying on the desk: they were fake boots, disconnected from his body. At the sight of boots on a desk and a person behind it, we amodally complete the image by filling in the missing parts — we see boots, we see a person, we connect the boots with the person, Fig. 4: Boots, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 4: Boots, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 4: Boots, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) picks it up, and as he walks our perception changes: we realise that the phone is actually oversized [fig. 3]. Perceptual amodal completion is at the base of other gags. The East-Germany commander is seen reading a book in his office, his boots lying on the desk. We are distracted by the book’s title: ‘Hermann Goering’s Workout Book’, and we are led to think that this is the sight gag here. When the attendant enters the office, the commander stands up and his boots keep staying on the desk: they were fake boots, disconnected from his body. At the sight of boots on a desk and a person behind it, we amodally complete the image by filling in the missing parts — we see boots, we see a person, we connect the boots with the person, imagining legs that we do not actually see, as per the Law of Good Continuation. These gags make fun of our routinised perception: do not presume the presence of legs only because you see boots! [fig. 4] In a later scene, the protagonists knock at the door of the underground- movement headquarters. GESTALT AND AUDIOVISUAL HUMOUR We hear the whistle signal, the engine warms up, and we see, from inside the wagon, that the train is leaving the station… until we realise that it is actually the station that is leaving the train. The false movement is confirmed when the narration cuts to the exterior of the wagon and we see the station, bizarrely mounted on a wheeled platform, departing from the stationary train — to add more absurdism, a late-coming passenger chases the travelling station. Other gags in Top Secret! deceive the viewer by playing on the flatness of the bidimensional film image, in a way similar to Kanizsa’s paintings. As the leader of the underground fighters is observing through binoculars the meadow in front of the prison they are about to infiltrate, we cut to a point-of-view shot: we see the typical reverse-eight-shaped black matte of binoculars framing the meadow, in which some cows are roaming. Suddenly, the cows jump over the black matte as if it were a fence, tilting our perception of the different planes of 106 Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Moment the space, as in a trompe-l’oeil [fig. 2]. the space, as in a trompe-l’oeil [fig. 2]. In another scene, the perceptual prank is played through forced perspective — the same trick used to make objects or people look bigger or smaller depending on their position between the camera and the set, a sort of Ames 107 Ci é & Ci l 22 38 2022 ISSN 2036 461X Fig. 2: Trespassing Cows, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 2: Trespassing Cows, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 2: Trespassing Cows, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) 107 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X room, as employed in Darby O’ Gill and the Little People (Robert Stevenson, 1959).47 The scene starts with a phone ringing in a large salon with people in the background. The phone is very close to the camera and appears magnified because of its placement. The East-Germany commander walks to the phone to Fig. 3: Big Phone, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) Fig. 3: Big Phone, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) 108 Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Fig. 5: Tall/short, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) GESTALT AND AUDIOVISUAL HUMOUR A little sliding window opens on the very top of the door and a man peeks outside suspiciously, asking for the password. Given the high position of his head, we prepare ourselves to see an abnormally tall person. As the newcomers provide the correct password, the door is open… to 109 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X reveal that the peeking man was in fact a ‘little person’, evidently standing on a high stool. Because of our routinary amodal completion process, we imagined a person as tall as the door window, filling the non-visible parts supposed to lead up to the visible head [fig. 5]. The types of sight gags that can be called perceptual pranks are not as Audissino, The Aha, Ha! Moment Fig. 5: Tall/short, from Top Secret! (David Zucker, Jim Abrahams, Jerry Zucker, 1984) 110 Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Moment common as audiovisual puns and the ‘regular’ sight gags, but when one finds them, these are ideal for the application of Gestalt, because they rely more on the primary process than on the secondary process: they do not disrupt our knowledge of norms and conventions; they disrupt our perception. THE ‘AHA, HA! MOMENT’ As a conclusion, an important question of Humour Theory might be tackled, even if only tentatively: Why we often laugh when faced with audiovisual humour, multistable figures, or perceptual pranks? In the case of the Royal Couple Vase, the first reaction is one of weirdness, and weirdness can be a threat to normalcy and stableness, so we muster psychic energies to solve the problem. When the weirdness is found to be innocuous and deliberately created to play on our perception, then a re-configuration of the energies happens and comic amusement is the effect of this sudden mental switch. This comic amusement might produce the physical reaction of a smile or even a burst of laughter. Why? Release Theories concentrated on this: What is laughter? This is admittedly ‘something left unexplained by the […] Incongruity Theories’.48 The Incongruity Theories focus on the mental processes, and the psychic reward is a ‘mental pleasure’ similar to the one gained from solving puzzles, ‘but when we engage in genuine puzzle solving […] we aim at discovering the right answers and take pleasure in that, whereas with things such as jokes, we are happy — really happy — with the wrong answers’, notes Noël Carroll.49 From the angle of Release Theories, the physical reward is the pleasure deriving from a discharge of pent-up energy. As a conclusion, an important question of Humour Theory might be tackled, even if only tentatively: Why we often laugh when faced with audiovisual humour, multistable figures, or perceptual pranks? In the case of the Royal Couple Vase, the first reaction is one of weirdness, and weirdness can be a threat to normalcy and stableness, so we muster psychic energies to solve the problem. When the weirdness is found to be innocuous and deliberately created to play on our perception, then a re-configuration of the energies happens and comic amusement is the effect of this sudden mental switch. This comic amusement might produce the physical reaction of a smile or even a burst of laughter. Why? Release Theories concentrated on this: What is laughter? THE ‘AHA, HA! MOMENT’ This is admittedly ‘something left unexplained by the […] Incongruity Theories’.48 The Incongruity Theories focus on the mental processes, and the psychic reward is a ‘mental pleasure’ similar to the one gained from solving puzzles, ‘but when we engage in genuine puzzle solving […] we aim at discovering the right answers and take pleasure in that, whereas with things such as jokes, we are happy — really happy — with the wrong answers’, notes Noël Carroll.49 From the angle of Release Theories, the physical reward is the pleasure deriving from a discharge of pent-up energy. Release Theories posit that laughter is a discharge of energy: when we are confronted with a puzzling situation, we muster psychic energies to face the potential problem, and when we realise that the puzzling situation is nothing serious, then the accumulated energy is released in the form of the laughter. Quoting John Dewey, Morreall succinctly expresses the point: ‘the laugh is thus a phenomenon of the same general kind as the sigh of relief’.50 In Gestalt terms, an energy pattern in the brain is suddenly reconfigured into a different one. When the new macro-configuration stabilises, the sudden switch from one macro-configuration to the other creates a release of the energy summoned for the problem-solving effort. A burst of laughter ensues: it is a type of problem solving in which we experience a ‘Aha, Ha! Moment’. Release Theories are largely dismissed nowadays because of the mental model they posit, ‘based on an outdated hydraulic theory of the mind’,51 and they have a ‘tendency […] to proliferate unwarranted mental entities and/or processes’.52 The critique about ‘outdated theories of the mind’ and ‘unwarranted mental processes’ reminds of the principal critique against Gestalt theory, and specifically against the already-mentioned psychophysical isomorphism.53 The cognitive psychologist Alan J. Parkin sums it up in these words: ‘While Gestalt demonstrations are very powerful, the theory that went along with them 111 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X was rather weak. […] Their principal idea was that of isomorphism, in which a particular Gestalt was thought to set up a corresponding electrical force in the brain which served as the basis for perception’.54 The energy fields that flow in the brain posited by Gestalt’s psychophysical isomorphism might look, to the contemporary eye, quite similar to the ‘hydraulic’ theory of the mind on which the Release Theories are based. THE ‘AHA, HA! MOMENT’ Energy patterns in the brain are triggered and shaped in specific ways by the external stimuli, and the configuration of such energy patterns in our brain determines the configuration in which the stimuli are organised into shapes and objects in our resulting perception. In his ‘maluma/takete’ experiment, Köhler demonstrated that the perception of a ‘Roundness’ Gestalt is share by both a round figure and the non-word ‘maluma’, and the perception of a ‘Sharpness’ Gestalt is shared by a spiky and angular figure and the non-word ‘takete’.55 Both image and sound are perceived as curvy or spiky, respectively. Apparently, certain stimuli, whether visual or aural, activate the same energy patterns in the brain, and such energy patterns produce the same specific perception in either modality. The Gestaltists could never demonstrate this theory convincingly. Yet, the way in which we react to multistable figures seems to suggest that there seems to be some sudden switch in perception that has little or nothing to do with cognition, exactly like an energy field that suddenly changes configuration: when we flip from one configuration to the other, there is like a mental ‘click’ that commutates the configurations. Indeed, some current neurological studies explain the shifts in multistable perception on the ground of ‘perceptual alternations [that] derive from the autonomous oscillations of a circuit within the visual areas’.56 It is not my intention here to defend psychophysical isomorphism or outdated ‘hydraulic’ views of the mind but to submit that it would be perhaps enriching to integrate the Incongruity Theory with the Release Theory, to explain the ‘Aha, Ha! Moment’ as a sort of tickling of our brain induced by the sudden shift of energy fields. I am not the first one to propose such integration. For example, an attempt to reconsider Release Theory from a cognitivist’s perspective was made by Noël Carroll: ‘when presented with an anomaly — such as the punchline of a joke — one is affronted with a challenge, an incongruity which may be appraised as threatening, annoying, in need of a solution, or amusing, the difficulty is removed. From being primed for effort, a sense of effortlessness, ease, and relaxation ensues. Audissino, The Aha, Ha! Moment THE ‘AHA, HA! MOMENT’ An initial intuition that something is being demanded of us disappears, resulting in relief’.57 We do not have to worry about finding a solution for the anomaly because the anomaly is there just for fun, and hence we experience a ‘mental experience of being unburdened cognitively’.58 What I propose here is an approach to audiovisual humour that employs Gestalt Theory not only as an analytical tool but also as a sort of Release Theory, to be integrated to the more diffused approaches based on Cognitivism and Incongruity Theory. As in the case of film music in my previous works, I see Gestalt as a fruitful addition that can add new implements to our film-analysis toolbox. Instead of discarding tools because of their supposed obsolescence or because of current fads in the academe, it is more productive to enlarge the set of tools at our disposal. 112 Audissino, The Aha, Ha! Moment Moreover, the more resources we can mobilise to study humour, the better it is, given humour’s centrality in our lives and its socio-cultural importance. Not dissimilarly to multistable and impossible/unthinkable figures, humour often constitute a challenge not only to our normal understanding of things — ‘to disrupt the heuristics we deploy in everyday life’ and to single out the ‘cognitive bugs’59 — but also to our normal perception of things. Besides procuring us comic amusement, humour has also a central function as a ‘source of social information about the norms that govern the culture we inhabit’ because it ‘alerts us to the relevant social norms and serve to reinforce them. […] In some cases, humour may even function to enforce norms — to serve as a corrective’.60 By contravening the expected results and disrupting the norms, humour highlights those norms and expectations that are often so common that become invisible to us in everyday life. This social function of making us look at everyday life from another perspective is also the key social function of all the arts according to the Russian Formalists. As explained by Kristin Thompson: Art is set apart from the everyday world, in which we use our perception for practical ends. We perceive the world so as to filter from it those elements that are relevant to our immediate actions. […] Films and other artworks, on the contrary, plunge us into a non-practical, playful type of interaction. THE ‘AHA, HA! MOMENT’ They renew our perceptions and other mental processes because they hold no immediate practical implications for us. […] The nature of practical perception means that our faculties become dulled by the repetitive and habitual activities inherent in much of daily life. Thus art, by renewing our perceptions and thoughts, may be said to act as a sort of mental exercise, parallel to the way sports is an exercise for the body.61 As Viktor Shklovsky puts it, ‘as perception becomes habitual, it becomes automatic’ and ‘habitualization devours work, clothes, furniture, one’s wife, and the fear of war. And Art exists that one may recover the sensation of life; it exists to make one feel things, to make the stone stony’.62 What Art does is ‘defamiliarise’ the world for us, making our perception of it ‘roughened’ so that habitualisation and automatisms are removed and we can appreciate the world anew. By aesthetically transforming the ‘materials’ of the world and making fun of them, humour too operates such defamiliarisation and constitutes an indispensable instrument not only for the cognitive consolidation of the societal cultural norms but also to ‘break the glass armour of familiarity’ of our routinised perception. 113 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Notes ic-Wilson, The Musicality of Narrative Film (Basingstoke: Palgrave MacMillan, 2015).ii 7 Danijela Kulezic-Wilson, The Musicality of Narrative Film (Basingstoke: Palgrave MacMillan, 2015). 8 Cognitivism, more than a ‘specific theory’, has been called a ‘program’ for the difficulty to establish a unity of and general consensus on the doctrines at its base: see Currie, 106. 8 Cognitivism, more than a ‘specific theory’, has been called a ‘program’ for the difficulty to establish a unity of and general consensus on the doctrines at its base: see Currie, 106. zsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 5.i ( 9 Kanizsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 5. 10 On fabula and syuzhet in films see David Bordwell Narration in the Fiction Film (Madison WI: Univers 9 Kanizsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 5. 10 On fabula and syuzhet in films, see David Bordwell, Narration in the Fiction Film (Madison, WI: Universit ) Kanizsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 5. 10 On fabula and syuzhet in films, see David Bordwell, Narration in the Fiction Film (Madison, WI: University of Wisconsin Press, 1985), 49–62. 10 On fabula and syuzhet in films, see David Bordwell, Narration in the Fiction Film (Madison, WI: University of Wisconsin Press, 1985), 49–62. 12 Kanizsa, 4. 13 Kevin J. Donnelly, Occult Aesthetics: Synchronization in Sound Film (Oxford and New York: Oxford University Press, 2014), 18–19. 14 Emilio Audissino, Film/Music Analysis. A Film Studies Approach (Basingstoke: Palgrave Macmillan, 2017). 14 Emilio Audissino, Film/Music Analysis. A Film Studies Approach (Basingstoke: Palgrave Macmillan, 2017). 15 Audissino, ‘A Gestalt Approach to the Analysis of Music in Films’, Musicology Research, 2.1 (20 Audissino, A Gestalt Approach to the Analysis of Music in Films , Musicology Research, 2.1 (2017), 69 88. 16 The word ‘gestalt’ is sometimes translated as ‘form’ but I see this as potentially confusing with the more vague concept of ‘form’ that is used in everyday parlance — the outer aspect of something, or the ‘vehicle’ of contents in the form/content-split discourses — or the ‘form’ as used in formalist approaches. I employ the term ‘configuration’ to translate Gestalt’s specific concept of a dynamic process of organisation and reciprocal relation amongst the parts of a system. Notes 1 Noël Carroll, Humour (Oxford and New York: Oxford University Press, 2014), 37. 1 Noël Carroll, Humour (Oxford and New York: Oxford University Press, 2014), 37. 2 For example, Meraj Dhir, ‘A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies ed. by Scott Higgins (New York: Routledge, 2011), 89–106. 2 For example, Meraj Dhir, ‘A Gestalt Approach to Film Analysis’, in Arnheim for Film and Media Studies, ed. by Scott Higgins (New York: Routledge, 2011), 89–106. 3 I would like to thank the editors of this issue and one of the two anonymous peer-reviewers for their constructive criticism and suggestions. 3 I would like to thank the editors of this issue and one of the two anonymous peer-reviewers for their constructive criticism and suggestions. Kanizsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 3. 4 Gaetano Kanizsa, Organization in Vision: Essays on Gestalt Perception (New York: Praeger, 1979), 3. 5 See Gregory Currie, ‘Cognitivism’, in A Companion to Film Theory, ed. by Toby Miller and Robert Stam (Malden, MA: Blackwell, 2004), 105–122. 5 See Gregory Currie, ‘Cognitivism’, in A Companion to Film Theory, ed. by Toby Miller and Robert Stam (Malden, MA: Blackwell, 2004), 105–122. 6 Psychophysical Isomorphism is a central, though controversial, tenet of Gestalt, according to which the energy patterns in the brain configure in such a way that a correspondence exists between the perceptual 6 Psychophysical Isomorphism is a central, though controversial, tenet of Gestalt, according to which the energy patterns in the brain configure in such a way that a correspondence exists between the perceptual phenomena and the mental processes — more on this below. On Psychophysical Isomorphism, see Abraham S. Luchins and Edith H. Luchins. ‘Isomorphism in Gestalt theory: Comparison of Wertheimer’s and Kohler’s concepts’, Gestalt Theory, 21.3 (1999), 208–234. On ‘mirror neurons’ — neurons that fires both when an action is performed and when the same action is merely observed — see Giacomo Rizzolatti, Luciano Fadiga, Leonardo Fogassi and Vittorio Gallese, ‘Resonance Behaviors and Mirror Neurons’, Archives italiennes de biologie, 137.2 (1999), 85–100. The relation between the two is surveyed, for example, in Morris N. Eagle and Jerome C. Wakefield, ‘Gestalt Psychology and the Mirror Neuron Discovery’, Gestalt Theory, 29.1 (2007), 59–64, and Carmelo Calì, ‘Isomorphism and Mirror Neurons’, Gestalt Theory, 29.2 (2007), 168–173. Audissino, The Aha, Ha! Moment 12 Kanizsa, 4. Notes Reidel, 1985); Delia Chiaro, The Language of Jokes: Analyzing Verbal Play (New York: Routledge, 2006).i 24 Carroll defends it as ‘the most fruitful’, after having traced the deficiencies of the competing theories — Carroll, Humour, 8. 25 Norman R. F. Maier, ‘A Gestalt Theory of Humour’, British Journal of Psychology, 23 (1932), 69–74 (69). 25 Norman R. F. Maier, A Gestalt Theory of Humour’, British Journal of Psychology, 23 (1932), 69–74 (69). 26 Ivana Bianchi, Erika Branchini, Carla Canestrari and Roberto Burro, ‘On Pleasures of the Mind Related to Humour and Insight Problem Solving: An Investigation of People’s Awareness of What They Like and Why’, Journal of Cognitive Psychology (2022) <doi: 10.1080/20445911.2022.2047058>. Other articles in the past had already connected insight and problem solving with humour: for example, Ori Amir and others, ‘Ha Ha! Versus Aha! A Direct Comparison of Humor to Nonhumorous Insight for Determining the Neural Correlates of Mirth’, Cerebral Cortex, 25.5 (2015), 1405–1413, and Aaron Kozbelt and Kana Nishioka, ‘Humor Comprehension, Humor Production, and Insight: An Exploratory Study’, Humor — International Journal of Humor Research, 23.3 (2010), 375–401. 27 Steve Neale and Frank Krutnik, Popular Film and Television Comedy (New York and London: Routledge, 1990), 48–51. 28 Carroll, ‘Notes on the Sight Gag’, in Id., Theorizing the Moving Image (Cambridge: Cambridge University Press, 1996), 146. 29 Leonard Maltin, Of Mice and Magic: A History of American Animated Cartoons (New York: New American Library, 1987), 291. 29 Leonard Maltin, Of Mice and Magic: A History of American Animated Cartoons (New York: New American Library, 1987), 291. 30 Giorgio Biancorosso, ‘The Harpist in the Closet: Film Music as Epistemological Joke’, Music and the Moving Image, 2.3 (2009), 11–33. 30 Giorgio Biancorosso, ‘The Harpist in the Closet: Film Music as Epistemological Joke’, Music and the Moving Image, 2.3 (2009), 11–33. 31 The scene is analysed in terms of ‘syn-diegetic perspective’ in Emile Wennekes, ‘Out of Tune? Jazz, Film and The Diegesis’, in Cinema Changes: Incorporations of Jazz in the Film Soundtrack, ed. by Emile Wennekes and Emilio Audissino (Turnhout: Brepols, 2019), 3–18 (12–14). 31 The scene is analysed in terms of ‘syn-diegetic perspective’ in Emile Wennekes, ‘Out of Tune? Jazz, Film and The Diegesis’, in Cinema Changes: Incorporations of Jazz in the Film Soundtrack, ed. by Emile Wennekes and Emilio Audissino (Turnhout: Brepols, 2019), 3–18 (12–14). Notes This distinction is also essential to clarify that I do not claim that the overall form of a film should be equated to the concept of ‘gestalt’: the film has its own formal system (in formalist terms) and to analyse the functions and effects of the devices within the film form, I employ the Gestalt-inspired micro-configuration/macro-configuration method to study the function and effect of the single device at hand. 17 This type of fusion is similar to the ‘widow concept’ discussed by Sergei Eisenstein in The Film Sense, trans. and ed. by Jay Leda (New York: Meridian Books, 1957), 7–8. The Kuleshov effect can also be interpreted as two micro-configurations (Close-Up of Mozhukhin observing something x Detail Shot of the observed object/person) producing a novel macro-configuration: Mozhukhin is hungry; Mozhukhin is mournful; Mozhukhin is lustful. On the Kuleshov Effect, see Kuleshov on Film: Writings of Lev Kuleshov ed. by Ronald Levaco (Berkeley and Los Angeles: University of California Press, 1974), 200. y g y Kellogg, Fundamentals of Cognitive Psychology (Los Angeles and London: Sage, 2012), 246. 18 Ronald T. Kellogg, Fundamentals of Cognitive Psychology (Los Angeles and London: Sage, 2012), 246. 19 J. Burton Cunningham and James N. MacGregor, ‘Productive and Re-productive Thinking in Solving Insight Problems’, Journal of Creative Behavior, 48 (2014), 44–63. While they are often considered synonyms in the post-structuralist or culturalist approaches, where films are ‘read’, I distinguish ‘film analysis’ — which also considers ‘film comprehension’ — from ‘film interpretation’: ‘interpretation is only one part of 18 Ronald T. Kellogg, Fundamentals of Cognitive Psychology (Los Angeles and London: Sage, 2012), 246. 19 J. Burton Cunningham and James N. MacGregor, ‘Productive and Re-productive Thinking in Solving Insight Problems’, Journal of Creative Behavior, 48 (2014), 44–63. While they are often considered synonyms in the post-structuralist or culturalist approaches, where films are ‘read’, I distinguish ‘film analysis’ — which also considers ‘film comprehension’ — from ‘film interpretation’: ‘interpretation is only one part of 114 Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Moment analysis’, Kristin Thompson, Breaking the Glass Armor: Neoformalist Film Analysis (Princeton: Princeton University Press, 1988), 34n25. 20 Wolfgang Köhler, The Task of Gestalt Psychology (Princeton: Princeton University Press, 1969), 143– 144, 147–153. 21 Carroll, Humour, 4. , , 22 John Morreall, Comic Relief: A Comprehensive Philosophy of Humor (Chichester: Wiley-Blackwell, 2009), 10–11. 23 Victor Raskin, Semantic Mechanisms of Humor (Dordrecht, Boston and Lancaster: D. Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 37 Ibidem, 41–45. Notes 32 Philipp Sterzer, Andreas Kleinschmidt and Geraint Rees, ‘The Neural Bases of Multistable Perception’, Trends in Cognitive Sciences, 13.7 (2009), 310–318 (310). 32 Philipp Sterzer, Andreas Kleinschmidt and Geraint Rees, ‘The Neural Bases of Multistable Perception’, Trends in Cognitive Sciences, 13.7 (2009), 310–318 (310). 33 Andrew J. Parker and Kristine Krug, ‘Neuronal Mechanisms for the Perception of Ambiguous Stimuli’, Current Opinion in Neurobiology, 13.4 (2003), 433–439 (433). 33 Andrew J. Parker and Kristine Krug, ‘Neuronal Mechanisms for the Perception of Ambiguous Stimuli’, Current Opinion in Neurobiology, 13.4 (2003), 433–439 (433). 34 Reproduced in Benjamin B. Lahey, Psychology: An Introduction (New York: McGraw-Hill, 201 35 K i 21 22 35 Kanizsa, 21–22. 36 Ibidem, 41. 38 Multistability has been employed elsewhere to study verbal and visual comedy: for example, respectively, Daniel Kjellander, ‘Gold Punning: Studying Multistable Meaning Structures Using a Systematically Collected Set of Lexical Blends’, Lexis Journal in English Lexicology, 14 (2019), 1–28; Karin Kukkonen, ‘Adventures in Duck-Rabbitry: Multistable Elements of Graphic Narrative’, Narrative, 25.3 (2017), 342–358. 39 On Zucker-Abrahams-Zucker, see Audissino, ‘New Hollywood’s “Zany Godards”: A “Shirley” Serious Assessment of Zucker-Abrahams-Zucker’, in New Wave, New Hollywood: Gender, Reassessment, Recovery and Legacy, ed. by Nathan Abrams and Gregory Frame (New York: Bloomsbury Academic, 2021), 79–100, and Audissino, ‘Police Squad!. The Zucker-Abrahams-Zucker Style VS the Substance of Traditional Television’, in Substance/Style: Moments in Television, ed. by Sarah Cardwell, Lucy Donaldson and Jonathan Bignell (Manchester: Manchester University Press, 2022), 178–201. 41 Of course, one might argue that, since we are dealing with a Marx Brothers film, expectations are oriented towards abnormality rather than normality, so the appearance of the sea seal might not be entirely unexpected. Nevertheless, the humorous effect is produced precisely because the normal amodal completion of the event constitutes the term of reference that anchors our perception of abnormality in the 115 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X deviant development of the scene. 55 Köhler, Gestalt Psychology: The Definitive Statement of the Gestalt Theory (New York: Liveright, 1947), 224–225. 56 David A. Leopold and Nikos K. Logothetis, ‘Multistable Phenomena: Changing Views in Perception’, Trends in Cognitive Sciences, 3.7 (1999), 254–264 (254). Rhythm Beyond the Cinematic Medium/ The Pixel Beyond the Movie Theatre1 Sharon Jane Mee, University of New South Wales Keywords Rhythm Shimmer Pixel Ethics Digital Hostility DOI 10.54103/ 2036-461X/17923 Gregory J. Seigworth and Melissa Gregg write about Roland Barthes’s splendid notion of ‘shimmer’: an ‘exhaustively nuanced space’ that may be inventoried as patho-logies (by which to contemplate pathos) of bodies (human and nonhuman). In Alex Garland’s 2018 film Annihilation, a refracting effect — the Shimmer — which has appeared around a lighthouse and is slowly spreading outwards, is being studied. A group of female scientists enter the Shimmer and begin to inventory the strange organic duplicates of form within it. These organic structures, while extraordinarily nuanced, are also patho- logies of organic life as they are refracted by the Shimmer. This article will consider the ‘exhaustively nuanced space’ of cinema and its patho-logies via the conditions of the rhythm of the pixel in cinema, and beyond, in social media. In an examination of the rhythm of the pixel beyond the cinematic medium, I consider the energetic ‘becoming’ of the spectator/operator and the digital image (text and image in social media) as they act in relation. In an examination of the rhythm of the pixel beyond the movie theatre, I consider the infinite intensities in the aisthetic encounter of body and text/image in social media and its correlation to the politics of a mass-art. Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License censed under a Creative Commons Attribution 4.0 International License An ‘exhaustively nuanced space’ is the way Roland Barthes describes ‘shimmer’ in his series of published lectures, The Neutral: Lecture Course at the Collège de France (1977–1978).2 In The Affect Theory Reader, Gregory J. Seigworth and Melissa Gregg outline the affects that would constitute an ‘inventory of shimmers’ in ‘neutrally inflected, immanent pathos or “patho-logy”’.3 In Alex Garland’s 2018 film Annihilation, the Shimmer is a refracting effect that causes the area around a lighthouse to become populated with organic duplicates of existing organic structures. The Shimmer in Annihilation allows for a way of thinking about the shimmering energetic plane in and of the digital image. My interest is in how ‘shimmer’ as an ‘extreme changeability of affective moments, a rapid modification’4 characterizes an aisthetic, as well as ethical, encounter with the image, and how such an encounter may be considered in spectator/ operator interactions with media texts beyond cinema. deviant development of the scene. 42 ZAZ and Tati are compared in Audissino, ‘Police Squad!’, 186–187. 43 Bordwell, 306. 44 Thompson, 251. p , 45 The comedic mechanics of non-sensical accumulation are exemplified in Audissino, ‘New Hollywood’s “Zany Godards”’, 86. 46 Ibidem 95 45 The comedic mechanics of non-sensical accumulation are exemplified in Audissino, ‘New Hollywood’s “Zany Godards”’, 86. 46 Ibidem 95 46 Ibidem, 95. Ibidem, 95. 47 Fiona M. C. Dorward and Ross H. Day, ‘Loss of 3-D Shape Constancy in Interior Spaces: The Basis of the Ames-Room Illusion’, Perception, 26.6 (1997), 707–718. 48 M ll 16 47 Fiona M. C. Dorward and Ross H. Day, ‘Loss of 3-D Shape Constancy in Interior Spaces: The Basis of the Ames-Room Illusion’, Perception, 26.6 (1997), 707–718. 48 Morreall, 16. 49 Carroll, Humour, 36. 50 Quoted in Morreall, 17. 51 Morreall, 23. 52 Carroll, Humour, 41. , , 53 Some of the misunderstandings of Gestalt Theory are discussed in Riccardo Luccio, ‘Gestalt Psychology and Cognitive Psychology’, Humana Mente, 17 (July 2011), 67–128. 53 Some of the misunderstandings of Gestalt Theory are discussed in Riccardo Lucc and Cognitive Psychology’, Humana Mente, 17 (July 2011), 67–128. g y gy y 54 Alan J. Parkin, Essential Cognitive Psychology (Hove and New York: Psychology Press, 2000), 7.i 54 Alan J. Parkin, Essential Cognitive Psychology (Hove and New York: Psychology Press, 2000), 7. 55 Köhler, Gestalt Psychology: The Definitive Statement of the Gestalt Theory (New York: Liveright, 1947), 224–225. g y gy y gy 55 Köhler, Gestalt Psychology: The Definitive Statement of the Gestalt Theory (New York: Liveright, 1947), 224–225. 56 David A. Leopold and Nikos K. Logothetis, ‘Multistable Phenomena: Changing Views in Perception’, Trends in Cognitive Sciences, 3.7 (1999), 254–264 (254). 57 Carroll, Humour, 41. 58 Ibidem. 59 Ibidem, 70. 61 Thompson, 8–9. 62 Quoted in ibidem, 10. 116 Audissino, The Aha, Ha! Moment Audissino, The Aha, Ha! Moment Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Rhythm Beyond the Cinematic Medium/ The Pixel Beyond the Movie Theatre1 Sharon Jane Mee, University of New South Wales With the rise of nationalist movements and authoritarian governments, and digital hostility in social media, a return to the study of the sensory and sensuous body is more important than ever, for in our aisthetic encounters 121 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X we may apprehend an ethics beyond a politics of inadequate ideas. Referring to Gilles Deleuze’s citation of the Second World War as ‘a violent encounter to thought’, Nadine Boljkovak, in her book Untimely Affects: Gilles Deleuze and an Ethics of Cinema, points to Deleuze’s contention that the ‘war machine’, beyond its violence, comprises ‘revolutionary movements’ such as are found in art’s creative invention and resistances.5 Thinking about affect’s potential — and the creativity and resistance that it inspires — this article seeks to examine social media text and image through infinite intensities by which we may recognize the conditions for a patho-logical interaction in this ‘exhaustively nuanced space’.6 p g y p This article will consider relations of affect in the development of cinema to the digital image and beyond, in social media, via the rhythm of the pixel. In a consideration of the pixel beyond the movie theatre, that is, in the relocation of the pixel from cinema to social media, it is necessary to recognize the affective and energetic relations that exist between social media text and image and social media operator. The rhythm of the pixel in both cinema and social media generates an energetic relation between the media text and the spectator/ operator. I want to consider this energetic relation as an ethical one through the media text and the spectator/operator’s ‘capacities to affect and be affected’.7 To think about relations of affect in the rhythm of the pixel beyond the cinematic medium, I first establish what this means for cinema. I analyse Annihilation to understand the affective and energetic force of the pixel in cinema that acts in relation. To consider the rhythm of the pixel beyond the cinematic medium is to consider an affective acting in relation. Such an acting in relation is an energetic exchange between media text and spectator/operator in a mutual ‘becoming’.8 In my consideration of the place of affect in these encounters, my intention is to locate an ethics of care, compassion, and empathy in our engagement with cinema and beyond, in social media. Mee, Rhythm Beyond the Cinematic Medium ANNIHILATION I think as she was dying, part of her mind became part of the creature that was killing her.’ In consideration of the refraction of DNA through the medium of the Shimmer in Annihilation, this article will examine the refraction of affect in cinema and beyond, in social media, via the ‘exhaustively nuanced space’ of what Barthes calls ‘shimmer’.9 What is also refracted through this ‘exhaustively nuanced space’ is the (human and nonhuman) patho-logies of our digital interactions by which media text and spectator/operator engage in a mutual ‘becoming’.10 AESTHETICS AND ETHICS The aesthetics, and even ethics, of the digital image (text and image in social Fig. 1: The Shimmer in Annihilation (Alex Garland, 2018) Fig. 1: The Shimmer in Annihilation (Alex Garland, 2018) Fig. 1: The Shimmer in Annihilation (Alex Garland, 2018) mutations were subtle at first. More extreme as we grew closer to the lighthouse. Corruptions of form, duplicates of form.’ Lomax: ‘Duplicates?’ Lena: ‘Echoes.’ Lomax: ‘Is it possible these were hallucinations?’ Lena: ‘I wondered that myself, but they were shared among all of us. It was dream-like.’ Lomax: ‘Nightmarish?’ Lena: ‘Not always. Sometimes it was beautiful.’ However, it is Josie who more fully explains the Shimmer and the organic structures that are present within it when she says to Lena: ‘The Shimmer is a prism, but it refracts everything, not just light and radio waves, animal DNA, plant DNA, all DNA.’ The refracting effect of the Shimmer is observed when Cass is taken by a bear-like creature and after she is found dead, the creature returns with a growl that sounds like Cass’s death cries. Josie reflects upon it: ‘It was so strange hearing Sheppard’s voice in the mouth of that creature last night. I think as she was dying, part of her mind became part of the creature that was killing her.’ In consideration of the refraction of DNA through the medium of the Shimmer in Annihilation, this article will examine the refraction of affect in cinema and beyond, in social media, via the ‘exhaustively nuanced space’ of what Barthes calls ‘shimmer’.9 What is also refracted through this ‘exhaustively nuanced space’ is the (human and nonhuman) patho-logies of our digital interactions by which media text and spectator/operator engage in a mutual ‘becoming’.10 ANNIHILATION In Garland’s Annihilation, Lena (Natalie Portman) is an academic who works in biology at John Hopkins University. After her husband, Kane (Oscar Isaac), is quarantined at a science facility after a military mission, Lena decides to join the next mission into what is called the Shimmer (fig. 1). The mission is to reach the purported source of the Shimmer — the lighthouse — enter the lighthouse, acquire data, and return. As Dr Ventress (Jennifer Jason Leigh) explains to Lena, the Shimmer is: ‘A religious event, an extra-terrestrial event, a higher dimension. We have many theories, few facts.’ Dr Ventress’s party consists of five women, all scientists — herself, Cass Sheppard (Tuva Novotny), Anya Thorensen (Gina Rodriguez), Josie Radek (Tessa Thompson), and Lena. Once inside the Shimmer, the group of women find mutated organic structures — flowers, plants, and animals. The Shimmer provides the conditions and process (is the patho-logy) for the blooming of life. Lena explains the organic structures to Lomax (Benedict Wong) at the science facility on her return: ‘The 122 Mee, Rhythm Beyond the Cinematic Medium mutations were subtle at first. More extreme as we grew closer to the lighthouse. Corruptions of form, duplicates of form.’ Lomax: ‘Duplicates?’ Lena: ‘Echoes.’ Lomax: ‘Is it possible these were hallucinations?’ Lena: ‘I wondered that myself, but they were shared among all of us. It was dream-like.’ Lomax: ‘Nightmarish?’ Lena: ‘Not always. Sometimes it was beautiful.’ However, it is Josie who more fully explains the Shimmer and the organic structures that are present within it when she says to Lena: ‘The Shimmer is a prism, but it refracts everything, not just light and radio waves, animal DNA, plant DNA, all DNA.’ The refracting effect of the Shimmer is observed when Cass is taken by a bear-like creature and after she is found dead, the creature returns with a growl that sounds like Cass’s death cries. Josie reflects upon it: ‘It was so strange hearing Sheppard’s voice in the mouth of that creature last night. AESTHETICS AND ETHICS The aesthetics, and even ethics, of the digital image (text and image in social media) — as an affective and energetic force — can be found in the organic rhythms of life. In an early scene in the film, Lena describes the evolution of a cell/of all life to her classroom of students: This is a cell. Like all cells it is born from an existing cell. By extension, all cells were ultimately born from one cell. A simple This is a cell. Like all cells it is born from an existing cell. By extension, all cells were ultimately born from one cell. A simple 123 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X organism alone on Planet Earth, perhaps alone in the universe. About 4 billion years ago, one became two, two became four. Then 8, 16, 32. The rhythm of the dividing pair, which becomes the structure of every micro blade of grass, sea creature, plant creature, and human. The structure of everything that lives and everything that dies. […] The cell we are looking at is from a tumour. The rhythm of the dividing pair is an organic rhythm (fig. 2). Thus, the rhythm of the dividing pair has a ‘thisness’ found in relations of movement and affect. Such a ‘thisness’ is the way Deleuze and Félix Guattari describe a body as a ‘mode of individuation very different from that of a person, subject, thing, or substance’.11 Deleuze and Guattari write: ‘They are haecceities in the sense that they consist entirely of relations of movement and rest between molecules or particles, capacities to affect and be affected.’12 Thus, a body, for Deleuze and Guattari, is defined by haecceities: A body is not defined by the form that determines it nor as a determinate substance or subject nor by the organs it possesses or the functions it fulfils. AESTHETICS AND ETHICS On the plane of consistency, a body is defined only by a longitude and a latitude: in other words, the sum total of the material elements belonging to it under given relations of movement and rest, speed and slowness (longitude); the sum total of the intensive affects it is capable of at a given power or degree of potential (latitude).13 The organic structures in the Shimmer are mutations of form precisely because they are first of all a relation: the refraction of ‘animal DNA, plant DNA, all DNA’. The voice of the bear-like creature is the sign of a relation and an intensive affect having taken place: the sonic intensities of Cass’s death-cries have affected the bear-like creature in the same moment that Cass has been affected by the bear-like creature, which has also resulted in her death. Equally, it could be said that the rhythm of the pixel has a ‘thisness’ found in relations of 124 Mee, Rhythm Beyond the Cinematic Medium Fig. 2: The rhythm of the dividing pair in Annihilation (Alex Garland, 2018) Fig. 2: The rhythm of the dividing pair in Annihilation (Alex Garland, 2018) Fig. 2: The rhythm of the dividing pair in Annihilation (Alex Garland, 2018) Fig. 2: The rhythm of the dividing pair in Annihilation (Alex Garland, 2018) 124 Mee, Rhythm Beyond the Cinematic Medium 124 Mee, Rhythm Beyond the Cinematic Medium movement and affect. The intensities of the digital image affect the spectator/ operator, and the expression of the affect in the spectator/operator is the sign of a relation and an intensive affect having taken place. For cinema, ‘capacities to affect and be affected’ are found in the ‘automatic movement’ of the movement- image.14 As Deleuze writes: It is only when movement becomes automatic that the artistic essence of the image is realized: producing a shock to thought, communicating vibrations to the cortex, touching the nervous and cerebral system directly.15 Thus, what is found in the ‘automatic movement’ of cinema — in the vibrations of movement that ‘touch[…] the nervous and cerebral system directly’16 — is a communicating ripple of affective intensities ‘producing a shock to thought’.17 Deleuze gives as examples the work of Dziga Vertov, Sergei Eisenstein, Abel Gance, and Élie Faure. AESTHETICS AND ETHICS 38 2022 · ISSN 2036-461X Kurosawa’s Cobweb Castle (also known as Throne of Blood, 1957).28 Such an encounter is an aisthetic encounter: that is, an encounter with sensation prior to our recognition of it, where subject and object are instead engaged in a mutual ‘becoming’.29 As Boljkovak writes: ‘This shattering of stable constructions by force enables intensive perception and new approaches to life, seeing and being.’30 Haecceities of ‘movement and rest, speed and slowness’ while ‘unthinkable in thought’ ‘touch[…] the nervous and cerebral system directly’.31 In the aisthetic encounter, a body — defined not by ‘form’, ‘substance or subject’ — has the potential for ‘becoming-intense, becoming-animal, becoming-imperceptible’.32 In Annihilation, the voice of a bear becomes Cass’s voice, leaves grow from the skin of Josie’s arms and as she disappears amongst a stand of human- shaped trees we understand that she becomes a tree, and energy pours from Dr Ventress’s mouth until she becomes the imperceptible energy that swirls around Lena to form a throbbing vortex. Kurosawa’s Cobweb Castle (also known as Throne of Blood, 1957).28 Such an encounter is an aisthetic encounter: that is, an encounter with sensation prior to our recognition of it, where subject and object are instead engaged in a mutual ‘becoming’.29 As Boljkovak writes: ‘This shattering of stable constructions by force enables intensive perception and new approaches to life, seeing and being.’30 Haecceities of ‘movement and rest, speed and slowness’ while ‘unthinkable in thought’ ‘touch[…] the nervous and cerebral system directly’.31 In the aisthetic encounter, a body — defined not by ‘form’, ‘substance or subject’ — has the potential for ‘becoming-intense, becoming-animal, becoming-imperceptible’.32 In Annihilation, the voice of a bear becomes Cass’s voice, leaves grow from the skin of Josie’s arms and as she disappears amongst a stand of human- shaped trees we understand that she becomes a tree, and energy pours from Dr Ventress’s mouth until she becomes the imperceptible energy that swirls around Lena to form a throbbing vortex. Cinema provides a valuable resource for challenging ways of thinking. Aisthetic encounters with media texts, bodies, characters, and social and cultural situations, can provoke consideration of how we can engage ethically with others and the world. AESTHETICS AND ETHICS In Expressionism in Philosophy: Spinoza, Deleuze invokes what Baruch Spinoza says in his book, Ethics: ‘We do not even know of what a body is capable.’33 In consideration of ‘intrinsic determinations’ such as intensities of colour,34 we can begin to understand an ethics whereby the expression of a body may affect other bodies. While the rhythm of the dividing pair sets ‘intrinsic determinations’ or ‘intensive qualities’ for a body that may be affected by other bodies/the world,35 it also announces the potential whereby the expression of a body may also affect other bodies. According to Spinoza’s Ethics, it is in the expression of, and relations between, bodies where communities are formed. As Bruce Baugh writes, ‘a community or an association, corresponds to a collective power of being affected, and results in collective or communal affects’.36 It is through intensities of colours and the qualities, sounds and textures of moving bodies, that cinema can express the power that bodies have to affect other bodies, and also, an ethical engagement with others and the world. My article seeks to understand ‘communal affects’ through intensity’s difference in cinema and beyond, in social media.37 AESTHETICS AND ETHICS In Vertov’s 1929 film Man with a Movie Camera, the use of shot and montage constructs a ‘rhythmic montage’.18 The operation of Eisenstein’s montage is not simply one of the ‘communication of movement in images’, but of the development of montage ‘from the image to thought’.19 Montage has the capacity to produce a ‘shock to thought’, which gives rise to what Deleuze calls the ‘spiritual automaton’ in the spectator.20 The ‘spiritual automaton’ does not come about through ‘logical or abstract’ thought by ‘formally deducing thoughts from each other’ to think ‘determinate substance or subject’.21 Just as a body has ‘capacities to affect and be affected’,22 ‘[a]utomatic movement gives rise to a spiritual automaton in us, which reacts in turn on movement’.23 That is, the ‘spiritual automaton’ comes about in ‘the circuit into which they [the spectator] enter with the movement-image, the shared power of what forces thinking and what thinks under the shock; a nooshock’.24 The intensive vibrations of the movement-image ripple outwards, causing change, like the rippling outwards of the Shimmer’s refractions that is causing, as Lena says, ‘giant waves in the gene pool’. The ‘shock to thought’ of the movement-image can be conceived as an encounter with energetic and material qualities in the image/world that ‘gives rise to sensibility’.25 As Deleuze writes in Difference and Repetition: Something in the world forces us to think. This something is an object not of recognition but of a fundamental encounter. […] The object of encounter […] really gives rise to sensibility with regard to a given sense. It is not an aisthêton but an aisthêteon. It is not a quality but a sign. It is not a sensible being but the being of the sensible. It is not the given but that by which the given is given. It is therefore in a certain sense the imperceptible [insensible].26 When ‘something in the world forces us to think’ it is to think the ‘unthinkable in thought’.27 That is, it is a ‘thinking’ in haecceities — an encounter with the sensible, and even imperceptible — ‘the grey, the steam and the mist’ in Akira 125 Cinéma & Cie vol. 22 no. THE AISTHETIC ENCOUNTER AND MASS-ART How do we understand the aisthetic encounter that attends mass-art as entertainment? In this article, the convergence is represented by the affective force of cinema in communication with its spectators via a sensory manifold. I contend that the image and the spectator are components in the flow of energy and are simultaneously invested: in the circuit of the movement-image, the image and the spectator each have ‘capacities to affect and be affected’.38 And yet, precisely because of this affective relation between the spectator and the image, mass spectatorship retains heterogeneity. As Deleuze writes: ‘What 126 Mee, Rhythm Beyond the Cinematic Medium theatre and especially opera had unsuccessfully attempted, cinema achieves (Battleship Potemkin, October): to reach the Dividual, that is, to individuate a mass as such, instead of leaving it in a qualitative homogeneity or reducing it to a quantitative divisibility.’39 The aisthetic encounter with the image is a heterogeneous experience. The viewer of cinema thinks and feels intensities in a particular way. As Deleuze notes in Expressionism in Philosophy: ‘Intensive quantity is infinite, and the system of essences an actually infinite series. We are here dealing with infinity “through a cause.”’40 On the other hand, the capacity of bodies ‘to be affected’ can only be in ‘a very great number of ways’.41 Thus, the particularity of intensities thought and felt by the viewer is of intensities in the aisthetic encounter — the event as ‘cause’ — for spectators of mass art. In light of these intensive quantities, we may understand the cinematic event as ‘cause’ for the conditions, or patho-logies, that arise in the encounter between body and image/world. THE AISTHETIC ENCOUNTER AND MASS-ART body in its affective ‘becoming’.49 In Annihilation, the ‘transition into something else’ of the body in its affective ‘becoming’ is even an annihilation of the self. CINEMA BEYOND THE CINEMATIC MEDIUM: THE SHIMMER Beyond the cinematic medium of celluloid, ‘shimmer’ characterizes the pixel in digital cinema. In The Cinema Effect, Sean Cubitt writes of the pixel: ‘Movement starts in non-identity, the unstable zero pixel at origin.’50 In Annihilation, the Shimmer refracts; that is, the Shimmer is the deflection of light waves, radio waves etc. as it passes through different mediums.51 In his series of lectures published as The Neutral, Barthes writes of the ‘shimmer’ as an ‘exhaustively nuanced space’ ‘whose aspect, perhaps whose meaning, is subtly modified according to the angle of the subject’s gaze’.52 As a ‘conjunction of intellect and affect’, Barthes further writes about the ‘shimmer’ that it is a ‘hyperconsciousness of the affective minimum, of the microscopic fragment of emotion […] which implies an extreme changeability of affective moments, a rapid modification, into shimmer’.53 Seigworth and Gregg develop Barthes’s notion of shimmer in ‘An Inventory of Shimmers’: ‘It becomes then a matter of accounting for the progressive accentuation (plus/minus) of intensities, their incremental shimmer: the stretching of process underway, not position taken.’54 Like the ‘shimmer’ as ‘process’ rather than ‘position taken’, the pixel ‘starts in non-identity’.55 For Cubitt, ‘pixels are temporal, not spatial. That cinematic present, like the point of origin of graphs, can be given a number: zero. Zero is not a quantity so much as a relation.’56 It is the energetic relation between the image and the viewer that I am interested by which we can describe the digital image. I am particularly interested in the way the energy of the pixel — as a ‘stretching of process underway’ — acts in relation.57 Rosalind E. Krauss characterizes the energetic renewal of each pixel that makes up the image of the television set as the electric ‘pulse’ of the (analogue) televisual image.58 Seigworth and Gregg’s description of ‘shimmer’ as a ‘progressive accentuation (plus/minus) of intensities’ is much like the on/off of pixels that Krauss describes, where plus/minus determines the progress of pulsing intensity and its relation.59 The digital image, composed of moving (vibrating) and energetic pixels and sound, has the force of an energetic field. The digital image does not have the indexical recuperability of the photographic image, rather, in its moving pixel terrain, it has the behaviour and expression of an opening out of the energetic plane. Garland’s film Annihilation demonstrates the force of intensity that opens out the body to the image/world. THE AISTHETIC ENCOUNTER AND MASS-ART According to Seigworth and Gregg in their introduction to The Affect Theory Reader, patho-logies are accounted for in an ‘inventory of shimmers’: What should follow as critical practice, Barthes argued, is a neutrally inflected, immanent pathos or “patho-logy” that would be an “inventory of shimmers, of nuances, of states, of changes (pathè)” as they gather into “affectivity, sensibility, sentiment,” and come to serve as “the passion for difference.” 42 In this ‘exhaustively nuanced space’ ‘of shimmers, of nuances, of states, of changes (pathè)’ as they are inventoried, we may further consider the viewer’s relation to — and the patho-logies that would be an inventory of — the shimmers, nuances, states, and changes of the pixel in the digital image.43 And yet, in thinking about cinema as the ‘cause’ for infinite patho-logies, Eisenstein’s work on pathos in cinema suggests something more about the empathic bond that is formed between the film and the spectator. Eisenstein notes that this bond of pathos is not one of mimesis, that is, of ‘impelling the spectator to reproduce the perceived action, outwardly’.44 Rather, Eisenstein suggests that ‘the affect of a work of pathos consists in whatever “sends” the spectator into ecstasy […] ex-stasis — literally, “standing out of oneself”, which is to say, “going out of himself”, or “departing from his ordinary condition”’.45 In this sense, ex-stasis in cinema, for Eisenstein, implies more than a patho-logy in the ‘departure from a condition’.46 As Eisenstein writes: ‘To go out of oneself inevitably implies a transition into something else, to something different in quality, to something opposite to what was.’47 In Annihilation, the refraction of Lena’s DNA for the formation of the Lena double (Kristen McGarrity) implies a transition of Lena out of herself. The refractions caused by the Shimmer are a ‘breaking up’, deflection or a ‘change in direction’ of DNA as in the Oxford English and Spanish Dictionary definition of refraction: ‘light, radio waves, etc. being deflected in passing obliquely through the interface between one medium and another or through a medium of varying density’.48 The ‘transition into something else’, it could also be said, is a ‘power or degree of potential’ of the 127 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X body in its affective ‘becoming’.49 In Annihilation, the ‘transition into something else’ of the body in its affective ‘becoming’ is even an annihilation of the self. CINEMA BEYOND THE CINEMATIC MEDIUM: THE SHIMMER When Lena enters the lighthouse, she finds a deep pit surrounded by a coral-like webbing and makes her way inside. She finds Dr Ventress in a cave at the bottom of the pit. As Dr Ventress says to Lena before energy begins to pour from her open mouth: ‘Our bodies and our minds will be 128 Mee, Rhythm Beyond the Cinematic Medium fragmented into their smallest parts until not one part remains: annihilation.’ fragmented into their smallest parts until not one part remains: annihilation.’ Dr Ventress’s body disperses in a cloud of swirling energy and, as the energy swirls around Lena, it forms a vortex that opens like an eye in front of Lena (fig. Dr Ventress’s body disperses in a cloud of swirling energy and, as the energy swirls around Lena, it forms a vortex that opens like an eye in front of Lena (fig. Dr Ventress’s body disperses in a cloud of swirling energy and, as the energy swirls around Lena, it forms a vortex that opens like an eye in front of Lena (fig. Dr Ventress’s body disperses in a cloud of swirling energy and, as the energy swirls around Lena, it forms a vortex that opens like an eye in front of Lena (fig. 3). The vortex is composed of throbbing energetic and material particles. The throbbing vortex draws a drop of blood from Lena’s eye, and, within the opening of the vortex, the blood cells divide and multiply until a Lena double is formed. 3). The vortex is composed of throbbing energetic and material particles. The throbbing vortex draws a drop of blood from Lena’s eye, and, within the opening of the vortex, the blood cells divide and multiply until a Lena double is formed. Cinema is found in these throbbing energetic and material particles. In a 1927 issue of Close Up, the poet and cineaste H. D. refers to cinema as having the therapeutic powers of a mind cure.60 H. D. describes the experience of cinema spectatorship: ‘We depended on light, on some sub-strata of warmth, some pulse or vibration [...] We sank into this pulse and warmth and were recreated.’61 Cinema is found in these throbbing energetic and material particles. In a 1927 issue of Close Up, the poet and cineaste H. D. refers to cinema as having the therapeutic powers of a mind cure.60 H. D. CINEMA BEYOND THE CINEMATIC MEDIUM: THE SHIMMER describes the experience of cinema spectatorship: ‘We depended on light, on some sub-strata of warmth, some pulse or vibration [...] We sank into this pulse and warmth and were recreated.’61 The encounter with cinema is an encounter with vibrations of energy — as light — that ‘recreates’, which we can see extending, for H. D., from the nineteenth- century belief in the restorative powers of electricity. It is interesting to note the similarities in the mind cures alluded to in H. D.’s description of cinema as ‘pulse or vibration’ and what Deleuze says about the vibrations of ‘automatic movement’ that ‘touch[…] the nervous and cerebral system directly’.62 The behaviour and expression of an opening out of an energetic plane in cinema and beyond, in the digital image, coincides with what Seigworth and Gregg call a ‘bloom-space’ for ‘affectivity, sensibility, sentiment’.63 They write: ‘In fact, as much as anything, perhaps that is what such a “neutral” bloom-space offers: the patho-logy of a body intersecting with the pedagogy of an affective world.’64 What is found in the patho-logy of a body in its intersection with the world, indeed, at the intersection of spectator/operator and media text, is, as Barthes writes, ‘the passion for difference’,65 or what Deleuze calls ‘infinity “through a cause”’.66 Thinking about cinema beyond the cinematic medium of celluloid is a thinking about event as opposed to narrative.67 The digital event is a relation. Cubitt writes: ‘The verb “relates”, however, should be understood to mean “establishes Fig. 3: The throbbing vortex of energetic and material particles in Annihilation (Alex Garland, 2018) 129 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X a relationship”, not as “tells a story.”’68 Working within the digital event as a relation, the encounter with the digital image is an encounter with the energy of the pixel. The pixels that form the throbbing vortex in Annihilation, alongside the pulsing electronic soundscape takes cinema beyond the cinematic medium to something like a music video. As Cubitt writes: ‘It is important to recognize that narrative is neither primary nor necessary to cinema, and it forms no part of any putative essence of the medium.’69 The importance of the digital event as a medium is of relation and not of story. In Annihilation, the Shimmer refracts. CINEMA BEYOND THE CINEMATIC MEDIUM: THE SHIMMER Like the energy of the pixel that acts in relation, what is found inside the Shimmer is an acting in relation via duplicates of form and refractions of movement and sound. The Shimmer is a reification of Jenelle Troxell’s explanation of how Henry Wood’s 1893 manual Ideal Suggestion through Mental Photography: A Restorative System for Home and Private Use describes: ‘invisible threads, which connect us with each object which makes up our environment. Vibrations are ever passing over these connections, backward and forward, and it is for us to control their purpose and quality.’70 Like the vibrations of ‘automatic movement’ that ‘touch[…] the nervous and cerebral system directly’,71 the ‘invisible threads, which connect’ in Annihilation are of movement, force, and pressure. When a Lena double is formed in the pit beneath the lighthouse, Lena runs for the door. The Lena double in mirroring her movements, produces a corresponding pressure against the door, such that Lena is pressed between the door and the Lena double. It is only when Lena releases her own pressure against the door and falls that the Lena double falls with her. However, this acting in relation is also what leads to the Lena double’s demise. In corresponding movement with the Lena double, Lena places a grenade between their hands — a gift given — and pulls the pin with her thumb. She runs as it explodes. Fire engulfs the Lena double. As the lighthouse catches alight, the Shimmer surrounding the lighthouse dissipates and the organic structures begin to collapse. THE PIXEL BEYOND THE MOVIE THEATRE With the rise of nationalist movements and authoritarian governments that give way to digital hostility in social media, we can begin to consider the energetic power of the pixel beyond the movie theatre. Indeed, in 1985 in Cinema 2: The Time-Image (translated into English in 1989) Deleuze identified the effects of the rise of nationalist movements in the treatment of the cinema spectator: Cinema is dying, then, from its quantitative mediocrity. But there is a still more important reason: the mass-art, the treatment of the masses, which should not have been separable from an accession of the masses to the status of the true subject, has degenerated into state propaganda and manipulation, into a kind of fascism which brought together Hitler and Hollywood, Hollywood and Hitler.72 130 Mee, Rhythm Beyond the Cinematic Medium How might the effects of cinema as mass-art be aligned with social media? What is interesting to consider is the ways that social media may be aligned with ‘bad cinema’, which, according to Deleuze, represents violence to produce shock. Deleuze writes: ‘The shock would be confused, in bad cinema, with the figurative violence of the represented instead of achieving that other violence of a movement-image developing its vibrations in a moving sequence which embeds itself within us.’73 We could, however, ask of Deleuze what happens when the ‘figurative violence of the represented’ and the ‘violence of a movement- image’ are both at once in the image? 74 What happens when the ‘violence of a movement-image’ — the vibrations of the energetic pixel ‘touching the nervous and cerebral system directly’ — is the affect of the image (as well as text in social media), precisely because it appears in a violent representation?75 This is to extend the consideration of ‘bad cinema’ and indeed, social media, beyond the ‘violence of the represented’ to consider its thought and felt vibrations: vibrations that constitute, for Deleuze, the ‘automatic movement’ of the movement- image.76 The importance for me in this argument, is the way by which violence is perpetuated in social media then, not simply in a representation of violence, but in the thought and felt vibrations of violence as an outcome of the energetic relation in digital communication. THE PIXEL BEYOND THE MOVIE THEATRE Certainly, social media has the potential for representing violence, such as the violence in the case of the raping of legba and Starsinger by Mr Bungle in the text-based virtual world LambdaMOO.77 The ‘violence of the movement-image’ in social media is also in the rhythm of vibrations and the energetic relation — the ‘shock to thought’ of text and image.78 Such a ‘shock to thought’ is as Deleuze notes: ‘a recognition of powerlessness […]. What cinema advances is not the power of thought but its “impower.”’79 The aisthetic encounter, in its sounds, textures, rhythms, movements, and affects, is the source of an ‘unthinkable in thought’.80 As Deleuze writes: ‘if it is true that thought depends on a shock which gives birth to it (the nerve, the brain matter), it can only think one thing, the fact that we are not yet thinking, the powerlessness to think the whole and to think oneself, thought which is always fossilized, dislocated, collapsed’.81 In Annihilation, thinking is not found in a thinking of the whole from the outside, but in the encounter: ‘Unfathomable mind’, Dr Ventress says with her eyes sealed shut by skin to Lena. ‘We spoke. What was it we said? That I needed to know what was inside the lighthouse. That moment’s passed. It’s inside me now.’ The affective nature of ‘bad cinema’ — and the same could be said of social The affective nature of ‘bad cinema’ — and the same could be said of social media — suggests a philosophising potential as it relates to the kind of subject- spectator/operator generated in the encounter; that is, in the energetic relation of the pixel in communication with its spectators/operators. Just as Kieran J. O’Meara contends that feminism should be understood as a tradition rather than an ideology, thinking and feeling with cinema, or social media for that matter, cannot be found in an ‘all-encompassing logic of life and history’.82 The pixel communicates. However, what the pixel communicates is an affective force in the aisthetic encounter of body and image. In our aisthetic encounters we may apprehend an ethics beyond a politics of inadequate ideas. As O’Meara writes: 131 Cinéma & Cie vol. 22 no. DIGITAL HOSTILITY Like mass spectatorship in cinema, it is possible to see how social media retains heterogeneity. The particularity of image and text is the cause of infinite intensities in the aisthetic encounter. The iterative and participatory qualities of social media — which is also the cause of the energetic relation in social media — means that the pixel is more event than narrative.85 For multi-user dimensions (MUDs) or MOOs (MUD, Object-Orientated) on the internet such as LambdaMOO, textual descriptions of the virtual world and the commands given for how you want your character to appear and act, puts your character in energetic relation with other characters. However, the energetic relation in social media — via the affective force of the virtual and energetic pixel — is one of emotional entwinement with our real-life selves. Thus, we may think about the affective force of virtual intensities in the way that Boljkovak writes: Deleuze again insists upon an act of replaying or redoubling, upon foldings, unfoldings and refoldings that expose not only the actual events of our lives but also their underlying virtual intensities and affective significances. To counter-actualise, then, is to refold, break open and recombine thought, not to sense a totalising, homogeneous world but to strive to explore fragmentary, imperceptible relationships, to become imperceptible, neither actual nor virtual, this nor that, but always becoming, differing.86 Deleuze again insists upon an act of replaying or redoubling, upon foldings, unfoldings and refoldings that expose not only the actual events of our lives but also their underlying virtual intensities and affective significances. To counter-actualise, then, is to refold, break open and recombine thought, not to sense a totalising, homogeneous world but to strive to explore fragmentary, imperceptible relationships, to become imperceptible, neither actual nor virtual, this nor that, but always becoming, differing.86 Such ‘fragmentary, imperceptible relationships’ are the stuff of haecceities in the aisthetic encounter — of sounds, textures, rhythms, movements, and affects that do violence to thought.87 In Annihilation, Lena describes to her husband, Kane, the rhythm of the dividing pair by which the cell becomes immortal and never dies, whereby the cell is coded with its own destruction — a fault in the genes with old-age as the result. Equally, our interaction with social media could be said to be coded with its own self-destruction wherein digital hostility arises. Self-destruction is the sentiment with which the film Annihilation sets its biological premise. THE PIXEL BEYOND THE MOVIE THEATRE 38 2022 · ISSN 2036-461X ‘the Feminist tradition challenges us to think corporeally, to consider life as a bodied subject, where norms collide to cluster around our bodied existence, and how our experiences of these bodies encounter “the political”’.83 Sensory images do not produce metaphorical allusions or engage in politically divisive debate, but ‘demonstrate’ the power of affect in their political dimension.84 An attention to the aisthetic encounter suggests a kind of affirmative (rather than divisive) politics, whereby care, compassion, and empathy may be considered as an outcome of the affective intensities and connectives of the movement- image. Mee, Rhythm Beyond the Cinematic Medium DIGITAL HOSTILITY As Dr Ventress says to Lena: ‘Almost none of us commit suicide, and almost all of us self-destruct in some way, in some part of our lives. We drink or we smoke. 132 Mee, Rhythm Beyond the Cinematic Medium We destabilize the good job or the happy marriage. These aren’t decisions, they are impulses. […] Isn’t self-destruction coded into us, programmed into each cell?’ The ‘violence of a movement-image’ is a violence to ourselves — as one of self-destruction; however, it is also the vehicle for violence by and to others.88 Deleuze writes: ‘the movement-image was from the beginning linked to the organization of war, state propaganda, ordinary fascism, historically and essentially’.89 Moving from mass-art to ubiquitous social media and the effect of digital hostility on bodies, I want to argue that the violence of the movement- image when imposed by others can also be recognized as wrapped up in a politics of inadequate ideas when affect is exchanged for passion. Greta Olson writes in her essay ‘Love and Hate Online: Affective Politics in the Era of Trump’ about how the Trump campaign inspired hate as well as love in Trump’s followers: the Trump campaign was an affective one.90 As Olson writes: ‘political sentiments are determined by viscerally experienced sentiments and a physically imagined sense of rightness or wrongness, rather than one that is worked out through rational means’.91 In a politics of inadequate ideas it would not simply be affective engagement that forestalls such ideas — in fact, they may be made up of it — but rather an ethics that extends from ‘capacities to affect and be affected’.92 We must be careful to note that, as Deleuze writes: An affection is not a passion, except when it cannot be explained by the nature of the affected body: it then of course involves the body, but is explained by the influence of other bodies. Affections that can be completely explained by the nature of the affected body are active affections, and themselves actions.93 Where Trump’s campaign is considered to be an affective one that inspired hate as well as love, to confuse affect with the passion inspired is to limit the ethical relation in affect. DIGITAL HOSTILITY The Trump campaign was built on a politics of inadequate ideas by conjuring a divisive binary of winners and losers — ‘us’ versus ‘them’ (where the ‘them’ is often the media).94 What is refracted through our digital interactions in social media is patho-logies of passion found in divisive debate. In Spinoza’s Ethics, it is not simply that the body has a power for being affected: The more power a thing has, or the greater its power of existence, the greater number of ways in which it can be affected. Bodies are affected by different things, and in different ways, each type of body being characterised by minimum and maximum thresholds for being affected by other bodies: what can and what cannot affect it, and to what degree.95 What can be noted is that, as Baugh writes: ‘a body’s power of acting and being affected’ is also a ‘relation of parts’.96 In consideration of what defines a body’s potential, Deleuze writes: ‘A body’s structure is the composition of its relation. What a body can do corresponds to the nature and limits of its capacity to be affected.’97 For a community, as Baugh writes, this is the ‘collective power of being affected, and results in collective or communal affects’.98 Thus, a 133 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X thinking and feeling with affect — the ‘unthinkable in thought’99 of affect — is not a depiction of a particular kind of speech act as exemplified by Andrew Anglin when he writes: ‘One of the unifying marks of the Alt-Right sensibility is the assumption that no speech act is beyond the pale.’100 A thinking and feeling with affect is an ethical engagement for a harmonious collective. In her book Untimely Affects, Boljkovak considers the events of the Holocaust and Hiroshima in an examination of films by Chris Marker and Alain Resnais. Boljkovak describes a kind of ‘creative becoming’ through art’s resistance to violence.101 She writes: ‘As Deleuze and Guattari repeatedly insist, destructive forces double each possibility for flight; caution must be taken to pursue the line of greatest resistance and creativity.’102 Thus, there is another way by which we might consider mass-art as a ‘war machine’. Notes 1 I would like to thank Luke Robinson for endorsing the publication of my article and Gabrielle Lowe for her feedback on my article, as well as the anonymous peer-reviewers for their very helpful comments on an earlier draft of this article 2 Roland Barthes, The Neutral: Lecture Course at the Collège de France (1977–1978), trans. by Rosalind E. Krauss and Denis Hollier (New York: Columbia University Press, 2005), 51. 2 Roland Barthes, The Neutral: Lecture Course at the Collège de France (1977–1978), trans. by Rosalind E. Krauss and Denis Hollier (New York: Columbia University Press, 2005), 51. 2 Roland Barthes, The Neutral: Lecture Course at the Collège de France (1977–1978), trans. by Rosalind E. Krauss and Denis Hollier (New York: Columbia University Press, 2005), 51. 3 Gregory J. Seigworth and Melissa Gregg, ‘An Inventory of Shimmers’, in The Affect Theory Reader, ed. by Melissa Gregg and Gregory J. Seigworth (Durham, NC and London: Duke University Press, 2010), 1–25 (11); quoting from Barthes, 77. 3 Gregory J. Seigworth and Melissa Gregg, ‘An Inventory of Shimmers’, in The Affect Theory Reader, ed. by Melissa Gregg and Gregory J. Seigworth (Durham, NC and London: Duke University Press, 2010), 1–25 (11); quoting from Barthes, 77. 4 Barthes, 101. 5 Nadine Boljkovac, Untimely Affects: Gilles Deleuze and an Ethics of Cinema (Edinburgh: Edinburgh University Press, 2013), 18, 13; quoting from Gilles Deleuze, Negotiations, 1972–1990, trans. by Martin Joughin (New York: Columbia University Press, 1995), 172. 5 Nadine Boljkovac, Untimely Affects: Gilles Deleuze and an Ethics of Cinema (Edinburgh: Edinburgh University Press, 2013), 18, 13; quoting from Gilles Deleuze, Negotiations, 1972–1990, trans. by Martin Joughin (New York: Columbia University Press, 1995), 172. 6 Barthes, 51. 7 Gilles Deleuze and Félix Guattari, A Thousand Plateaus: Capitalism and Schizophrenia, trans. by Brian Massumi (Minneapolis and London: University of Minnesota Press, 1987), 261. Barthes, 51. 7 Gilles Deleuze and Félix Guattari, A Thousand Plateaus: Capitalism and Schizophrenia, trans. by Brian Massumi (Minneapolis and London: University of Minnesota Press, 1987), 261. 8 Deleuze and Guattari, 232–309. , 9 Barthes, 51. It is thought-provoking to compare Barthes’s notion of ‘shimmer’ with the way that Deborah Bird Rose describes ‘shimmer’ through the event of ‘reciprocal capture’ in which ‘different ways of being and doing find interesting things to do together’. Deborah Bird Rose, ‘Shimmer: When All You Love is Being Trashed’, in Arts of Living on a Damaged Planet, ed. DIGITAL HOSTILITY Vertov’s Man with a Movie Camera is just one example of a ‘revolutionary movement’ in cinema’s creative invention.103 Revolution is also the revolving action of transport and industrial machinery in Vertov’s film and entails new perceptive capacities for the spectator. However, ‘revolutionary movements’ also require resistance. Deleuze writes: ‘The work of art is not an instrument of communication. […] The work of art strictly does not contain the least bit of information. To the contrary, there is a fundamental affinity between the work of art and the act of resistance.’104 When considering affect and the body’s potentials, it is worth considering what the body is open to, or indeed, where there is resistance. As Seigworth and Gregg write: ‘affect is persistent proof of a body’s never less than ongoing immersion in and among the world’s obstinacies and rhythms, its refusals as much as its invitations’.105 Beyond a politics of inadequate ideas, we can take affect’s ‘demonstration’ to mean a revolution of sorts: a revolution by which care, compassion, and empathy may be considered as a resistance — or refusal — to hostility.106 For Boljkovak, art’s potential is found in Marker’s ‘things that quicken the heart’: perhaps an energetically pulsating pixel by which we may consider affect in all its ethical compassion.107 134 Mee, Rhythm Beyond the Cinematic Medium , 14 Ibidem, 261; Gilles Deleuze, Cinema 2: The Time-Image, trans. by Hugh Tomlinson and Robert Galeta (Minneapolis: University of Minnesota Press, 1989), 156. 15 j , 31 Deleuze and Guattari, 260; Deleuze, Cinema 2, 168, 156. 12 Ibidem. 18 Anna Lawton, ‘Rhythmic Montage in the Films of Dziga Vertov: A Poetic Use of the Language of Cinema’, Pacific Coast Philology 13 (Oct 1978), 44–50 (49). 13 Ibidem, 260. Ibidem. 70 Troxell, 356; quoting from Henry Wood’s Ideal Suggestion through Mental Photography: A Restorative System for Home and Private Use (Boston, MA: Lee and Shepard, 1893). 71 Deleuze, Cinema 2, 156. 72 Ibidem, 164. Mee, Rhythm Beyond the Cinematic Medium Notes by Anna Tsing, Heather Swanson, Elaine Gan and Nils Bubandt (Minneapolis: University of Minnesota Press, 2017), G51–G63 (G51). In her analysis, Rose gives the definition of the Australian Indigenous Yolngu term bir’yun as: ‘shimmer, the ancestral power of life [that] arises in relationship and encounter’ (Rose, G52–53). Rose writes: ‘The term bir’yun — which does not distinguish between domains of nature and culture — is characteristic of a lively pulsating world, not a mechanistic one. Bir’yun shows us that the world is not composed of gears and cogs but of multifaceted, multispecies relations and pulses’ (Rose, G55). 10 Barthes, 51; Deleuze and Guattari, 232–309. 11 Deleuze and Guattari, 261. 12 Ibidem. 13 Ibidem, 260. b de , 60 14 Ibidem, 261; Gilles Deleuze, Cinema 2: The Time-Image, trans. by Hugh Tomlinson and Robert Galeta (Minneapolis: University of Minnesota Press, 1989), 156. 15 D l Ci 2 156 14 Ibidem, 261; Gilles Deleuze, Cinema 2: The Time-Image, trans. by Hugh Tomlinson and Robert Galeta (Minneapolis: University of Minnesota Press, 1989), 156. 15 Deleuze, Cinema 2, 156. 16 Ibidem, 156. 17 Ibidem. 18 Anna Lawton, ‘Rhythmic Montage in the Films of Dziga Vertov: A Poetic Use of the Language of Cinema’, Pacific Coast Philology 13 (Oct 1978), 44–50 (49). 19 D l Ci 2 157 18 Anna Lawton, ‘Rhythmic Montage in the Films of Dziga Vertov: A Poetic Use of the Language of Cinema’, Pacific Coast Philology 13 (Oct 1978), 44–50 (49). 19 Deleuze, Cinema 2, 157. 20 Ibidem, 156. 21 Ibidem, 156; Deleuze and Guattari, 260. 22 Deleuze and Guattari, 261; Deleuze, Cinema 2, 156. 23 Deleuze, Cinema 2, 156. 24 Ibidem, 156. Ibidem, 156. 25 Gilles Deleuze, Difference and Repetition, trans. by Paul Patton (New York: Columbia University Press, 1994), 139. 26 Ibidem 139–40 25 Gilles Deleuze, Difference and Repetition, trans. by Paul Patton (New York: Columbia University Press, 1994), 139. 26 Ibidem, 139–40. 27 Deleuze, Cinema 2, 168. 28 Ibidem, 169. 29 Deleuze and Guattari, 232–309. 30 Boljkovak, 18. 31 Deleuze and Guattari, 260; Deleuze, Cinema 2, 168, 156. 135 135 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X 32 Deleuze and Guattari, 260, 232. 32 Deleuze and Guattari, 260, 232. 32 Deleuze and Guattari, 260, 232. Deleuze and Guattari, 260, 232. 33 Gilles Deleuze, Expressionism in Philosophy: Spinoza, trans. by Martin Joughin (New York: Zone Books, 1990), 226. 34 , , 33 Gilles Deleuze, Expressionism in Philosophy: Spinoza, trans. by Martin Joughin (New York: Zone Books, 1990), 226. 33 Gilles Deleuze, Expressionism in Philosophy: Spinoza, trans. by Martin Joughin (New York: Zone Books, 1990), 226. 34 Deleuze, Expressionism in Philosophy, 196. 35 Ibidem. Ibidem. 36 Bruce Baugh, ‘Body’, in The Deleuze Dictionary, ed. by Adrian Parr (Edinburgh: Edinburgh University Press, 2005), 35–37 (37). 37 B h 37 Ibidem. 36 Bruce Baugh, ‘Body’, in The Deleuze Dictionary, ed. by Adrian Parr (Edinburgh: Edinburgh University Press, 2005), 35–37 (37). 37 3 36 Bruce Baugh, ‘Body’, in The Deleuze Dictionary, ed. by Adrian Parr (Edinburgh: Edinburgh University Press, 2005), 35–37 (37). 37 Baugh, 37. 37 Baugh, 37. 38 Deleuze and Guattari, 261. 39 Deleuze, Cinema 2, 162. 40 Deleuze, Expressionism in Philosophy, 197. 40 Deleuze, Expressionism in Philosophy, 197 41 Ibidem, 218. 42 Seigworth and Gregg, 11; quoting from Barthes, 77. 43 Barthes, 51, 77. , , 44 Jenelle Troxell, ‘Mind Cure and Ecstasy on the Pages of Close Up’, Screen 58.3 (Autumn 2017), 349–71 (364); quoting from Sergei Eisenstein, ‘Methods of Montage’, in Film Form, ed. and trans. by Jay Leyda (New York: Harcourt Brace, 1977), 80. 44 Jenelle Troxell, ‘Mind Cure and Ecstasy on the Pages of Close Up’, Screen 58.3 (Autumn 2017), 349–71 (364); quoting from Sergei Eisenstein, ‘Methods of Montage’, in Film Form, ed. and trans. by Jay Leyda (New York: Harcourt Brace, 1977), 80. 45 Troxell, 364; quoting from Sergei Eisenstein, ‘The Structure of the Film’, in Film Form, ed. and trans. Jay Leyda (New York: Harcourt Brace, 1977), 166. 45 Troxell, 364; quoting from Sergei Eisenstein, ‘The Structure of the Film’, in Film Form, ed. and trans. Jay Leyda (New York: Harcourt Brace, 1977), 166. 46 Troxell, 364; quoting from Eisenstein, ‘The Structure of the Film’, 167. 47 Ibidem. 48 ‘Refraction’, in Oxford English and Spanish Dictionary <https://www.lexico.com/definition/refraction> [accessed 9 March 2021]. 48 ‘Refraction’, in Oxford English and Spanish Dictionary <https://www.lexico.com/definition/refraction> [accessed 9 March 2021]. 49 Troxell, 364; quoting from Eisenstein, ‘The Structure of the Film’, 167; Deleuze and Guatta 49 Troxell, 364; quoting from Eisenstein, ‘The Structure of the Film’, 167; Deleuze and Guattari, 260. 32 Deleuze and Guattari, 260, 232. 50 Sean Cubitt, The Cinema Effect (Cambridge, MA and London: The MIT Press, 2004), 41. Troxell, 364; quoting from Eisenstein, The Structure of the Film , 167; Deleuze and Guattari, 260. 50 Sean Cubitt, The Cinema Effect (Cambridge, MA and London: The MIT Press, 2004), 41. 50 Sean Cubitt, The Cinema Effect (Cambridge, MA and London: The MIT Press, 2004), 41. 50 Sean Cubitt, The Cinema Effect (Cambridge, MA and London: The MIT Press, 2004) 51 ‘Refraction’, in Oxford English and Spanish Dictionary 52 Barthes, 51. 53 Barthes, 101. 54 Seigworth and Gregg, 11. 55 Ibidem, 11; Cubitt, 41. 56 Cubitt, 33. 57 Seigworth and Gregg, 11. 58 Krauss writes: ‘Or, and why not, there is the television set itself, this impassive eye that nonetheless exudes a constant visual beat since its image is produced by an electric current scanning upward along the hundreds of lines that cross the screen, generating an “image” through the continual renewal of its pulse, becoming all the more apparent when the set goes out of calibration and the whole image is wiped upward again and again as though pushed by an insistently reappearing black, horizontal bar’. (Rosalind E. Krauss, The Optical Unconscious [Cambridge, MA and London: The MIT Press, 1993], p. 202). 61 Ibidem; quoting from H. D., ‘Cinema and the Classics III: The Mask and the Movietone’, Close Up 1.5 (1927), 23. 62 Troxell, 354; quoting from H. D., 23; Deleuze, Cinema 2, 156. 63 Seigworth and Gregg, 9; Barthes, 77. 64 Seigworth and Gregg, 12. 65 Barthes, 77. 66 Deleuze, Expressionism in Philosophy, 197. 68 Ibidem. 69 Ibidem. 71 Deleuze, Cinema 2, 156. 72 Ibidem, 164. 136 136 Mee, Rhythm Beyond the Cinematic Medium Ibidem. 77 Julian Dibbell, ‘A Rape in Cyberspace. How an Evil Clown, a Haitian Trickster Spirit, Two Wizards, and a Cast of Dozens Turned a Database into a Society’, Village Voice, 23 December 1993, 36. , , Katleen Gabriels and Marjolein Lanzing also note how ‘instances of online vitriol can be conceptualized as onlife violence: violence that transgresses and affects both the offline and online world’. Katleen Gabriels and Marjolein Lanzing, ‘Ethical Implications of Online Vitriol’, in Violence and Trolling on Social Media: History, Affect, and Effects of Online Vitriol, ed. by Sara Polak and Daniel Trottier (Amsterdam: Amsterdam University Press, 2020), 197–214 (197). 74 Ibidem, 157. 73 Ibidem, 157. Ibidem. 77 Julian Dibbell, ‘A Rape in Cyberspace. How an Evil Clown, a Haitian Trickster Spirit, Two Wizards, and a Cast of Dozens Turned a Database into a Society’, Village Voice, 23 December 1993, 36. 75 Ibidem, 156–57. 104 Gilles Deleuze, ‘Having an Idea in Cinema (On the Cinema of Straub-Huillet)’, in Deleuze and Guattari: New Mappings in Politics and Philosophy, trans. by Eleanor Kaufman, ed. by Eleanor Kaufman and Kevin Jon Heller (Minneapolis: University of Minnesota Press, 1998), 14–19 (18). 105 , , Katleen Gabriels and Marjolein Lanzing also note how ‘instances of online vitriol can be conceptualized as onlife violence: violence that transgresses and affects both the offline and online world’. Katleen Gabriels and Marjolein Lanzing, ‘Ethical Implications of Online Vitriol’, in Violence and Trolling on Social Media: History, Affect, and Effects of Online Vitriol, ed. by Sara Polak and Daniel Trottier (Amsterdam: Amsterdam University Press, 2020), 197–214 (197). 76 Ibidem. 32 Deleuze and Guattari, 260, 232. Katleen Gabriels and Marjolein Lanzing also note how ‘instances of online vitriol can be conceptualized as onlife violence: violence that transgresses and affects both the offline and online world’. Katleen Gabriels and Marjolein Lanzing, ‘Ethical Implications of Online Vitriol’, in Violence and Trolling on Social Media: History, Affect, and Effects of Online Vitriol, ed. by Sara Polak and Daniel Trottier (Amsterdam: Amsterdam University Press, 2020), 197–214 (197). 79 Deleuze, Cinema 2, 156, 166. 80 Ibidem, 168. 81 Ibidem, 167. , 82 Kieran J. O’Meara, ‘On Feminism as Tradition, Not Ideology’, poLit: Discussion Political Thought (10 March 2021), <https://thinkpolit.blogspot.com/2021/03/on-feminism-as-tradition-not-ideology. html?fbclid=IwAR346cmixPHN40632He6uUixqtbc_pT69jTCWkh3nV5NxPzn1FT9-DCSsTE&m=1> [accessed 15 March 2021] (para 9 of 9) 82 Kieran J. O’Meara, ‘On Feminism as Tradition, Not Ideology’, poLit: Discussion Political Thought (10 March 2021), <https://thinkpolit.blogspot.com/2021/03/on-feminism-as-tradition-not-ideology. html?fbclid=IwAR346cmixPHN40632He6uUixqtbc_pT69jTCWkh3nV5NxPzn1FT9-DCSsTE&m=1> [accessed 15 M h 2021] ( 9 f 9) 82 Kieran J. O’Meara, ‘On Feminism as Tradition, Not Ideology’, poLit: Discussion Political Thought (10 March 2021), <https://thinkpolit.blogspot.com/2021/03/on-feminism-as-tradition-not-ideology. html?fbclid=IwAR346cmixPHN40632He6uUixqtbc_pT69jTCWkh3nV5NxPzn1FT9-DCSsTE&m=1> [accessed ] ( ) 15 March 2021] (para. 9 of 9). 83 O’Meara, para. 9 of 9. 84 Deleuze, Cinema 2, 183. , , 85 Greta Olson, ‘Love and Hate Online: Affective Politics in the Era of Trump’, in Violence and Trolling on Social Media: History, Affect, and Effects of Online Vitriol, ed. by Sara Polak and Daniel Trottier (Amsterdam: Amsterdam University Press, 2020), 153–77 (173). 85 Greta Olson, ‘Love and Hate Online: Affective Politics in the Era of Trump’, in Violence and Trolling on Social Media: History, Affect, and Effects of Online Vitriol, ed. by Sara Polak and Daniel Trottier (Amsterdam: Amsterdam University Press, 2020), 153–77 (173). 86 Boljkovac, 20. 87 Boljkovac, 20, 29. 88 Deleuze, Cinema 2, 157. 89 Ibidem, 165. 90 Olson, 160. 91 Ibidem, 154. 92 Deleuze and Guattari, 261. 93 Deleuze, Expressionism in Philosophy, 218–19. 94 Ibidem, 162–63. 95 Baugh, 36. 96 Ibidem, 36–37. 97 Deleuze, Expressionism in Philosophy, 218. 98 Baugh, 37. 99 Deleuze, Cinema 2, 168. 100 Olson, 172; quoting from Andrew Anglin, ‘A Normie’s Guide to the Alt-Right’, Daily Stormer, 31 August 2016. 2016. 101 Boljkovac, 13. 102 Ibidem, 21. 103 Ibidem, 13; quoting from Deleuze, Negotiations, 172. 104 Gilles Deleuze, ‘Having an Idea in Cinema (On the Cinema of Straub-Huillet)’, in Deleuze and Guattari: New Mappings in Politics and Philosophy, trans. by Eleanor Kaufman, ed. by Eleanor Kaufman and Kevin Jon Heller (Minneapolis: University of Minnesota Press, 1998), 14–19 (18). 105 Seigworth and Gregg, 1. 106 Deleuze, Cinema 2, 183. 107 Boljkovac, 93. 137 137 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X REVI EWS/ COM PTES- REVI EWS/ COM PTES REVI EWS/ COM PTES- RENDUS COM Daniel Morgan The Lure of the Image: Epistemic Fantasies of the Moving Camera Oakland: University of California Press, 2021, pp. 244 DOI 10.54103/2036-461X/17978 If film is an inherently ephemeral object, the mobile camera is perhaps its most elusive aspect. Accordingly, Daniel Morgan’s theorization of camera movement in The Lure of the Image explores a topic film theory has hitherto largely ignored in favor of Ontology and Montage. The book sets out to articulate a systematic account of camera movement, venturing beyond Film Studies’ disciplinary boundaries while keeping a close eye on film itself. It comprises seven chapters and is divided into two parts. Morgan engages with an impressive number of scholars and (both high-brow and low-brow) films, drawing from a wide range of sources, including filmmaking manuals. His prose is devoid of jargon and illustrates theoretical concepts with analytical precision. In addition, Morgan builds his arguments through the close study of film, interweaving careful descriptions of the way the camera articulates spatial arrangements and interpretations tying aesthetic operations to narrative meaning. film style), and Terence Malick. In contrast, the final chapter returns to theory, shifting its focus to the proliferation of camera movement and cameras themselves in digital cinema. Rather than taking the most obvious route, Morgan aptly downplays the rhetorical calls for a paradigmatic shift by gesturing towards the use of animation techniques throughout the pre-digital 20th century — “this long history matters […] New technologies of the moving camera do not necessarily produce new conceptual problems” (pp. 221-222) — from Star Wars (1977) to early phantom rides. The chapter’s unexpected turns encapsulate the book’s conceptual richness, as Morgan jumps (swish-pans?) from discussions on New Media and military drones to stereoscopy, Late Godard’s 3D films, Orson Welles and cinéma vérité. work is licensed under a Creative Commons Attribution 4.0 International License The author begins his discussion by offering a survey of the existing scholarship on the subject, situating himself in relation to Patrick Keating’s examination of camera movement and the ideas about it in classical Hollywood.1 Morgan’s key insight is that “not all the ideas are good ones” (p. 4) and the all-pervasive notion of the camera- eye is a particularly bad — or “deeply flawed” (p. 5) — one. The book’s opening movement endeavors to erode this fatal misconception from its dominant position by unraveling the While the first three chapters raise the book’s film-theoretical stakes, the second section mobilizes its newly minted conceptual tools to authorial case studies centered on canonical directors such as Fritz Lang (examined comparatively alongside Guru Dutt), Max Ophüls (a privileged site in discussions about virtuosic 141 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X conflation of theories of camera movement and point of view. The chief target of his critique here is phenomenologically-inflected film theory, which Morgan characterizes as reductively built around a specific formal operation: subjective shots in which the camera moves forward. As he rightly puts it, while certain narrative films operate according to the conventions of point of view, this is not always the case. Rather than uphold film as an embodied experience, Morgan puts our ability to identify with the camera into doubt and gestures towards many instances in which we sympathize with the characters on screen — Morgan mentions mirror neurons in passing but the topic warrants a deeper engagement with neuroimaging. In addition, he upholds shots in which characters presumably ‘doing’ the looking emerge in the frame — but fails to mention Roberto Rossellini’s fascist war film Un Pilota Ritorna’s (1942) mobilization of this aesthetic operation notwithstanding the implications that its panning shot of a Prison-of- war camp would have in relation to Neorealism and, as a consequence, to Bazinian and Deleuzian film theory — or are revealed to have not been ‘with’ the camera, as in Horror films tricking us into believing ‘subjective’ shot approaching an oblivious victim belongs to a serial killer. develops this model only after a lengthy meta- analysis of the reception of an ‘infamous’ shot in Gillo Pontecorvo’s Kapo (1960) in French film criticism. This allows him to dispel Brechtian, Platonic, and Barthesian anxieties underpinning politicized cinephobia (p. 40). work is licensed under a Creative Commons Attribution 4.0 International License It is interesting that Morgan’s discussion of the aestheticization of the dead body does not consider Pontecorvo’s framing of tortured bodies accompanied by a liturgical tune in The Battle of Algiers (1966) and thus its possible links to Catholic iconography, an intertext corroborating his relativization of bad taste as context-specific. In place of identification, the book invites us to think about our relation to the worlds films construct in terms of imagination, “buttressing” Richard Wolheim’s conceptualization of the “internal spectator” (p. 76) who is attuned to diegetic characters without being aligned to their optical perspective, with Cristopher McCarrol’s Sartrean work on “observer memories… in which we see ourselves from the outside” (pp. 80-81).2 This Frankensteinian “path around” the impasse of identification (p. 81) may initially appear convoluted but is immediately clarified through the analysis of two film moments featuring camera movement which “attune” us to the characters’ experience (p. 82). At the same time, by resorting to Wollheim and especially McCarrol’s work, Morgan undermines his self-professed attempt to produce an organic theory stemming from the critical analysis of films (an approach advocated by Dudley Andrew) in opposition to the reductive imposition of frameworks from above. In fact, Morgan too ultimately relies parasitically on exterior conceptualizations — in this case developed in Art History and cognitive science via the Philosophy of Mind. The fantasy of an unmediated approach to film remains liable to the same critique Hegel moved against empiricism for (unconsciously) reading the subject into the object rather than the unmediated object itself. According to Morgan, rather than identify with the camera, we merely desire to be with the camera while knowing full well that this is impossible, a process he describes as an epistemic fantasy. This immediately raises the stakes as it questions much that has been written about film. Morgan takes the work of various film scholars to task for relying all too heavily (explicitly or implicitly) on the assumption that the camera’s position corresponds to that of the spectator. He makes a persuasive claim and reiterates it across various paragraphs. At the same time, this repetition slows down the flow of his argument. We remain bogged down in the pars destruens long after recognizing the need for an alternative model, gripped by a sense of totalizing skepticism. work is licensed under a Creative Commons Attribution 4.0 International License Indeed, Morgan With regards to empiricism, the book would benefit from a deeper engagement with the science of vision and the phenomenon of motion 142 The Lure of the Image The Lure of the Image sickness. If we genuinely identified with the moving camera, would we not feel sick? This is not unusual in first-person and third-person videogames, and while Morgan mentions videogames in passing, he does not consider this distinction. In addition, moments of cinephilic appreciation — such as the description of a crane shot as “gorgeous” (p. 236) — feel unnecessary, and Morgan could have dedicated a few words to dispel Lacan’s theorization of the mirror stage rather than bypass him via Ovid. Further research could be developed in dialogue with work emerging outside the boundaries of Anglophone film scholarship, such as Masaki Kondo’s Derridean analysis of the Cartesian logic of Samuel Becket’s film about eyes and non-being FILM (1965).3 Nonetheless, the book is quite brilliant. It is both innovative and rooted in tradition, remaining deeply committed to film as film. Accordingly, it should be required reading for any film theory course which aims to (re)think “seriously” (p. 244) about camera movement and, more generally, film aesthetics. Antonio Patrick D’Amico [Yale University] Notes ¹ Patrick Keating, The Dynamic Frame. Camera Movement in Classical Hollywood (New York: Columbia University Press, 2019). ² Cristopher McCarrol (Remembering from the Outside: Personal Memory and the Perspectival Mind, Oxford: Oxford University Press, 2018). ³ Masaki Kondo, ‘The Eye and the Gaze: Peering into Samuel Beckett’s Film’, Screen, 61.3 (2020), 423–435 (427). 143 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Enfin le cinéma! Arts, images, spectacles en France (1833–1907) sous la direction de Dominique Païni, Paul Perrin, Marie Robert Paris: Musée d’Orsay / Réunion des Musées Nationaux – Grand Palais, 2021, pp. 331 DOI 10.54103/2036-461X/17979 DOI 10.54103/2036-461X/17979 ‘It is not a question here of telling the story of the invention of cinema but rather of evoking what it invents: the modern spectator that, for the most part, we are still’ (p. 26): the unequivocal statement by film theorist and historian Dominique Païni – curator, together with Paul Perrin and Marie Robert, of the catalogue of the exhibition Enfin le cinéma! Arts, images, spectacles en France (1833-1907), which recently took place at Musée d’Orsay, makes explicit, from the very beginning, the underlaying assumption that guides the text’s structure. The adoption of an alphabetical order by the editors of the book is the pretext for the organization of an atlas of modernity: a portolan chart drawing the tangle of routes that crosses ‘a bit haphazardly’, not without risks, and drifts towards the unknown ‒ a pêle mêle, to use Païni’s words (p. 15) ‒, the handful of years during which the new techniques of the modern observer arose. the sensorial richness of universal exhibitions is what emerges from the words and images of the volume. Subjected to the optical and motion stresses provoked by the new urban landscape, illuminated day and night by electric light and accelerated by modern modes of transportation, modern women and men demanded a visual entertainment equal to the urban phantasmagoria. The convergence of scientific discoveries, technical achievements, industrialization and rising capitalism, the desire for ever more extreme sensory stimulations and the availability of a portion of free time that had previously been unknown to peasant living led to the invention of optical instruments capable of opening the gaze towards the boundless amplitudes of the world or, on the contrary, concentrating it into an unprecedented, spectacular extraordinariness. This provided the thrills and shocks that constituted the armamentarium of effects aimed at producing that aesthetic of astonishment which, according to Tom Gunning, among others, characterized the production of moving images until 1907.4 work is licensed under a Creative Commons Attribution 4.0 International License Félix Valloton and Pierre Bonnard, abandoning the classical framing, break down the vision by highlighting its episodic and fragmentary nature, transforming painting and print into a proto- cinema, a synchronic repertory of the attractions that make Paris the city-spectacle par excellence, where everything is on display (on this subject: Discontinuité by Isabelle Cahn, p. 86 and Nabi by Mathias Chivot, p. 170). Moreover, Parisian architecture, the epitome of which is the Eiffel Tower, is a tool for the creation of a composite horizon of views that constantly allude to its outliers, as in Henri’s lithographs and plates (see Hors-champ by Marie Robert, p. 118). The defining apparatus makes use of concepts consolidated in the field of Film Studies, such as, for example, screen, space and montage, (respectively: Écran by Vanessa R. Schwartz, p. 92; Espace by Michel Frizot, p. 102 and Montage by François Albera, p.158); these are always illuminated by a multidisciplinary gaze that gives an account of the mutual influence between arts, languages and techniques on which the aesthetics of modernity and the new way of looking pivot. The body, the inescapable protagonist of the cinematographic language — as much in its presence as in its noisy absence — is narrated as a comic agent (Corps Comique, by Laurent Guido, p. 62) and as a pathological patient (Corps Pathologique by Rae Beth Gordon, p. 68); it is described as colonized by the Western imperialist gaze (Exhibitions by Stéphane Tralongo, p. 109) or by the equally imperialist male eye when it turns to the scrutiny of the female body as a sexual object, further eroticized through the cinematic mediation of keyhole masks or binocular lenses (Voyeur by Marie Robert, p. 286). The lemma ‘montage’, for example, is not only described as intrinsic to the film, but is used to determine a new mode of fruition: the expository form of the screening program, which lines up heteroclite fragments. Movement, one of the main categories guiding the book, is investigated The lemma ‘montage’, for example, is not only described as intrinsic to the film, but is used to determine a new mode of fruition: the expository form of the screening program, which lines up heteroclite fragments. work is licensed under a Creative Commons Attribution 4.0 International License By the beginning of the XIX century, the modern metropolis had grown thanks to the engineering of iron as a building material, granting new views above and beyond human perception; simultaneously the urban fabric became lightened and enriched with new recadrages due to the replacement of masonry with large glass panels. A city intersected by passages and railways, lifted by the Eiffel Tower, intoxicated by The book includes a large number of renowned scholars from various disciplines engaged in writing short essays which compose a heteroclite lemmary. The entries of this abecedary account not only for this technical marvel, but also for the 145 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X by Paul-Louis Roubert; Populaire by Valérie Vignaux, Hors-champ by Marie Robert, p. 118; Immersion by Livio Belloï, p. 208, Temps by Michel Frizot, p. 272; Vue by Érik Bullot, p. 292). social construction of modernity, its less obvious manifestations that are nevertheless crucial in outlining the profile of a demanding and shrewd audience, still open to wonder and amazement for the sake of discovery and sensation. The theatre of the world, understood as an inventory of images and repertoire of experiences (see the entry compiled by Paul Perrin Inventaires, p. 134 and Jean François Staszak’s definition of Monde, p. 154), is Paris: the city-screen portrayed in its discontinuity by the Nabis painters. As the editors affirm, in the opening texts of the volume, it is not a question of reconstructing the umpteenth history of cinema or sanctioning its ultimate truth, but of recovering the dimension of movement, one that is enthusiastic and often fortuitous, at times even frightening; that is understood not as mere mobility but in terms of change, the true index of modernity. Out of time and off the screen, the world continues to happen and cinema, within the boundaries traced by the luminous rectangle of the frame, resigns itself to intercepting a constant absence, chasing after what Jacques Aumont calls ‘the feeling of an interminable anecdote’ (p. 40), that happens a little further away or a little before, perhaps a little after. work is licensed under a Creative Commons Attribution 4.0 International License Movement, one of the main categories guiding the book, is investigated – in painting, photography, as well as in the early Lumière’s vedutist cinema – as a fortuitous and uncontrollable accident: the unexpected that frees bodies from the stasis of Étienne-Jules Marey’s chrono-photography and captures the instantaneous nature of action (see, for example, the entries Aléa by Jacques Aumont, p. 39; Animé And if cinema is the place where death seems to be defeated, at least for the duration of a projection, the body is precisely that battlefield where the miracle of animation is accomplished. The cinema of origins takes up the myth of Pygmalion and Galatea, one of the iconographic 146 Enfin le cinéma! Enfin le cinéma! themes dearest to sculpture, and elaborates it thanks to its media specificity. (see Sculpture by Leah Lehmbeck, p. 242). of arrival that is also a roaring restart; at the same time it seems to indicate the resolution of an enigma or the occurrence of an alchemical transmutation. An end title, rather than a header, which follows that garble of events that constitute the emergence of modernity, whose brightest manifestation remains cinema. Pictorial and proto-cinematographic naturalism is complementary to spectacle, the artifice which aims to entertain the masses. The depiction of the act of seeing, traces of scrutinies and evidences of cinetisme are the counterpart of another colourful, blithe, abundant progeny: the one of the dispositives amusant, of the cheerful or gloomy technologies of vision which determine — or are determined by — the inception of the modern spectator. The book, which can be intended as an autonomous publication, one that is not necessarily entangled with the Parisian exhibition, depicts the city as the perfect example of an intrinsically inter-media text, and indeed the appendix traces back the biographies of those inventors, actors, directors who wove the mesh of modernity into the new urban context. Tricks borrowed from prestidigitation, as in the case of Méliès, the use of colour and light in the composition of panoramas and dioramas, stage construction in studios, the fairground attractions, Loïe Fuller’s dance and, earlier, the plethora of optical toys and pre-cinematographic devices barely managed to satiate the optical hunger of the public at the turn of the nineteenth and twentieth centuries (see, among others, the entries Attractions by Martin Barnier, p. 53; Dessin by Dominique Willoughby, p. 78; Lanternes by Ségolène le Men, p. work is licensed under a Creative Commons Attribution 4.0 International License 147; Studios by Brian Jacobson, p. 262; Trucs by Frank Kessler and Sabine Lenk, p. 276). The repertoire of images belongs to a basin which is, for the most part, urban: street views, perspectives taken from the top of modern construction, boulevard audiences and theatrical spectatorship, storefront windows and department store’s display of goods, wall affiches, crowded film stills or empty studios in the outskirts of Paris, harbours and train stations: these are the great part of the buzzy visual population which inhabits the pages. The epitome of the cityscape’s cruciality as the most powerful cinema incubator is the cover image: Henry Rivière’s photograph of a couple caught entering a train station. They are not crossing the threshold of a salle, a proto-cinema theatre, they are entering the realm of movement, and, as modern subjects, they are most likely capable of grasping the spectacle of movement. Only there, in the end, at that stage of awareness, lies the cinema. Page after page, entry after entry, we observe the shaping of an atlas that is susceptible to continuous amendments and subsequent hybridizations, as if cinema — ‘a technical precipitate’ in Paini’s words — could avoid taxonomies and rigid classifications and could be grasped, albeit for an instant, only in the breadth of a never-too-stringent, evocative map. Anna Franceschini [Università IULM, Milano] The volume is characterised by a composite structure that pushes us to move back and forth between the pages, definitions and tropes, creating a short circuit between the linearity that characterizes the classic construction of a book and the impossibility of harnessing cinema in a single trajectory. In short, the volume itself is in motion. This begins with the title, which defines, with a joyful assertiveness marked by exclamation, the conclusion of a process, a point Notes 4 T. Gunning, ‘An Aesthetic of Astonishment: Early Film and the (In) credulous Spectator’, Art and Text, 34, (1989), 114-33. 147 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X La haute et la basse définition des images. Photographie, cinéma, art contemporain, culture visuelle sous la direction de Francesco Casetti et Antonio Somaini Milano-Udine: Éditions Mimésis, 2021, pp. 363. DOI 10.54103/2036-461X/17980 Anyone who has delivered an article or volume with illustrations to a publisher will sooner or later have had one or more images sent back to them, accompanied by the prompt reprimand: ‘the definition is too low. Please resend the file in higher definition’. The frustration of the author – who may have thought they could get away with a quick screenshot in .png format or who found themselves needing to reproduce an image that exists only as a lightweight .jpeg on the web – is usually followed by a frantic series of attempts to improve the source image by using image enhancement or image upscaling software (such as DeepImage), upgrading the original information so as to achieve an editorially acceptable result. Philosophy (Ernst Cassirer), phenomenological psychiatry (Erwin Straus) and cultural anthropology (Robert Hertz) have laid the foundations of an investigation that aims to explore those value investments and show their natural and cultural roots. The volume La haute et la basse définition des images. Photographie, cinéma, art contemporain, culture visuelle, edited by Francesco Casetti and Antonio Somaini, effectively collaborates with this investigation at the level of the theory and practice of images, with particular (but not exclusive) reference to technical and digital images, contributing to a problematization of that same polarity and the values associated with it (rich/poor, clear/ confused, precise/imprecise etc.). work is licensed under a Creative Commons Attribution 4.0 International License As the editors argue in their Introduction, the distinction between the definition of high and low takes on a meaning that is not only technological, but also aesthetic, epistemological, economic and political. At the technological level, the progress in devices for recording, encoding, transmission and manipulation of images constantly reshapes the high-low relationship. At the aesthetic level, the possibility of appreciating or not appreciating certain details of the image, according to its resolution, has an impact both on our sensory experience (aesthetics as aisthesis) and on our Low is bad, high is good. And not only in the merely numerical and quantitative sense of measuring the pixels contained in a digital file. The definition of images thus adapts to the millenary tendency to load the fundamental relations of space (high/low, but also right/left and front/back) — relations that are rooted in the anthropos as an oriented and situated body — with axiological and symbolic values: ‘to start off on the right/wrong foot’, ‘to report a sinister’, ‘to be always one step ahead’, ‘low blow’, ‘to walk tall’, ‘State of right’ (and not of wrong)... 149 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X artistic experience (aesthetics as art theory); it also has an impact to the point that we might even question whether we are dealing with the same image and the same work in the case of different definitions of the same one. At an epistemological level, the level of knowledge to which we have access depends directly on the informational content of the image. At the economic level, financial investments in increasingly sophisticated technologies make the pace of device obsolescence ever faster (‘today’s high definition inevitably tends to become tomorrow’s low definition’). At the political level, the distinction between high and low definition negotiates the dialectic between the regimes of visibility and invisibility, with inevitable consequences for information accessibility (the investigations conducted by Eyal Weizman and the Forensic Architecture group he coordinates at Goldsmiths in London are a striking example of this with regard to military/civilian opposition). surfaced in Plato’s Parmenides. La haute et la basse définition des images Rosa Cinelli/Ph.D. Thesis Abstract1 Ph.D. in co-direction: Università di Milano ERC Advanced Project “An-Iconology. History, Theory and Practices of Environmental Images” Université Côte d’Azur Extended reality research and creative Center xr²c² Rosa Cinelli/Ph.D. Thesis Abstract1 Ph.D. in co-direction: Università di Milano ERC Advanced Project “An-Iconology. History, Theory and Practices of Environmental Images” Université Côte d’Azur Extended reality research and creative Center xr²c² DOI 10.54103/2036-461X/17982 be defined as a visualization, composition, or assemblage: all of these are complex concepts entailing different theories and archaeologies. A new type of imaginary challenges the traditional tools commonly used to describe photographic and filmic images, such as, for instance, Peirce’s very concept of the index.3 While it is true that a complete feeling of scepticism towards the image — often prophesied by the detractors of the digital revolution — has not completely taken over, it is possible to state that the procedure which allows for an image to be regarded as a visual fact seems more often to derive from a context-based rhetorical mechanism4 than to be guaranteed by the technical genesis of the image. This seemingly very subtle change, since it does not entail a radical transformation from the pragmatic point of view, is nevertheless a very theoretically rich node as it suggests a shift from the ontological to the rhetorical plane of discourse. Must the traditional ontology of the photographic image as a truthful — or even sacred — imprint be discarded for good? Throughout the history of mechanical images — the first of which is surely photography — the capacity of the image to adhere to its object or referent has influenced its positioning within aesthetic, artistic, and semiotic theories. Referentiality, understood as the ‘founding order of photography’,2 was considered the cornerstone of a precise ontological definition of the image and has contributed to creating a link between mechanically generated images and the realm of veridiction. Throughout the history of mechanical images — the first of which is surely photography — the capacity of the image to adhere to its object or referent has influenced its positioning within aesthetic, artistic, and semiotic theories. Referentiality, understood as the ‘founding order of photography’,2 was considered the cornerstone of a precise ontological definition of the image and has contributed to creating a link between mechanically generated images and the realm of veridiction. Nevertheless, today, new mediatic practices like Virtual Reality, CGI, and AI-powered images (such as machine vision) are increasingly challenging the epistemological paradigm of what counts as visual evidence. work is licensed under a Creative Commons Attribution 4.0 International License Once again, then, a dialectic that we could define as aesthetic- pragmatic, aimed at inducing in the observer a sensorimotor behavior of approaching or moving away from the image, and of lesser or greater perceptual integration on the part of the observer (a theme that would later become central to the mediological approach of McLuhan, not by chance a reader of Wölfflin). The volume brings together contributions from specialists in film theory, media and visual culture studies (in addition to the curators, Erika Balsom, Raymond Bellour, Emmanuel Burdeau, Enrico Camporesi, Arild Fetveit, Filippo Fimiani, Jacob Gaboury, André Habib, Frédéric Monvoisin, Roger Odin, Peppino Ortoleva, Marie Rebecchi, Lina Maria Stahl, Peter Szendy). And, significantly, it gives voice to artists (Hito Steyerl, Jacques Perconte, Thomas Hirschhorn) who have placed at the center of their practice and reflection the questions of image definition and of the multiple senses — from the aesthetic to the political — produced by the dynamics of compression/decompression, impoverishment and pixelization. These are levels that we can certainly distinguish in the analysis, but which are evidently intertwined in the concrete practices of the production and reception of images today. The contemporary situation, however, does not jump out of the blue; its archeology is inscribed in a longue durée that the editors propose to designate as the polarization between ‘neat’ and ‘flou’, crucial for the history of pre-digital images. They recall in this regard — in addition to the poetics of flou in the history of analogue cinema, and the difference between photographic pictorialism and New Objectivity — also the Renaissance opposition between linear perspective (based on the rigorous geometrization of the represented space) and aerial perspective (played instead on pictorial shading and atmospheric effects). We could add here that distinction between images to be observed from close up and images to be appreciated from afar, which was made famous by art historians such as Heinrich Wölfflin and Alois Riegl respectively for the transition from Renaissance to Baroque and the transition from Egyptian to late Roman art, but which had already Andrea Pinotti [Università degli Studi di Milano] 150 La haute et la basse définition des images PROJ ECTS ABS TRA CTS & PROJ ECTS ABS TRA CTS & Rosa Cinelli/Ph.D. Thesis Abstract1 Ph.D. in co-direction: Università di Milano ERC Advanced Project “An-Iconology. History, Theory and Practices of Environmental Images” Université Côte d’Azur Extended reality research and creative Center xr²c² From immersive and visual journalism to forensic practices and data-driven investigations, a vast panorama is taking shape in which photographic images are more and more blended with computer- based ones, creating uncanny configurations which are reshaping the regimes of visuality as well as our information economy. At times, this polymorphic class of composite images may sacred — imprint be discarded for good? Ethical concerns may also be raised when 153 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X this hybrid visual regime is considered in its socio-political agency. Contemporary journalism practices, such as visual journalismi5 and the emerging fields of forensic aesthetics6 are imposing new challenges to research. Making broad use of so-called ‘algorithmic’ devices,7 these experimentations are aspiring to confer full visibility to complex socio-political phenomena, ranging from humanitarian conflicts and migrations to the ecological crisis. Re-signifying what Paul Virilio already referred to as ‘the vision machine’,8 a very problematic posthuman dimension is added to the very human relevance of visual evidence. forms of empathy,14 raising again the question of the representability of others’ sufferings as well as issues in power and gaze distribution. When contemplating this spectre of practices, many questions can be raised: are we witnessing the affirmation of a new kind of visual regime regulating the realm of veridiction by simultaneously hyper-stimulating an empathic response and completely eluding the percipient subject? What kind of images are now worth believing in, at a time of an increasing hybridization between ‘traditional’ photography and new technological imageries? How do these new developments in the realm of the image affect the slippery relationship between the document and the artistic genre of the documentary? And more generally, is it still possible to talk about images at all, or should we give up this concept in favour of a different one? This ‘forensic turn’ in visual studies9 — or, rather, the ‘visual turn’ of forensics — is proposing revisions of aesthetics and media theories, since an important part of such investigations aims to make sensible and evidential what so far has been considered as a ‘mere’ thing, such as the dust that is lifted in an explosion, the cracks in the walls of a building, or the growth patterns of trees in forests.10 This doctoral project aims to try to answer these questions by considering photography’s episteme and its remediations in the contemporary media ecology. Cinelli, A World of Imprints Rosa Cinelli/Ph.D. Thesis Abstract1 Ph.D. in co-direction: Università di Milano ERC Advanced Project “An-Iconology. History, Theory and Practices of Environmental Images” Université Côte d’Azur Extended reality research and creative Center xr²c² Particular attention will be paid to the semiotic concept of index as well as to those of trace, imprint, and document. Drawing from visual culture studies and media-archaeology approaches, this project aims to articulate a theoretical framework that will fill these research gaps, and it intends to do so by adopting an interdisciplinary methodology combining the field of aesthetics, semiotics, and theory of photography. On the other hand, looking at what seems to constitute a more sensationalist pole of the scope, the experiences proposed by the field of Virtual Reality immersive journalism11 are characterized by the idea of reporting on news episodes, providing the viewer not only with a hyper-realistic rendering of their object but also to make him or her feel as if she or he were assisting at the scene of the event. The verisimilitude of sensorimotor movements allowed by the head-mounted device, the plausibility of the reconstruction and the emotional and empathic response of the experiencer confer a sense of quasi-reality that is linked to a very strong sense of presence. The feeling of almost ‘being there’12 suggested by these ‘out-of-frame’13 images creates a perpetually unfolding present, which seems to re-actualize the ‘has been’ of Barthesian memory with forms of immediacy: ‘I am there’, ‘that is’, ‘this is happening’. However, this sense of presence does not come without the risks of what has been regarded as ‘toxic’ 154 Cinelli, A World of Imprints Cinelli, A World of Imprints Notes 1 Ph.D thesis supervised by Barbara Grespi (Università di Milano) e Matteo Treleani (Université Côte d’Azur). For information: rosa.cinelli@unimi.it. 1 Ph.D thesis supervised by Barbara Grespi (Università di Milano) e Matteo Treleani (Université Côte d’Azur). For information: rosa.cinelli@unimi.it. 1 Ph.D thesis supervised by Barbara Grespi (Università di Milano) e Matteo Treleani (Université Côte d’Azur). For information: rosa.cinelli@unimi.it. 2 Roland Barthes, Camera Lucida, trans. By Richard Howard (New York: Hill and Wang, 198 3 B. Grespi, ‘L’evidenza dell’immagine. Postfotografia e idea documentaria’, Dalla parte delle immagini. Temi di cultura visuale, ed. by Barbara Grespi and Luca Malavasi (Milan: McGraw Hill, 2022), 61–104. 3 B. Grespi, ‘L’evidenza dell’immagine. Postfotografia e idea documentaria’, Dalla parte delle immagini. Temi di cultura visuale, ed. by Barbara Grespi and Luca Malavasi (Milan: McGraw Hill, 2022), 61–104. 4 André Gunthert, L’image partagée (Paris: Textuel, 2015). 5 Some examples are, The Citizen Evidence Lab held by Amnesty International, the visual journalism section in the New York Times online journal, or the website Bellingcat.com. 5 Some examples are, The Citizen Evidence Lab held by Amnesty International, the visual journalism section in the New York Times online journal, or the website Bellingcat.com. 6 Eyal Weizman, Forensic Architecture: Violence at the Threshold of Detectability (New York: Zone Books, 2017), Eyal Weizman and Matthew Fuller, Forensic Aesthetics. Conflicts and Commons in the Politics of Truth (London, New York: Verso, 2021). 6 Eyal Weizman, Forensic Architecture: Violence at the Threshold of Detectability (New York: Zone Books, 2017), Eyal Weizman and Matthew Fuller, Forensic Aesthetics. Conflicts and Commons in the Politics of Truth (London, New York: Verso, 2021). 7 Ruggero Eugeni, Capitale Algoritmico. Cinque dispositivi postmediali (più uno) (Brescia: Scholé, 2021). 7 Ruggero Eugeni, Capitale Algoritmico. Cinque dispositivi postmediali (più uno) (Brescia: Scholé, 2021). 8 Paul Virilio, The Vision Machine, trans. by Julie Rose (Bloomington: Indiana University Press, 1994). 8 Paul Virilio, The Vision Machine, trans. by Julie Rose (Bloomington: Indiana University Press, 1994). 9 Cf. David Houston Jones, Visual Culture and the Forensic. Culture, Memory, Ethics (London: Routlege, 2022); Thomas Keenan, Eyal Weizman, Mengele’s Skull. The Advent of a Forensic Aesthetics (London: Sternberg Press, 2012). 9 Cf. David Houston Jones, Visual Culture and the Forensic. Culture, Memory, Ethics (London: Routlege, 2022); Thomas Keenan, Eyal Weizman, Mengele’s Skull. The Advent of a Forensic Aesthetics (London: Sternberg Press, 2012). 10 Weizman, Forensic Aesthetics. 10 Weizman, Forensic Aesthetics. In-Transit Televisions: Productive Patterns and Urban Imageries of Mobility Emiliano Rossi/Ph.D. Thesis Abstract1 Università di Bologna DOI 10.54103/2036-461X/17981 From the early 2000s, in Italy, too, the installation of video-communication systems within transportation terminals (i.e., rail and underground stations, airports, waiting shelters, highway rest areas) has progressively affected the urban mediascape, posing new challenges for the design of public infrastructures. This doctoral thesis explores so-called ‘go-television’ screen networks located in Italian high-flow hubs, mainly — but not exclusively — in metropolitan areas: such real-time broadcasting circuits are conceived to entertain and inform spectators caught in the time of waiting and transit (e.g., ‘captive audiences’), through displays and video-totems which interact with synchronised and spatially assembled viewers. As a result, transportation facilities seem nowadays to be characterized by a combination of news stream, recorded image loops, audio announcements, advertisement, multi-screen live relays which became structural components of their genius loci: being containers of audiovisual experiences, these interstitial settings shed light on the multiple ways media interconnect with architectures of transport, forging what scholars have defined the ‘media-architecture complex’.² Outlined thus, go-TV stands for the result of collective efforts: not only there are specialized companies in this market, but transportation companies and local authorities are also included in the intricate production chain of the video contents taken into account in the thesis, blurring the line between editorial clips, marketing materials, traffic bulletins and public utility alerts directed to travellers. Through a mapping of the major national cases of in- transit television, the inquiry aims to deepen the commercial and professional practices implied in the functioning of such audiovisual channels: acting as a last-mile medium, go-TV appears to work as a window on the travelling habits of thousands of daily passengers as well as en- passant spectators, generating and reflecting a particular experience of mobility. This research attempts to situate its object of analysis within the Italian media environment, demonstrating how these networks have grown relevant in virtue of their complex creative drives, the strategies of the professionals involved as well as their proprietary assets and the various purposes of their wide range of stakeholders. First, the thesis positions go-television on a theoretical level, showing how mobility and visual cultures have interwoven since the beginning of the entertainment industry. The double logic of transportation as a material and symbolic carrier is deepened, given that television itself has repeatedly been assessed as a technology of dynamism. Notes 11 Some of the most famous examples are: Hunger in Los Angeles (Nonny De la Peña and others, 2012); Project Syria (Nonny De la Peña et al, 2014); The Protectors (Catherine Bigelow, 2017); Omni (Ai Wei Wei, 2017); Home After War (Gayatri Parameswaran, 2018); Reeducated (Sam Wolson, 2021). 12 Matthew Lombard and others, Immersed in Media. Telepresence Theory, Measurement & Technology (Berlin: Springer, 2015). 13 Cfr. Andrea Pinotti, Alla soglia dell’immagine (Torino: Einaudi, 2021), and Pietro Conte, Unframing Aesthetics (Milano-Udine: Mimesis, 2020). 14 Lisa Nakamura ‘Feeling Good about Feeling Bad: Virtuous Virtual Reality and the Automation of Racial Empathy’, Journal of Visual Culture, 19.1 (April 2020), 47–64. 14 Lisa Nakamura ‘Feeling Good about Feeling Bad: Virtuous Virtual Reality and the Automation of Racial Empathy’, Journal of Visual Culture, 19.1 (April 2020), 47–64. 155 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X In-Transit Televisions: Productive Patterns and Urban Imageries of Mobility Furthermore, this study deals with the connections between the small screen and the urban environment, reflected in the particular socio-topological status of go-TV’s 157 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X consumption settings: attention is reserved, therefore, to the ecological dimension of this medium, to its supposed space-making effects, to its site-dependence — or context-sensitiveness — which operates on a centripetal basis, anchoring the transmission of video contents to the specific spatial frames where they are received. Assuming that the impact of audiovisual media outside the home relies on such a site specificity, the second aim of the thesis is to provide a comprehensive definition of go-television, adequate for the whole range of actors entailed in its production. Third, considering that television developed for travel venues typically aggregates hybrid media forms, the project examines the mediological profile of go-TV: in spite of the growing importance of out-of-home communication, television studies have rarely dealt with video networks outside the domestic sphere (with the notable exception of Anna McCarthy’s inquiries),³ which remains a blind spot of scholarly research. Far from consisting in a mere relocation of TV sets away from the household, in-transit video screens seem to strengthen the televisual specificities at the core of their editorial (and professional) manufacturing, while fostering original devices of brand urbanism and geolocalized marketing, as well as inheriting some specific characteristics of cinema and radio (i.e. large screening surfaces, attractionality, voice- centrism and schedules mainly structured upon a ‘clock’ template). The following section of the thesis features an overview of the commercial and professional habits underlying this channel of distribution, as part of corporate trends which were sampled in the wake of media production studies: the mapping provides an insight on fourteen different companies operating in Italy (beside the profiles of ten now-defunct place media corporations), together with three case studies (Grandi Stazioni Media, Telesia, video portals onboard high-speed trains). These latter contents. In this respect, the inquiry integrates a cultural and industrial approach, and is the result of structured and informal interviews with insiders and company specialists, on-site observations (severely limited by the Covid-19 outbreak) and a review of promotional paratexts, trade press, consultancy reports, internal use documents and journalistic sources. In-Transit Televisions: Productive Patterns and Urban Imageries of Mobility The access, when possible, to semi-public documents as contracts, budget plans, financial statements and commercial agreements — in addition to industrial portfolios, consultancy reports and white papers issued by industrial forums — completed the methodological framework, which also included attendance to business fairs and public initiatives. One of the primary results emerging from this research is that, aligning itself to the ‘mobile privatization’, the ‘TV of the stations’ absorbs and re-mediates the context where it is positioned: the dissertation, therefore, deconstructs the main level on which mobility and its contraries are visualized and depicted on these circuits, exploiting the very same movement of spectators. From this perspective, the areas of in- betweenness examined act either as containers of in situ audiovisual experiences or as the subjects (content) of the messages shown by the screen themselves, reconfiguring notions of space, networks and border regimes. One of the arguments put forward is that, while negotiating distances and perceptions of speed through their moving contents, those video outlets embody and promote images of ‘squared mobility’ (i.e. mobility within mobility), generating multiple senses of place and offering a legitimating self-portrait of the experience of travel. Hence, mobility and its aesthetics work as a narrative trope, also when reduced to codified icons and electronic signs, essential as orientational and wayfinding appliances, rerouting passengers’ movement in public spaces. This fetishization of mobility is functional both for the tourism and transport industries, which capitalise their corporate images, becoming direct publishers of 158 Rossi, In-Transit Televisions televisual contents. In conclusion, this research points out how go-TV can be evaluated also by the prospects of public communication practices: due to the scheduling of collective interest inserts, in-transit video screens are increasingly called to reinvigorate civic engagement of citizen, as well as sustain the participatory processes of modern smart cities, rehabilitating the public role of the televisual medium. Notes 1 Supervisor: Prof. Luca Barra. For more information: emiliano.rossi5@unibo.it 1 Supervisor: Prof. Luca Barra. For more information: emiliano.rossi5@unibo.it cott McQuire, The Media City. Media, Architecture and Urban Space (London: Sage, 2008). 2 Scott McQuire, The Media City. Media, Architecture and Urban Space (London: Sage, 20 2 Scott McQuire, The Media City. Media, Architecture and Urban Space (London: Sage, 2008). 3 Anna McCarthy, Ambient Television. Visual Culture and Public Space (Durham: Duke University Press 2001) 3 Anna McCarthy, Ambient Television. Visual Culture and Public Space (Durham: Duke University Press, 2001). 4 John T. Caldwell, Production Culture. Industrial Reflexivity and Critical Practice in Film and Television (Durham: Duke University Press, 2008). See also Vicky Mayer, Miranda Banks and John T. Caldwell (eds.), Production Studies. Cultural Studies of Media (London: Routledge, 2009). In the Italian context, see Luca Barra, Tiziano Bonini and Sergio Splendore (eds.), Backstage. Studi sulla produzione dei media in Italia (Milan: Unicopli, 2016) and Marco Cucco and Francesco Di Chiara (eds.), “I media industry studies in Italia: nuove prospettive sul passato e sul presente dell’industria cine- televisiva italiana”, Schermi. Storie e culture dei media in Italia, 3.5 (2019). 159 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Contributors / Collaborateurs Maarten Coëgnarts holds a PhD in Film Stu dies and Visual Culture and an MA in Sociology (University of Antwerp, Belgium). Since 2010 he has researched the inter play between embodied cognition, metaphor and cinema. The results have been published in, among others, Image [&] Narra tive, Projections, Cinéma & Cie, Metaphor and Symbol and Metaphor and the Social World. He is co-editor of the book Embodied Cognition and Cinema (Leuven Uni versity Press, 2015) and author of the book Film as Embodied Art: Bodily Meaning in the Cinema of Stanley Kubrick (Acade mic Studies Press, 2019). Philippe Bédard Philippe Bédard Maarten Coëgnarts Emilio Audissino is Associate Professor at Linnaeus University, Sweden. His research interests are audiovisual production and style; screenwriting; neofor malist film and television analysis; comedy; horror; and sound and music for film and media. He is the author of The Film Music of John Williams. Revi ving Hollywood’s Clas sical Style (University of Wisconsin Press, 2014, second edition 2021) the first book-length study in English on the composer, and Film/Music Analysis. A Film Studies Approa ch (2017), a method to analyse music in films that blends Neoformali sm and Gestalt Theory. His current research fo cus is on comedy, with al ready-published articles on the film and television work of the Zucker-A brahams-Zucker trio, and a forthcoming handbook about music in comedy cinema, co-edited with Emile Wennekes.. recently completed a doc torate in film studies at Université de Montréal, where he wrote a dis sertation on the body-ca mera-space relations of select camera movement technologies throughout film history. His resear ch interests include film technology and aestheti cs, phenomenology, Vir tual Reality, virtual pro duction, GoPro cameras, camera movement and screendance. Along with Alanna Thain and Carl Therrien, he is the co-edi tor of an upcoming edited collection titled States of Immersion: Bodies, Me dia Technologies. 160 Contributors / Collaborateurs Contributors / Collaborateurs Adriano D’Aloia is Associate Professor of film and television stu dies at the Università Cattolica del Sacro Cuo re, Milan. He is a founding member of NECS - Euro pean Network of Cinema and Media Studies, and member of the editorial boards of Comunica zioni Sociali. Journal of Media, Performing Arts and Cultural Studies and Cinéma & Cie. Film and Media Studies Journal. On the issues inherent to his proposal, he recently published: “The Psycho logical Construction of Emotion. A Filmological Approach”, Comunicazio ni Sociali. Journal of Me dia, Performing Arts and Cultural Studies, 2, 2017: 265-275 and “Il catalogo musicale pop e il cinema delle emozioni: il caso Yuppi Du (1975)”, Scher mi. Stories and Cultures of Cinema and Media in Italy, 7, 2020: 47-71. is Adjunct Lecturer in Film Studies at the Uni versity of New South Wales, Australia. Her research applies post structuralist and posthu man-feminist approaches to questions of the sen sory and sensuous cine matic experience, and the aesthetics and ethics of rhythm, movement, and affect in experimental film and horror film. Her recent book is The Pulse in Cinema: The Aesthe tics of Horror (Edinbur gh UP, 2020) and she is co-editor of the collection Sound Affects: A User’s Guide (Bloomsbury, for thcoming). is Associate Professor of film and media stu dies at the Università di Bergamo, where he tea ches Visual culture and History of cinema. His research focuses on the aesthetic, intersubjective and enactive features of media experience. He is the author of Neurofilmo logy of the Moving Ima ge. Gravity and Vertigo in Contemporary Cinema (Amesterdam University Press, 2021) and curator of Rudolf Arnheim’s Ita lian writings on cinema (I baffi di Charlot. Scritti italiani sul cinema 1932- 1938, 2009). He is the co-editor with Ruggero Eugeni of Neurofilmolo gy. Audiovisual Studies and the Challenge of Neuroscience (Cinéma & Cie 22-23) and of a rea der on contemporary film theories (Teorie del cine ma. Il dibattito contempo raneo, 2017). 161 Cinéma & Cie vol. 22 no. 38 2022 · ISSN 2036-461X Ian Verstegen is Associate Professor in the Department of Com munication, Media, and Film at the University of Calgary. He is the au thor of Figure and Force in Animation Aesthetics (Oxford University Press, 2019). His articles have appeared, or are forthco ming, in the Journal of Cinema and Media Stu dies, The Velvet Light Trap, Animation, and the New Review of Film and Television Studies. is author of various arti cles and book chapters on cinema, narrative and aesthetics, and co-editor of volumes Compact Ci nematics (Bloomsbury, 2017) and Narrative Complexity: Cognition, Embodiment, Evolution (University of Nebraska Press, 2019). Since 2011 she has worked as lectu rer and assistant profes sor in film and media stu dies in the Netherlands and the United Kingdom. Her current research is focused on embodied and psychological aspects of moving image experien ce. is the Associate Director of Visual Studies at the University of Pennsylva nia. He works on early modern and modern art history, theory and hi storiography. His current writing and teaching are focused on the cognitive semiotics of images and pictures. Current writing projects center on the Vienna School of Art Hi story and an intellectual biography of Rudolf Ar nheim. He has written a series of works on art and psychology, including Ar nheim, Gestalt and Art: A Psychological The ory (Springer, 2005), Co gnitive Iconology: When and How Psychology Explains Images (Rodopi, 2014), and Arnheim, Ge stalt and Media: An Onto logical Theory (Springer, 2019). He is series editor of Routledge Studies and Research in the Psycho logy of Art. 162 Contributors / Collaborateurs Contributors / Collaborateurs
https://openalex.org/W4210887787	https://www.degruyter.com/document/doi/10.1515/omgc-2022-0006/pdf	English	null	Social Media and the Political Engagement of Young Adults: Between Mobilization and Distraction	Online media and global communication	2,022	cc-by	7,669	Online Media Glob. Commun. 2022; 1(1): 6–22 Jörg Matthes* Social Media and the Political Engagement of Young Adults: Between Mobilization and Distraction https://doi.org/10.1515/omgc-2022-0006 Received July 14, 2021; accepted October 22, 2021; published online February 8, 2022 https://doi.org/10.1515/omgc-2022-0006 Received July 14, 2021; accepted October 22, 2021; published online February 8, 2022 https://doi.org/10.1515/omgc-2022-0006 Received July 14, 2021; accepted October 22, 2021; published online February 8, 2022 Article Note: This article underwent single-blind peer review. *Corresponding author: Jörg Matthes, PhD (University of Zurich), Professor and Department Chair, Department of Communication, University of Vienna, Währinger Strasse 29, 1090 Vienna, Austria, E-mail: joerg.matthes@univie.ac.at. https://orcid.org/0000-0001-9408-955X Open Access. © 2022 Jörg Matthes, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Open Access. © 2022 Jörg Matthes, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Political Engagement of Young Adults 7 7 develop, describe, and empirically test a comprehensive model of how social media use has the potential to mobilize and to distract. develop, describe, and empirically test a comprehensive model of how social media use has the potential to mobilize and to distract. Keywords: social media, turnout, cross-national comparison, political participa- tion, entertainment, distraction, election Abstract Purpose: Scholars have expressed great hopes that social media use can foster the democratic engagement of young adults. However, this research has largely ignored non-political, entertainment-oriented uses of social media. In this essay, I theorize that social media use can significantly dampen political engagement because, by and large, young adults use social media primarily for non-political purposes, which distracts rather than mobilizes. Design/methodology/approach: I illustrate this argument using aggregate level data from the U.S., Germany, Switzerland, and Japan by comparing relative voter turnout and social media use data of young adults. Findings: Data suggest a so called Social Media Political Participation paradox in those countries: The gap in voter turnout between young adults and older gener- ations has not significantly decreased, despite a skyrocketing rise of social media use on the side of young adults, and the overwhelming research evidence that social media use fosters offline political participation. Implications: When trying to understand the implications of social media for democracy across the globe, entertainment-oriented content needs to be brought back in. Originality/value: This essay challenges the dominant research paradigm on so- cial media use and political participation. It urges future research to theoretically 8 for the democratically positive outcomes of social media use is simply over- whelming, particularly in cross-sectional survey research (Boulianne 2011; Chae et al. 2019; Skoric et al. 2016) and also with respect to young adults (Boulianne and Theocharis 2020). However, scholarship on the democratic outcomes of social media frequently seem to overlook the fact that social media are primarily used for entertainment and relational purposes, especially when it comes to young adults (Dimitrova and Matthes 2018; but see Skoric and Zhu 2016; Theocharis and Quintelier 2016). That is, the social media use of young people is clearly dominated by non-political content (Binder et al. 2021). Yet the vast majority of studies do not take these forms of exposure into account, eventually ignoring a large share of the diversity in content on social media. As a consequence, scholars have turned a blind eye on potentially distractive effects of social media use on political engagement, leading to a skewed overall picture of this research ﬁeld. In this conceptual paper, I take a different approach by theorizing that social media use can signiﬁcantly dampen political engagement. The main reason is that social media are primarily used for entertainment and social networking purposes, which has the potential to distract rather than mobilize (Heiss and Matthes 2021). In what follows, I will briefly summarize the literature on digital media and young adults’ political engagement. Then, using illustrative, aggregate level data from the U.S., Germany, Switzerland, and Japan, I will then describe the so called Social Media Political Participation Paradox, that is, the gap in voter turnout between young adults and older generations has not signiﬁcantly decreased, despite a skyrocketing rise of social media use, on the side of young adults, and the huge amount of studies suggesting a signiﬁcant relation between social media use and political participation. Then, I will develop a theoretical argument for why social media use is also likely to disengage, challenging the prevailing academic reading of the literature as well as the existing empirical evidence. 1 Introduction Around the globe, social media have become a centerpiece in young adults’ lives. Particularly with their smartphones, young adults can literally be on social media 24/7, permanently connected to the world and their peers (Vorderer and Kohring 2013). In fact, when comparing the current young generation to their older coun- terparts, there is a fundamental difference in media use behaviors: While young adults, aged 16–25, rely on digital platforms or messenger services, such as Facebook, TikTok, YouTube, Twitter, Instagram, WhatsApp, or WeChat, to get the news, the older generation is much more likely to be exposed to traditional news sources such as television or newspapers. At the same time, there are dozens of studies around the globe demonstrating that, traditionally, young adults are less interested in traditional politics compared to older generations (Delli Carpini 2017), less likely to vote, and generally less politically sophisticated (Binder et al. 2021). In short, political parties had been, before the emergence of digital media, struggling to reach out to the younger generation. Especially when it comes to traditional institutions, you adults are often described as detached and apathetic (e.g., Binder et al. 2021; Loader et al. 2014). Yet with social media, scholars have expressed great hopes regarding young adults’ democratic engagement (see Binder et al. 2021; Oser and Boulianne 2020): It has been argued that particularly social media can build new relationships be- tween political actors and young adults, enable social interaction about political topics, connect people, enhance political opinion expression, equalize engage- ment and generally foster participation as well as boost voter turnout or contribute to social cohesion (e.g., Boulianne 2011, 2015, 2020; Goh et al. 2019; Loader et al. 2014). So, with digital media, there are grounds to believe that the generational engagement gap may be reduced, and that young citizens could be reengaged into the political world. In fact, scholars working on digital media and political engagement have been fascinated by this idea, largely pointing to democratically welcomed outcomes of social media use, such as learning or participation. For instance, researchers observed a positive relationship between the frequency of social media use and protest participation among the youth (Valenzuala et al. 2014), and more generally, it has been found that political social media use is positively related to various forms of political participation (e.g., Ekström et al. 2014; Skoric and Zhu 2016). With recent meta-analyses on the topic, the evidence 8 J. Matthes 8 J. Matthes 9 Political Engagement of Young Adults party membership, and activism, which allows for inﬂuence in the political de- cision making process though protests represent distinct dimensions of political engagement (Ekman and Amnå 2012). Social media has given rise to entirely new forms of action and interaction that can only happen in a digitally networked space, such as online petitions or com- menting on politicians’ posts (Sloam 2014). Social networks allow for non- institutionalized and horizontal modes of engagement, which are often favored by young adults (Sloam 2014). Since such forms of political engagement only exist and can be exercised within social media, they also have to be treated as a separate subdimension of political engagement. Overall, the various forms of engagement can be described as either institutionalized (e.g., voting) or non- institutionalized (e.g., protest behavior). This distinction is relevant because “young adults are disproportionately more likely to participate through non- institutionalized means.” (Weiss 2020, p. 3), particularly in the online world. This is by no means a new insight. Since decades, scholars have bemoaned a participation gap between younger and older individuals (Quintelier 2007), as “in almost every election young people are the least likely to vote, and these participation rates are continuously declining” (Quintelier 2007, p. 165). For instance, in Austria, young people are allowed to vote at the age of 16, but nevertheless, their turnout rates are comparably low (Binder et al. 2021). Findings from other countries also suggest that young people have comparatively negative attitudes toward politics and low trust in the political system (Quintelier 2007; see Binder et al. 2021). But there is also hope. In this research area, “[y]outh’s digital media use is often seen as a partial remedy to the decline of youth participation in political and civic life” (Boulianne and Theocharis 2020, p. 112). The argument is that digital media is an important source of information for those not primarily interested in institutional politics. More speciﬁcally, it is generally assumed that social media fosters young adults’ political engagement because the networking function of social media helps young citizens to develop skills and psychological dispositions that promote ofﬂine participation (Kahne and Boyer 2018). Also, a seminal, cross- sectional study by Gil de Zuniga et al. (2012) suggests that social media indirectly promote participation by fostering opinion expression, which is a key driver of political participation. 2 Digital Media and Young Adults’ Political Engagement Political engagement, often also referred to as political participation, is under- stood as “actions or activities by ordinary citizens that in some way are directed toward influencing political outcomes in society” (Ekman and Amnå 2012, p. 287). This entails a diverse repertoire ranging from traditional (e.g., voting) and non- traditional (e.g., political online discussion; see Hopmann et al. 2015) forms of engagement to political consumerism (Skoric et al. 2016). Political engagement can be conceptualized along the lines of individual and collective actions (Adler and Goggin 2005). In addition, formal political participation, such as voting or 10 J. Matthes 10 J. Matthes 10 In addition, it has been theorized that social media fosters incidental exposure to political information, which leads to learning effects, and ultimately, an in- crease in traditional forms of political participation (Matthes et al. 2020). Furthermore, as explained by Boukes (2019), social media increase the opportu- nities for citizens to learn because “never before has so much information, mostly without extra ﬁnancial costs, been so close at hand for citizens” (p. 39). In line with this argument, the timeline structure of social networking platforms can lead to a “by-product” learning mechanism through which knowledge gaps are reduced (Chadwick 2012), ultimately leading to political participation. And, more germane to young adults, the Impressionable Years Hypothesis (Sears and Levy 2003) suggests that young people are more susceptible to social media effects compared to adults, as political efﬁcacy and engagement are not fully consolidated during adolescence (e.g., Eckstein et al. 2012). That is, when exposed to social media, the notion of efﬁcacy can be strengthened in young adults, leading to participation. So overall, the dominant argument is that social media use can foster soft— that is, non-institutionalized—forms of engagement in a first step, such as online political expression or low-effort forms of political participation. This, in turn, will then shape classical, institutionalized types of political engagement in a second step. As Bode et al. (2014) have put it, “When adolescents use social networking sites – something many of them do in their daily lives – in a new, politically oriented way, it may actually increase their likelihood of participating in politics in other ways.” (pp. 424–425). In addition, social media can also directly lead to a rise in ofﬂine participation among young adults (Boulianne and Theocharis 2020). This theory, in line with the available meta-analyses, leads to the clear prediction that social media use should increase not only non-institutionalized forms of partici- pation, but also—indirectly or directly—traditional forms such as voting. The available “ﬁndings offer a strong, conclusive statement that online and ofﬂine forms of engagement are highly correlated; youth engage in both environments” (Boulianne and Theocharis 2020, p. 123). But how is this observable at the aggre- gate level when it comes to the institutionalized forms of participation, such as voting in national parliamentary elections? As another indirect effect of social media on political participation, it has been argued that some forms of social media use, such as news sharing, can strengthen social relationships and increase social cohesion, which in turn, may be the driver for collective action and group engagement (e.g., Goh et al. 2019; Hwang and Kim 2015). Similarly, social media shapes civic attitudes, and such “civic identity is a central factor that fosters civic engagement because it helps individuals to see society as a construction of human actors with political and moral goals” (Chen et al. 2015, p. 445). 11 Political Engagement of Young Adults Political Engagement of Young Adults the past decades, social media use of young adults, unlike older generations, increased from basically zero to very high levels. That is, assuming there is a signiﬁcant small to medium effect size for the relationship between political social media use on online participation, and a medium to large effect size for the rela- tionship between online and ofﬂine participation (Boulianne and Theocharis 2020), we would expect that, in the past decades, the generational gap should have decreased. 3 The Generational Political Participation Gap To reiterate, the previously available literature unequivocally suggests that social media use, particularly political forms of use, can foster online and offline political engagement (e.g., Boulianne and Theocharis 2020). With these insights at hand, the traditional generational gap between the young and older generations in terms of institutionalized participation needs to be revisited. One could argue that, over 12 increase in the generational gap, rather than a decrease. Although these are only aggregate level data, they do not suggest that social media use—emerging in the last two decades—has completely changed the picture. increase in the generational gap, rather than a decrease. Although these are only aggregate level data, they do not suggest that social media use—emerging in the last two decades—has completely changed the picture. Data from the United States suggest a similar, albeit not identical picture. As can be seen in Figure 2, the generational gap in voter turnout is clearly visible over time, although young individuals slightly increased in turnout (e.g., from 44.30% in 2008 to 48% in 2020), whereas turnout for older generations remained largely at the same level. However, a fundamental shift in turnout when it comes to young adults is clearly not visible in the data, the generational gap is still evident. In Switzerland (Selects 2019), 28% of young adults aged 18–24 participated in the national parliamentary election in 1999 (35% in 2003), 33% did so in the last election in 2019. By contrast, 57% of adults aged 65–74 participated in the election in 1999 (54% in 2003), and 62% did so in the last election in 2019. Again, a clear generational gap that has been consistent over time. Finally, looking at data from Japan, the turnout of people aged 20–24 was 35.3% in 2012 and 30.69% in 2017. These numbers, however, were signiﬁcantly higher for individuals aged 65–69 (77.15% in 2012 and 73.35% in 2017), 70–74 (76.47% in 2012 and 74.13% in 2017), and 75–79 (71.02% in 2012 and 70.26% in 2017). This suggests a huge generational gap when it comes to participation in national elections in those countries, and there is no clear indication the gap has decreased over the years. So overall, these aggregate data deliver a clear message for these four coun- tries, none of which has witnessed huge protest movements outside traditional party politics. Young adults are significantly less likely to vote in national elections compared to older generations, and it seems this gap has not decreased over the Figure 2: Voter Turnout in National Elections and Young Adults Social Media Use over Time, USA. Source for voting: U.S. Census Bureau, Current Population Survey, Source for social media use: Pew Research Center, 2021. 3.1 The Social Media Political Participation Paradox To test this idea, I draw on official voter data (i.e., not self-report survey data) from the National Parliamentary Elections in Germany. As can be seen in Figure 1, as can be expected, social media use of young individuals has increased from zero in the early 2000s to more than 80% at the time of the last election in 2017. So, in terms of the meta-analyses reported above and although such aggregate level analyses do not allow causal conclusions, one of the theorized drivers of engagement has witnessed a skyrocketing increase. But paradoxically, as visible in Figure 1, there were no signiﬁcant increases in voter turnout for individuals aged 18–24 over the years. By contrast, in 1983, young individuals showed a turnout of 84.30 (18–20 year old) and 81.50 (21–24 year old)% in the national election. In 2017, these numbers were signiﬁcantly and substantially lower (69.90% and 67.00% respec- tively). That is, if anything changed, the numbers are more suggestive of an Figure 1: Voter Turnout in National Elections and Young Adults Social Media Use over Time, Germany. Source for social media data: Koch and Frees (2007), source for election data: Bundeswahlleiter (2017). Figure 1: Voter Turnout in National Elections and Young Adults Social Media Use over Time, Germany. Source for social media data: Koch and Frees (2007) source for election data: Bundeswahlleiter Figure 1: Voter Turnout in National Elections and Young Adults Social Media Use over Time, Germany Figure 1: Voter Turnout in National Elections and Young Adults Social Media Use over Time, Germany. Source for social media data: Koch and Frees (2007), source for election data: Bundeswahlleiter (2017). Figure 1: Voter Turnout in National Elections and Young Adults Social Media Use over Time, Germany. Source for social media data: Koch and Frees (2007), source for election data: Bundeswahlleiter (2017). Germany. Source for social media data: Koch and Frees (2007), source for election data: Bundeswahlleiter (2017). 12 J. Matthes J. Matthes J. Matthes 13 Political Engagement of Young Adults past decades, as one would expect from the enthusiastic voices in research on social media use and political participation. In fact, the findings appear somewhat paradox: The social media use has increased from zero to almost 100% in the last two decades, which should, considering the effect sizes observed in previous research, also become visible at the aggregate level. So, on the one hand, we learn from previous research that social media significantly fosters political participation, online and offline (e.g., Boulianne and Theocharis 2020), on the other hand, we don’t see a higher turnout compared to older generations, who are much less likely to use social media compared to their younger counterparts. And even if they did, we would expect an increase in turnout for this group as well, which has not been the case. This phenomenon can be called the Social Media Political Participation Paradox. In short, at the aggregate level, it appears as if social media has not changed a thing, although social science research suggests quite the opposite. Figure 2: Voter Turnout in National Elections and Young Adults Social Media Use over Time, USA. Source for voting: U.S. Census Bureau, Current Population Survey, Source for social media use: Pew Research Center, 2021. J. Matthes 14 participation efforts of young adults are often short-lived, they can rise to sub- stantial amounts during times of protest, but remain low in times of national elections. It would sufﬁce if one of the appraisals is negative, as for instance, when other goals appear to be more important in an behavioral situation. Obviously, typical self-report survey studies cannot fully grasp the process described in the model because the behavioral situation is hardly taken into account in the typical research designs and the processes can hardly be measured in retrospect. Second, it has been argued that social media is more likely to impact non- institutionalized forms of participation, rather than institutionalized ones (Sloam 2014). That is, social media may engage young people politically, but that doesn’t necessarily make them more likely to participate in elections. In other words, social media can have the potential to engage, but such engagement may be short-lived, conditional, and bound to speciﬁc topics such as environmental engagement, animal rights, or social protest. Third, and more importantly, when looking at the democratically relevant effects of social media use, scholars rarely consider (or control) non-political, entertainment-oriented content (Boulianne and Theocharis 2020). Non-political, entertainment-oriented content can be information on lifestyles, products, leisure, sports, or social relationships on social media (see Hanitzsch and Vos 2018). Yet when we estimate the effects of political social media use without at least con- trolling non-political forms, we may end up with erroneous conclusions about the mobilizing potential of social media. Political contents and entertainment- oriented contents are simultaneously present on social media. A typical news- feed completely mixes both. As expressed by Boulianne and Theocharis (2020), “purely social-, entertain- ment-, and leisure-oriented activities carried out on digital media do not necessarily mobilize individuals for civicor political action.” (p. 114)Entertainment-oriented use of social media may serve the purpose to create an “emotional relief generated by temporarily recreating or recessing from daily routines” (Buzeta et al. 2020, p. 81). When compared to political social media use, entertainment-oriented use accounts for a large proportion of everyday Internet use, especially among adolescents (Büchi etal.2016). For instance,ina recentovertime experimentusing behavioral browsing data, Wojcieszak et al. (2021) found that news websites comprised only 3.54% of the browsing behavior. The authors conclude that “news content is nearly unnoticeable in the context of overall information and communication ecology of most in- dividuals” (p. 8). 3.2 Explaining the Paradox Of course, on the methodological the most obvious explanation is ecological fal- lacy. We can’t derive any assumptions on individual level effects when looking at aggregate level data. Ultimately, no causal claims can be made, nor can we say anything about intraindividual change or processes. There may also be simulta- neous, competing processes, and third variables cannot be controlled at all. Yet what we learn is that there is a generational gap in participation at the aggregate level which is, by and large, rather substantial. So at best, the data reported above can be understood as anecdotal evidence. Yet on theoretical grounds, the ﬁndings could inspire us to elaborate on why social media use may not automatically lead to strong shifts in institutionalized forms of political participation. Several reasons can be found for this in the literature. On the theoretical side, the recent Social Media Political Participation model (Knoll et al. 2020) holds that social media can foster participation only when a chain of subsequent conditions are met. According to this model, young adults have to be (intentionally or inci- dentally) exposed to political content on social media, they need to appraise po- litical content on social media as relevant (as compared to other content that is simultaneously present), there needs to be a discrepancy between a current state and a future, desired state, they need to believe that a participation goal is attainable, and this goal must then be dominant in a real behavioral situation, in which other behavioral goals may be present as well (see Knoll et al. 2020). If one of the conditions is not met, social media use may not increase ofﬂine forms of political participation, according to the model. This model can explain why 14 J. Matthes Political Engagement of Young Adults Political Engagement of Young Adults Political Engagement of Young Adults Bowyer (2018), observed that non-political content can serve as a gateway to various forms of civic and political participation. Yet other studies found no such gateway effects. In a cross-sectional study conducted in the Netherlands, Bakker and de Vreese (2011) observed non-political social media use had negative con- sequences for political participation. Additionally, data collected by Chan et al. (2012) suggest that the effect of Weibo use on the willingness to express one’s political views was weakened when entertainment motives prevailed. An experi- ment by Theocharis and Lowe (2016) even suggests negative consequences of Facebook use for participation because it can distract from politics. So taken together, only a few studies looked into the effects of non-political social media use on participation, some suggest that entertainment activities on social media may serve as a gateway to participation (Kahne and Bowyer 2018), others speak of distraction from politics (Bakker and de Vreese 2011; Boukes 2019; Chan et al. 2012; Theocharis and Lowe 2016). Besides adding entertainment-oriented exposure as a predictor, it is also important to understand how entertainment-oriented and political uses of social media interact. If we accept both types of uses as separate dimensions, we arrive at four different types of audience members: “the Avoiders” (political use: low and entertainment-oriented use: high), “the Inactive” (political use: low and entertainment-oriented use: low), “the Distracted” (political use: high and entertainment-oriented use: high), and “the Focused” (political use: high and entertainment-oriented use: low). This typology of ideal groups is depicted in Figure 3 (see Matthes et al. 2021). For the purposes of the present paper, the theoretically most relevant group are “the Distracted”. I use this term because I theorize that high loads of enter- tainment content may potentially distract the processing of political content. There are several theoretical reasons for that. As suggested by the priming literature (Higgins 1996), the accessibility of concepts can drive cognitions and behaviors. Thus, when young adults evaluate the importance of concepts, they do not use all of the information they have available in memory. Instead, they often rely highly accessible information (Iyengar and Kinder 1987). Here, accessibility refers to the ease or speed with which available information can be retrieved from memory. Information that is recently and frequency activated, is more likely to be temporary accessible (Arendt and Matthes 2014). Despite the relevance of non-political content on social media, scholars have hardly looked at the relationship of entertainment-oriented use of social media and political participation (Skoric et al. 2016). As one rare exception, Kahne and 15 Political Engagement of Young Adults At the same time, of course, individuals’ cognitions are also driven by their basic political and social orientations which are be chronically accessible. But the inﬂuence of chronically accessible concepts can be weakened when individuals are frequently and recently exposed to other considerations. When, for instance, people are exposed to entertainment-oriented content, these non-political considerations become cognitively accessible. That is, when young adults are permanently confronted with non-political content, as for 16 J. Matthes 16 J. Matthes J. Matthes 16 Political Use Political Use Entertainment-oriented Use Entertainment oriented Use Figure 3: A typology of users based on the two dimensions entertainment-oriented use and political use of social media. Political Use Political Use Entertainment-oriented Use Entertainment oriented Use Figure 3: A typology of users based on the two dimensions entertainment-oriented use and political use of social media. 17 Political Engagement of Young Adults holds that the goal to vote needs to be dominant in a real behavioral situation. When other goals are present as well, such as visiting friends or going shopping, then young adults will only cast their vote when this goal is dominant, that is, more important than other goals. So again, the argument is that non-political content on social media can shift the appraisal of goals. What is more, we know from entertainment research that particularly hedonic entertainment content has a high absorption potential, and based on that, it can interfere with the cognitive elaboration of political content, “because it absorbs attentional resources to a degree that interferes with further elaboration” (Bartsch and Schneider 2014, p. 376). Also, on a perceptual level, non-political content is often perceived as more eye-catching as compared to political content. The reason is that the former typically relates to close ties such as friends and family and it is often associated with immediate positive gratiﬁcations. Moreover, not all individuals may be exposed to political and non-political content on social media simultaneously. “The Avoiders”, albeit high in general social media use, may try to bypass exposure to political content, as for instance when an “individual exposes him- or herself to a limited amount of news because other content has more appeal to him or her, many algorithms will make future content decisions in favor of other content, e.g., entertainment, and give news stories a lower priority” (Skovsgaard and Andersen 2020, p. 466). Young adults interested in non-political contents may curate their newsfeed on SNS platforms in ways which exposes them to high degrees of entertainment-oriented content with low likelihood of exposure to political contents. This may lead to low-effort, feel- good types of engagement at best, making high-effort, ofﬂine political participa- tion less likely. But still, but even for news avoiders, scholars have argued that social media has the potential to foster participation because people can be accidentally exposed to political information in their newsfeeds. So even though young adults may not want to see political information, they may see it based on their social environments and networks (Matthes et al. 2020). This exposure to political information may then lead to learning, and ultimately, participation. Figure 3: A typology of users based on the two dimensions entertainment-oriented use and political use of social media. instance, about friends, family, movies, or sports, this content gets situated at the top of the head, making it more cognitively accessible when making judgments about political matters, including engagement. That means, non-political content on social media competes with (and may even impede) the accessibility of political content. The Social Media Political Participation model (Knoll et al. 2020) holds that political content on social media can only affect ofﬂine political participation when the content is appraised as relevant. However, when young adults are primarily exposed to non-political content, this may shift the perceived relevance of political considerations. In other words, when there is an abundance of entertaining, non-political news, the current political issues may appear less severe, and therefore, also less personally rele- vant. As a consequence, young adults may engage less with political content, which is a prerequisite to political participation. By the same token, the model While scholars agree on the importance of incidental exposure for participatory out- comes, they have, at the same time, entirely ignored the opposite logic: Social media also exposed individuals accidentally to non-political information. Such incidental exposure to non-political content “can have important effects on po- litical outcomes such as learning or participation. The more people are confronted with non-political content (without actively looking for it), the more they are potentially distracted from their primary political processing goal” (Matthes et al. 2020, pp. 1137–1038). So as much as incidental exposure to political content can engage, so can incidental exposure to non-political content distract and 18 J. Matthes J. Matthes J. Matthes 18 disengage. Unfortunately, while there are dozens of studies on the former phenomenon, we lack studies on the latter. disengage. Unfortunately, while there are dozens of studies on the former phenomenon, we lack studies on the latter. 3.3 Testing the Paradox The paradox described here is situated at the aggregate level. However, additional evidence is needed to explain why this paradox occurs. For this, it is therefore important to look at the outcomes of entertainment-oriented content at the indi- vidual level. That is, we should not only model political media use as a predictor of participatory outcomes, but simultaneously access (or at least control) exposure to non-political content. These two dimensions can then not only be used as focal predictors, they may also interact in a regression model (see Figure 3). High levels of entertainment-oriented non-political content on social media may have a “vampire” effect, when young adults are simultaneously exposed to political content on social media as well. Some young adults, those with high levels of political interest and sophistication, may primarily be exposed to political content on social media, which in fact, can be theorized to increase levels of online and ofﬂine political participation (Knoll et al. 2020). These conjectures suggest that social media use, as a generic category, is of limited use in global research on digital media. A similar argument can be made for the measurement of political participa- tion. Oftentimes, scholars create participation indices by averaging several, distinct acts, such as wearing buttons of a party, sharing personal political ex- periences on social media, signing a petition, or voting in an election (see for a discussion, Theocharis and van Deth 2018). These measures blur the differences between institutionalized and non-institutionalized forms of participation. Differentiating between these forms beyond the classic online versus ofﬂine notion, however, is crucial to understand the political consequences of social media use. In addition to that, it is important to note that the typical survey approach used in political communication scholarship has its limitations as well. The reason is that self-report measures of social media use are largely biased (Scharkow 2019). Also, typical experiments use forced-exposure settings, which cannot be compared to news reception situations in the real world (Wojcieszak et al. 2021) Thus, to empirically examine the paradox, future studies need to rely on more naturalistic settings, such as web-tracking data or mobile experience sampling. With such measures, we can more precisely estimate the amount of exposure to political and non-political content. 19 Political Engagement of Young Adults 4 Conclusion With the emergence of social media, great hopes have been expressed across the globe that young adults may reengage into traditional politics, directly due to the various activities happening on social media, and indirectly by shaping low-effort forms of online-engagement in the first place, which is then assumed to shape offline participation in a second step. In fact, the available evidence clearly sug- gests that social media use, particularly political one, has an impact on offline forms of participation (Boulianne and Theocharis 2020). However, aggregate level data from Germany, the U.S., Switzerland, and Japan indicate that generational gaps between younger and older adults have not or only marginally decreased. Although there was an unprecedented up rise of social media use over the past decades (including an increase in political uses), voter turnout did not change signiﬁcantly, and was substantially lower as compared to the older generation. At the same time, the entire body of research on social media and political partici- pation would suggest an increase in participatory activities of young adults. In explaining this paradox, this paper pointed to the potentially distracting functions of social media, mainly due to non-political, entertainment-oriented content. 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https://openalex.org/W2267352312	https://europepmc.org/articles/pmc4734642?pdf=render	English	null	Integration and Validation of the Genome-Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism	PloS one	2,016	cc-by	13,598	RESEARCH ARTICLE OPEN ACCESS Citation: Tomàs-Gamisans M, Ferrer P, Albiol J (2016) Integration and Validation of the Genome- Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism. PLoS ONE 11(1): e0148031. doi:10.1371/journal.pone.0148031 Integration and Validation of the Genome- Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism Màrius Tomàs-Gamisans, Pau Ferrer, Joan Albiol* Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain * joan.albiol@uab.cat * joan.albiol@uab.cat * joan.albiol@uab.cat * joan.albiol@uab.cat Motivation Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as dis- covering new properties of metabolic networks, predicting new targets for metabolic engi- neering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterolo- gous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented. Editor: Wagner L. Araujo, Universidade Federal de Vicosa, BRAZIL Received: August 26, 2015 Accepted: January 12, 2016 Published: January 26, 2016 Copyright: © 2016 Tomàs-Gamisans 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. Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain Introduction Genome-Scale metabolic models (GEMs) have become one of the most useful and widely employed tools in systems biology in the last fifteen years [1]. Since the first genome based met- abolic model was presented [2], a huge number of models have been developed for a broad variety of species, from archaea and bacteria, to higher eukaryotes [3]. These models link geno- type with phenotype; thus, they can predict the behavior of an organism under certain environ- mental conditions [4–6]. GEMs have been applied for both descriptive and predictive purposes. They have been used for multiple omics data integration [4, 7], for discovering meta- bolic network properties and organism capabilities as well as for comparing these capabilities between organisms. In addition they are commonly used for predicting metabolic engineering targets to improve growth and production of chemicals or recombinant proteins, thus making processes more efficient at industrial-scale [8–12]. Regarding the expression of heterologous proteins, Pichia pastoris has become more exten- sively used for industrial applications [13, 14]. It has many interesting properties that make it suitable for recombinant protein production. One of the main advantages is the availability of well-established protocols and techniques for its genetic manipulation [15, 16] that enables the expression and secretion of recombinant proteins, even those with complex post-translational modifications [17–19]. Moreover, there are a number of promoters available which are suitable for regulation with different carbon sources [20, 21]. Specifically, one of the most commonly used is the very powerful and tightly regulated AOX promoter, inducible with methanol [19]. Finally P. pastoris is able to grow up to high cell densities, resulting in high protein production yields [22, 23]. Due to the increasing interest in using this advantageous methylotrophic yeast, several efforts have provided tools to better understand its operation, from physiological char- acterization to metabolic modelling. The first metabolic models for P. pastoris were limited to the central carbon metabolism and were used for 13C flux data analysis [24–27]. In 2009, the genome of two different strains, DSMZ 70382 [28] and GS115 [29], was sequenced. More recently, a third strain (CBS7435) was also sequenced and annotated [30] showing some discrepancies with previously reported sequences. Once the genomic data was available, two GEMs were published simultaneously: iPP668 [31] corresponding to GS115 strain, and PpaMBEL1254 [32] based on the DSMZ 70382 genome. Integration, Upgrade and Validation of the P. pastoris' GEMs conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the iMT1026 model has shown a better performance than the previous existing models. conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the iMT1026 model has shown a better performance than the previous existing models. IPCRES network (Integrated Process and Cell Refactoring Systems for Enhanced Industrial Biotechnology - http://www.era-ib.net/ipcres). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. IPCRES network (Integrated Process and Cell Refactoring Systems for Enhanced Industrial Biotechnology - http://www.era-ib.net/ipcres). 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. Results Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Additionally, model file in SBML will be available from the BIOMODELS database (https://www.ebi.ac.uk/ biomodels-main/; accession number MODEL1508040001). In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental Funding: This work was supported by the project CTQ2013-42391-R of the Spanish Ministry of Economy and Competitiveness and the grant FPU12/ 06185 (M.T.) of the Spanish Ministry of Education, Culture and Sport. Also the authors would like to acknowledge partial support from the ERA-IB PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 1 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Introduction Two years later, a third model (iLC915) became available, also based on the GS115 genome [33]. Moreover, other GEMs derived by automatic application of reconstruc- tion algorithms are also available [34, 35] which will not be discussed in this paper due to their low level of curation and comprehensiveness. P. pastoris’ GEM models are potentially useful platforms for bioprocess design and optimi- zation, as well as for strain metabolic engineering [36, 37]. In fact, Nocon and co-workers [38] have already used PpaMBEL1254 to predict overexpression and deletion mutants to enhance recombinant protein production. Each one of the three currently available models is fully compartmentalized, but they differ in the number of reactions and metabolites. The first two models, iPP668 and PpaMBEL1254 have a comparable number of associated genes, reactions and metabolites, as well as similar 2 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs metabolite and reaction identifications and nomenclature. On the other hand, iLC915 incorpo- rates more P. pastoris specific gene-protein-reaction associations and hence, a larger number of genes, but several extracellular and nuclear reactions are missing. In general terms, these mod- els cover the same metabolic processes, but iLC915 is more detailed. Nevertheless, there still exist some critical issues in these models, such as missing and divergent information or reac- tions that require manual revision and curation. Similar to other organisms, such as Saccharomyces cerevisiae, new versions and updated models integrate previous versions and incorporate new features and information from newly published literature. In the case of S. cerevisiae, despite the existence of other versions, a con- sensus metabolic model was developed [39] and it was further developed and upgraded, being expanded and revised up to the 7th version [40–43]. In this work, we compare the models of P. pastoris and provide an upgraded version. As mentioned above two different strains were used to obtain these models. Nonetheless, there is a high degree of identity at the amino acid coding sequences level (93.7%) and functional anno- tation between the two genome sequences [32]. In addition, no differences were observed in reactions involved in the metabolization of the different carbon sources [44]. Therefore, a major objective of our study was to obtain a general model that can be applicable to both strains. Introduction Furthermore, an extensive analysis was performed on several pathways, comparing the three models and updating them with the newly published literature. Recent findings on P. pas- toris physiology and metabolism enabled to complete sphingolipid biosynthesis metabolism and glycosylation pathways, as well as the oxidative phosphorylation electron transport chain. Furthermore, we included different biomass compositions specific for each of the alternative carbon sources used. Finally, the model accuracy was tested in a variety of physiological conditions. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Materials and Methods Model merging For the model comparison, an initial step of metabolite nomenclature unification was required. In PpaMBEL1254 only the identifier (ID) was available in the SBML file, i.e. neither the com- plete name of metabolites nor any association to a reference database was included. These metabolite IDs, were mostly the standard IDs most commonly used and therefore also included in both in BiGG [45] and The SEED [46] reference databases. After this first metabolite parsing and renaming step was done, MetaNetX [47] database was used in order crosslink information from KEGG [48], ChEBI [49] and MetaCyc [50]. This step not only allowed unifying metabo- lite names but also to include its molecular formula and charge at pH 7.2. Once all metabolite names were unified, PpaMBEL1254 and iPP668 were compared using ModelBorgifier [51], thereby obtaining a first pre-merged model. In a second step, this merged model was compared to iLC915, generating a first draft of the consensus model. Due to impor- tant differences in model structure between iLC915 and the other models, a manual compari- son was necessary. This was performed by analyzing the structure of each of the remaining pathways or subsystems. Differences were resolved according to the available literature, com- paring the reactions with those included in two latest versions of the consensus S. cerevisiae GEMs [42, 43] or in another recently published S. cerevisiae GEM [52]. Divergences in gene assignments were resolved using P. pastoris or S. cerevisiae literature. The P. pastoris high qual- ity sequence annotation [30] and the automatic reconstructions for P. pastoris [34, 35] were also used to verify annotations and gene-reaction assignments from the previous models. Finally, pathway revamping and addition of new reactions was performed based on available yeast literature and metabolic pathways/reaction databases [45, 50, 53, 54]. 3 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs Eventually, the network was loaded into a convenient environment for debugging [55]. Thus, both COBRA [56] and RAVEN [57] toolboxes were used in order to ensure pathway connectivity and biomass formation. Duplicated reactions in the final model were deleted and blocked reactions were connected (gap filling) to the network when few steps were required. In addition, the elemental mass balance of each reaction was checked and corrected when unbal- anced. Materials and Methods Model merging The final model (S1 and S5 Files) can also be obtained in SBML format from BIOMO- DELS database with accession number: MODEL1508040001 [58]. The SBML model was generated with the RAVEN toolbox [57] and validated with SBMLeditor [59]. Biomass and recombinant protein composition The biomass reaction is defined by the sum of biomass components, grouped in macromole- cules (carbohydrates, proteins, lipids, DNA, RNA), essential cofactors and ATP consumption associated to growth. This equation was adapted depending on culture conditions or carbon source used in accordance to the available literature experimental data [60, 61]. In addition, composition of each macromolecule type, such as lipid and carbohydrate, was updated and extensively detailed due to the recently published detailed information of the specific composi- tion [62, 63]. See S2 File for details in composition and calculations. The model was also tested for the expression of two different recombinant proteins under different growth conditions: i) the antibody fragment 2F5 (FAB), expressed constitutively under the GAP promoter [64] and, ii) a Rhizopus oryzae lipase (ROL), regulated by the metha- nol inducible AOX promoter [65]. The dataset from the FAB-producing strain was used for simulations in oxygen limiting growth conditions [26, 66], whereas the dataset from the ROL producing strain was used in simulations for glycerol-methanol co-feeding experimental con- ditions [25, 61]. Reactions for heterologous protein production were included considering dif- ferent levels: DNA sequence, transcription and mRNA formation, as well as translation and protein formation. Similarly to PpaMBEL1254 and iLC915, a ratio of 1:100:105 between recom- binant DNA (gene copies), mRNA and heterologous protein was assumed, as described in [32]. These equations also include energetic requirements for polymer formation [67]. Details of DNA, RNA and amino acid composition of each protein, as well as equations for the biosyn- thesis of their components can be found in S3 File. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Model analysis and validation Model analysis and validation were performed using both RAVEN [57] and COBRA [56] tool- boxes as described below. Carbon assimilation capabilities were determined maximizing growth rate and arbitrarily constraining the carbon source influx to 10 mmolgDCW-1h-1 except were otherwise stated. Reaction essentiality was determined performing an additional set of simulations. The pro- cedure consisted on sequentially deleting each reaction of the model, maximizing biomass production and calculating the ratio of the resulting growth rate over the wild type result (GRKO = growthKO/growthWT). The ratios obtained allowed to classify each reaction into three categories: i) essential (GRKO = 0), ii) partially-essential (0 < GRKO < 1) iii) non-essen- tial (GRKO = 1). Evaluation of the effect of oxygen limiting conditions on glucose cultures was performed constraining glucose and oxygen uptake rates to the measured values [66]. For the glycerol: methanol experimental conditions, only glycerol and methanol uptake rates were constrained to the experimental values [24] while O2 uptake rate was left unconstrained. In all these cases biomass, CO2 and by-products were left as unconstrained positive values and therefore appeared as calculated products were necessary. In all these cases biomass production was the maximized objective function. Energy requirements Before the validation step, classic energetic parameters were estimated using experimental data. These parameters are the growth-associated maintenance energy (GAME) and non-growth- associated maintenance energy (NGAME). Both are represented as ATP consumption in the model. For the NGAME calculation, a classical approach was used [68]. For the glucose-limited cultivations, the glucose uptake rate (mmol glucosegDCW-1h-1) was represented against the specific growth rate using data from [24, 60, 69, 70]. The y-intercept of the linear regression line to this data corresponds to the amount of glucose needed for maintenance by non-growing cells. Using this value and the model, the NGAME can be calculated by maximization of the ATP turnover per mmol of glucose for the case of no biomass growth. Using the obtained value as fixed value for the non-growth associated maintenance, GAME is determined by adjusting the ATP consumption coefficient in biomass equation to fit biomass-substrate yields using experimental data (including CO2 and O2 constrains) from [60]. However, for the case of glycerol:methanol growth conditions NGAME was directly taken from the calculated values reported in [61]. This was necessary due to the range of cultivation conditions considered and the insufficient experimental data available. Using these values, and similarly to the glucose-only growth condition, the GAME values for glycerol:methanol 4 / 24 Integration, Upgrade and Validation of the P. pastoris' GEMs conditions were calculated by fitting the predicted values to the range of experimental bio- mass-substrate yields reported in [61]. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs Fig 1. Schematic overview of the major steps involved in the construction of P. pastoris GEM iMT1026. The process of GEMs integration started with the metabolite identification, unifying nomenclature and curation steps of iPP668, PpaMBEL1254 and iLC915. The continuation steps were performed on the resulting pre-merged model and subsequent drafts. Experimental data for model validation was taken from [25, 60, 61, 66]. Fig 1. Schematic overview of the major steps involved in the construction of P. pastoris GEM iMT1026. The process of GEMs integration started with the metabolite identification, unifying nomenclature and curation steps of iPP668, PpaMBEL1254 and iLC915. The continuation steps were performed on the resulting pre-merged model and subsequent drafts. Experimental data for model validation was taken from [25, 60, 61, 66]. doi:10.1371/journal.pone.0148031.g001 doi:10.1371/journal.pone.0148031.g001 Results and Discussion Model merging As described in Materials and Methods section and summarized in Fig 1, the generation of the new model consists of several steps. In the first step of reconciliation, PpaMBEL1254 and iPP668 were automatically compared using modelBorgifier [51]. After the initial pairing step, 75% of the complete set of reactions was identified as identical (exact coincidence) reactions. This pre-merged model was compared with iLC915 resulting in a low number of identical or equivalent reactions (36% of the complete set). Nevertheless, a larger number of reactions were comparable. Those mainly differ in having different stoichiometric coefficients, being assigned to different compartments, decomposed in multi-step reactions, or using alternative names for metabolites (different synonyms, generic names or corresponding to enantiomer compounds). The iPP668 and PpaMBEL1254 models were the first models to be published, and they are more similar to each other than to iLC915. Approximately 83% of the reactions from PpaMBEL1254 are present in iPP668 and 89% of the reactions in iPP668 are shared with PpaMBEL1254. Overall, models iPP668 and PpaMBEL1254 have a 75% of reaction identity. Furthermore, similar nomenclature and abbreviations are used in these two models. In addi- tion, model structure and detail are similar to iND750 [71], from S. cerevisiae, and those models in the BiGG database [45]. The third published model, iLC915, has many differences with the previous two models. Its nomenclature and structure is KEGG-based [48]. Therefore, its metabolites are fully proton- ated and many pathways include the same number of steps described in KEGG. That is, many condensed or simplified metabolic branches in the other two models appear decomposed as multi-step reactions in iLC915. Such reaction differences among the models are one of the major reasons for the low pairing of iLC915 with the other two models and seem to be the result of the main database or model scaffold used as basis for model reconstruction. 5 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Updated pathways As a result of the model comparison and merging step, some divergences in several pathways of the three existing models were evident. In addition, some of them were incomplete or mis- represented in all three models. Therefore we engaged in a curation step according to the recently published literature and database information. Nevertheless, some difficulties in gene and pathway verification were found. The fact that new genomes are usually automatically annotated, at least in an initial step, and that enzyme activities or functions are inferred by homology, propagates errors from already annotated sequences to the new ones [72]. This issue arises when there is limited biological knowledge of the organism. Furthermore, not only genome annotations are based in other organisms and sequences, but GEMs are also 6 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs commonly developed from previous existing models. As a result, annotation errors or misrep- resented pathways are also spread to the subsequent new/updated models. Moreover, as new GEMs are mostly based in pre-existing reconstructions, few new metabolic reactions are often incorporated in the novel GEMs versions. Consequently, the metabolic potential and biological diversity is often not fully reflected in the GEMs and the total number of enzymatic activities present in the existing models remains far below from the complete catalog of enzymatic steps described in the literature for each organism [3]. In the case of P. pastoris, the current annotation of its genome is rather limited [73], with most of all annotations being inferred by homology, mainly from S. cerevisiae. According to the best annotation available P. pastoris has 5040 protein-coding annotated genes of which only 3532 has been assigned an Ontology term and all but 21 annotations are automatically inferred [73]. Despite to the fact that P. pastoris and S. cerevisiae belong to the same family (Saccharomycetaceae), they present significant differences in their metabolic capabilities. Thus, besides P. pastoris well known additional pathways such as the methanol incorporation steps, other significant metabolic differences exist. More specifically, in this work, pathways such as sphingolipid biosynthesis, oxidative phosphorylation and glycosylation pathway, were adapted and redefined, as described below. Fatty acid biosynthesis Due to the limited information on specific fatty acid (FA) metabolic pathways in P. pastoris, it was assumed that most of S. cerevisiae fatty acid pathways were identical in the P. pastoris case. According to Hiltunen and co-workers [74], the latest version of yeast consensus model [43] and iTO977 model of S. cerevisiae [52], fatty acid biosynthesis takes place both in mitochondria and cytosol by fatty acid synthase (FAS) type II and I, respectively [75]. FAS type II has individ- ual enzymes for each reaction in fatty acid de novo biosynthesis and elongation. Despite it is well known that mitochondrial FAS type II synthetizes at least up to C8 fatty acid, some evi- dences suggest that this system can synthesize longer fatty acids [74, 76]. While in S. cerevisiae 7th version of the consensus model [43] mitochondrial biosynthesis is up to C8, this model also include reactions for up to C18 biosynthesis. On the other hand, cytosolic FAS is a complex formed by Fas1p and Fas2p within which the successive elongation reactions take place and only the final acyl-CoA is released [77]. The final products of this cytosolic complex are considered to be C14 to C18 acyl-CoAs, mainly because they are the main fatty acids found in P. pastoris [62]. The biosynthesis takes place inside the complex in a number of four step cyclic reactions for each acetyl-CoA added. Differ- ent number of cycles results in a range FA (C14, C16 and C18 acyl-CoAs). In addition to de novo biosynthesis, P. pastoris also has fatty acid elongation enzymes, which are able to extend C12-14 fatty acids and generate very long chain fatty acids (up to C26). Activation of free fatty acids (FFA) was considered to take place in cytosol only for C14, C16 and C18 FFA, as well as their respective acyl-CoA hydrolysis. Finally, only acyl-CoA desatura- tions were included (that is not acyl-ACP or FFA) according to the pathway defined in S. cere- visiae [43]. Fatty acid oxidation Two different transport mechanisms are commonly described depending on the FA chain length [78–80], both being closely coupled to its activation to acyl-CoA [81–85]: on the one hand, a simple diffusion and further activation of medium-chain fatty acids (up to C12 chain length) and, on the other hand, long and very long chain fatty acids are translocated as acyl- CoA concomitant with the corresponding ATP hydrolysis [86]. For the active transport 7 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs mechanism, the ATP has a cytosolic origin in ILC915, while in PpaMBEL1254 and iPP668 the required ATP is peroxisomal. According to [87, 88], peroxisomal ATP is only required for medium chain fatty acid activation, therefore long and very long chain fatty acids transport should be dependent on cytosolic ATP. Each cycle of β-oxidation is represented by 4 reactions. Nevertheless, for unsaturated fatty acid degradation, and due to its highly complex degradation steps which depend on the position of its double bounds [89, 90], lumped reactions up to the generation of acetyl-CoA were taken from iPP668. However, the desaturation reaction of C18:3 to C18:2 was taken from iLC915. Sphingolipid metabolism General sphingolipid biosynthetic pathways in yeast are partially homologous to S. cerevisiae and they are extensively described in the literature [91, 92]. Nevertheless, unlike S. cerevisiae, some yeasts such as P. pastoris, are able to produce glucosylceramides (GlcCer) from sphingoli- pid bases [93–96]. None of the three models include GlcCer biosynthesis. Ternes and co-workers [97, 98] iden- tified the gene role in GlcCer pathway and described the fatty acid specific composition in GlcCer and other sphingolipids, as well as the main chain sphingoid bases in P. pastoris. Sphingoid bases can be derived from palmitoyl-CoA and stearoyl-CoA. However, only a 5% of the detected species correspond to the last one. In fact, Ternes and co-workers [98] charac- terized sphingolipid composition assuming all the species were formed with a palmitoyl-CoA derived sphingoid base. This sphingolipid composition is in agreement with other literature sources [62, 99, 100]. As palmitoyl-CoA bases represents around 95% of sphingoid bases, only palmitoyl-CoA derived ones are taken into account in this model. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Oxidative phosphorylation There are P. pastoris specific traits in respiratory chain that should be included in the GEM. As an example, while complex I is not present in S. cerevisiae [117], it is described in P. pastoris [118, 119]. None of the previous P. pastoris models include respiratory complex I and so, an important proton translocation step was missing. In general, two main traits of oxidative phos- phorylation were deeply analyzed: the proton stoichiometry and the complexes integrating the electron transport chain. In this model the mitochondrial intermembrane space has been included. Hence proton translocation is assumed to occur from the mitochondrial matrix to the intermembrane space. Thus, protons pumped out from the mitochondria do not merge with the high amount of pro- tons from the cytosolic space. Regarding the electron transfer chain reactions, each of the previ- ous P. pastoris models shows a different stoichiometry for proton translocation. After a review of the stoichiometry and relevant literature it was decided to apply a stoichiometry that satisfies the H+ balance of the metabolites’ charged formula and including complex I stoichiometry con- siderations proposed by Wikström and Hummer [120]. This includes the complex I translocat- ing 4 H+ to the intermembrane space [120, 121]. In the present model, reactions for non H+ translocating outer and inner mitochondrial membrane NAD(P)H dehydrogenases (cytoplasmic side and matrix side) were also included. While inner NADH dehydrogenase appears in all three models, outer NADH dehydrogenase was only present in iPP668 and PpaMBEL1254, despite both dehydrogenases have been previ- ously reported [118, 122, 123]. We also included PAS_chr1-4_0299 putative NADPH dehydro- genase homologue to Kluyveromyces lactis [124, 125] and also included in its metabolic reconstruction [126]. On the other hand, complex III and IV are included in all three models, but several discrep- ancies exist on the details of the H+ balance due to the consideration of alternative metabolite’s molecular formulas or the result of using different criteria when considering chemical and translocated protons [127]. An additional trait for complex III equations is the complexity on the stoichiometric representation of Q-cycle [128, 129]. Therefore, in this model stoichiometric coefficients of equations for complex III and IV reactions were chosen with special attention to the proton balance. The selected stoichiometry was of 2H+/2e- for complex III and 4H+/2e- for complex IV according to recent literature [121, 128, 129]. One additional characteristic of P. Glycosylation pathways Protein glycosylation pathways are not accurately described in previous models of P. pastoris. Only iLC915 partially included N-glycosylation, O-glycosylation and glycosylphosphatidylino- sitol-anchor (GPI-anchor) biosynthesis pathways. However, compartmentalization of several reactions of this pathway also required revision. The first part of the N-glycosylation process is highly conserved among eukaryotes [101, 102]. It takes place in the cytosol up to the addition of 5 mannose residues (Man) forming (Man)5(GlcNAc)2(PP-Dol)1 oligosaccharide. At this point, the oligosaccharide is transferred to the endoplasmic reticulum (ER), where up to 9 Man and 3 glucose residues (Glc) are further added [103]. In the second and less conserved part of the pathway, the dolichol diphosphate attachment to the protein is represented by a pseudo-reaction forming the compound (Glc)3 (Man)9(GlcNAc)2(Asn)1 in which Asn represents an asparagine residue from the targeted protein. Once the oligosaccharide is attached to the Asn residue of the target protein, it is fur- ther modified by the removal of one Man. The resulting glycoprotein is transported to the Golgi Apparatus [104, 105]. There, an heterogeneous pattern of glycosylation has been observed corresponding to the different heterologous proteins expressed in P. pastoris [19, 106, 107]. As an example, differences in Man residues range from 6 to 18 [108–110] and even may include hypermanosylation [111]. Due to its complexity and variability, in this model an aver- age glycan is assumed to consist of (Man)9(GlcNAc)2(Asn). The resulting oligosaccharide con- tributes to the formation of a mannan (Man polymer represented by 1 mannose residue polymer) and a chitin (N-Acetylglucosamine polymer). Both contribute to the biomass forma- tion as a specific component of the carbohydrate fraction. Similarly to N-glycosylation in mannan formation, O-glycosylation is included assuming an average of 3 Man oligosaccharides [112–115]. O-glycosylation is also represented by a pseudo- 8 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs reaction forming the compound (Man)1(Ser/Thr)1 in which Ser/Thr represents a serine or threonine residue of a protein. Finally, GPI-anchor biosynthesis was also reviewed and compartments reassigned according to Orlean and Menon [116]. Oxidative phosphorylation pastoris is the presence of an alternative oxidase that could bypass complex III and IV in the respiratory chain [117, 130] which seems to be active only in certain growth conditions [117, 122, 131]. Although this oxidase was only included iLC915, our consensus model also incorporates this reaction in the electron transport chain module. Finally, the stoichiometry for ATP synthase was maintained as in PpaMBEL1254 and iPP668 (4H+/ATP, resulting in a final maximum theoretical stoichiometry of 2.5e-/ATP) as its H+ balance is in agreement with the available literature [132]. Other reviewed pathways Metabolization of some sugars was also updated. Sugars such as starch, maltose or cellobiose were able to be assimilated in some of the previous models. However, Kurtzman [44] and Nau- mov and co-workers [133] characterized substrate assimilation in P. pastoris and reported non 9 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs growth for these carbon sources. Consequently reasons for their metabolization were revised and discarded reactions are detailed in S4 File. L-rhamnose assimilation was only possible in PpaMBEL1254. However the specific metabolic steps included for its metabolization were not typical of yeast species. Therefore alternative reactions and genes associated to the metaboliza- tion of L-rhamnose were added as suggested in [134], also described similarly for P. stipitis [135, 136]. A full list of modifications from the original models, also including some additional path- ways and subsystems not discussed above such as phosphatidylinositol synthesis or transport reactions, is provided in S4 File. General characteristics of the model After following all the steps described above, an extended model (iMT1026) is obtained that integrates previous P. pastoris’ GEMs, which is provided in S1 and S5 Files and is also available at BIOMODELS database (MODEL1508040001) [58]. The general characteristics of the result- ing model are described in the following. In the first place this model includes an increased number of gene-protein-reaction relation- ships as can be seen in Table 1. Our final model incorporated 185 new reactions that didn’t appear in previous models and has 614 common reactions in all three models (Fig 2). Reactions appearing in Fig 2 as common to two or all three different models include those reactions that have been taken directly from the previous model. Therefore, they do not include those reactions that are either not the same but equivalent, have been decomposed in several reactions or are a combination of several other reactions. Thus, taking into account these multi-step, lumped or decomposed reactions, in the final model there are up to 721 equivalent reactions in common in all three models, 504 in two of the three models and 638 reactions in only one of the models, without any clear equivalence in any of the other two models. Maintenance and growth-associated ATP calculations As described in Materials and Methods, data from [24, 60, 69, 70] was used to calculate the NGAME. As an initial step, a value of 32 mol ATP/ mol glucose, was obtained by maximizing As described in Materials and Methods, data from [24, 60, 69, 70] was used to calculate the NGAME. As an initial step, a value of 32 mol ATP/ mol glucose, was obtained by maximizing Table 1. Comparison of the main features of iMT1026 and previous P. pastoris’ GEMs. iPP668 PpaMBEL1254 iLC915 iMT1026 Genes 668 540 915 1026 Metabolites a 1177 (684) 1058 (696) 1302 (899) 1689 (1018) Reactions 1354 1254 1423 2035 Cytosolic 623 604 790 1059 Mitochondrial 163 155 205 268 Peroxisomal 66 66 64 102 Extracellular 12 11 0 16 Endoplasmic reticulum 15 7 34 41 Golgi apparatus 4 8 4 13 Vacuolar 3 6 12 9 Nuclear 16 17 0 17 Intercompartmental/Transport 452 328 314 510 a Total number of metabolites, with compartment, and unique metabolites (in brackets). doi:10.1371/journal.pone.0148031.t001 Table 1. Comparison of the main features of iMT1026 and previous P. pastoris’ GEMs. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 10 / 24 Integration, Upgrade and Validation of the P. pastoris' GEMs Fig 2. Reactions from PpaMBEL1254, iPP668 and iLC915 included in iMT1026 model. doi:10.1371/journal.pone.0148031.g002 Fig 2. Reactions from PpaMBEL1254, iPP668 and iLC915 included in iMT1026 model. Fig 2. Reactions from PpaMBEL1254, iPP668 and iLC915 included in iMT1026 model. doi:10 1371/journal pone 0148031 g002 doi:10.1371/journal.pone.0148031.g002 doi:10.1371/journal.pone.0148031.g002 the ATP turnover using 1 mmol/(gDCWh) of glucose. The y-intercept from a representation of glucose uptake rate versus growth rate was 0.0878 mmol glucose/(gDCWh) which corre- sponds to 2.81 mmol ATP/(gDCWh) using the glucose-ATP conversion factor obtained in the initial step. This calculated glucose NGAME value is similar to the 2.3 mmol ATP/(gDCWh), previously proposed by [31] for glucose, and close to the NGAME estimated for Pichia (Schef- fersomyces) stipitis also growing in glucose [137]. the ATP turnover using 1 mmol/(gDCWh) of glucose. The y-intercept from a representation of glucose uptake rate versus growth rate was 0.0878 mmol glucose/(gDCWh) which corre- sponds to 2.81 mmol ATP/(gDCWh) using the glucose-ATP conversion factor obtained in the initial step. This calculated glucose NGAME value is similar to the 2.3 mmol ATP/(gDCWh), previously proposed by [31] for glucose, and close to the NGAME estimated for Pichia (Schef- fersomyces) stipitis also growing in glucose [137]. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Maintenance and growth-associated ATP calculations On the other hand, GAME for glucose was estimated by fitting the calculated biomass-sub- strate yields to experimental data. This way a value of 72 mmol ATP/gDCW was obtained. This amount of ATP associated to cell growth is also similar to 70.5 mmol ATP/gDCW calcu- lated previously for P. pastoris [33] and close to the experimentally calculated values for S. cere- visiae of 62–71 mmol ATP/gDCW [138, 139], and to the 69.2 mmol ATP/gDCW, in silico estimated [5]. For the case of the glycerol and methanol co-feeding cultivations, ATP maintenance val- ues calculated by Jordà and co-workers [61] were used as NGAME as available data was insufficient for a new determination. These values range from 4.5 to 5.7 mmol ATP/gDCW and are similar to 6 mmol ATP/gDCW, proposed in iLC915 [33]. For the different condi- tions tested, specific GAMEs were calculated by fitting the simulations to experimental data from [61]. These experimental data show that the ratio of the glycerol or methanol uptake rates with the growth rate is different for each pair of glycerol:methanol feeding conditions (80:20, 60:40 and 40:60, % w/w at 0.05 and 0.16 h-1 growth rates), therefore a specific GAME was calculated for each case. The obtained values ranged within the 69.8 and 125.6 mmol ATP/gDCW interval. These GAME values increase with the fraction of methanol in the mixed feeding and are in agreement with those calculated by Caspeta and co-workers [33], who estimated a maximum GAME for methanol as sole carbon source of 150 mmol ATP/ gDCW. 11 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs Carbon source assimilation The model agreement with P. pastoris utilization of different carbon sources was tested and compared to experimental data [44, 133]. A total of 47 carbon sources were evaluated (Table 2) using an in silico minimal medium, with ammonium, phosphate and biotin. The model suc- cessfully predicts carbon assimilation for all sources tested. Reaction essentiality An interesting trait of the obtained GEMs is the identification of those reactions critical or essential for biomass growth (essential reactions). Simulations to determine reaction essential- ity were performed for glucose in normoxia, limited oxygen and hypoxia conditions and also for mixtures of glycerol and methanol at different growth rates. No significant difference in reaction essentiality was observed in all the conditions tested. Thus, similar patterns of distri- bution of essential reactions in each pathway are observed for all the cases. The results, grouped into major metabolic pathways, are summarized in Fig 3 (and S1 Fig). From a global point of view, essential reactions represent a 15–16% of the total reactions, while 76–79% of the reac- tions are classified as non-essential. The remaining 6–9% are partially-essential and its deletion causes a decrease in growth rate. The results show that there are 312 condition independent essential reactions, which are essential in all the performed simulations. These essential reactions can be grouped in three main groups: in the first group, most of them are associated with lipid metabolism (40%), in the second group most reactions belong to the amino acid metabolism (18.6%) while the third group mostly includes cofactor related essential reactions as a 14.1% of all common essential reactions. Similarly to other GEM models, such as S. cerevisiae, extending the model and including more detailed biomass composition results in an increase of essential reactions directly linked to the biomass related metabolites. Nevertheless, this essentiality could be over- estimated in silico, due to the fact that in vivo systems are able to replace de missing species with other similar biomass components. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Model validation Experimental a In silico D-glucose + + DL-lactate + + Mannitol + + Ethanol + + Glycerol + + L-Rhamnose + + Methanol + + Succinate + + Trehalose + + Sorbitol + + Citrate v + D-Xylose - / + b + Xylitol - / + c + 5-keto-D-gluconate - - Arbutin - - Cellobiose - - D-arabinose - - D-Galacturonate - - D-gluconate - - D-glucono-1,5-lactone - - D-Glucosamine - - D-Glucuronate - - D-Ribose - - Erythrol - - Galactitol - - Galactose - - Hexadecane - - myo-Inositol - - inulin - - Lactose - - L-Arabitol - - L-Arabinose - - L-Sorbose - - L-Tryptophan - - Maltose - - Meleziose - - Melibiose - - methyl-alpha-D-glucoside - - N-acetylglucosamine - - Rafﬁnose - - Ribitol - - Saccharate - - Salicin - - Soluble starch - - Sucrose - - +, carbon assimilation and growth; -, no carbon assimilation; v, variable growth/non-growth experiments observed. a Experimental data from [44, 140]. b Described in [141]. c Described in [142]. doi:10.1371/journal.pone.0148031.t002 031 January 26, 2016 13 / 24 Table 2. Evaluation of the substrate assimilation capabilities in P. pastoris. Experimental a In silico D-glucose + + DL-lactate + + Mannitol + + Ethanol + + Glycerol + + L-Rhamnose + + Methanol + + Succinate + + Trehalose + + Sorbitol + + Citrate v + D-Xylose - / + b + Xylitol - / + c + 5-keto-D-gluconate - - Arbutin - - Cellobiose - - D-arabinose - - D-Galacturonate - - D-gluconate - - D-glucono-1,5-lactone - - D-Glucosamine - - D-Glucuronate - - D-Ribose - - Erythrol - - Galactitol - - Galactose - - Hexadecane - - myo-Inositol - - inulin - - Lactose - - L-Arabitol - - L-Arabinose - - L-Sorbose - - L-Tryptophan - - Maltose - - Meleziose - - Melibiose - - methyl-alpha-D-glucoside - - N-acetylglucosamine - - Rafﬁnose - - Ribitol - - Saccharate - - Salicin - - Soluble starch - - Sucrose - - +, carbon assimilation and growth; -, no carbon assimilation; v, variable growth/non-growth experiments observed. a Experimental data from [44, 140]. b Described in [141]. c Described in [142]. d i 10 1371/j l 0148031 t002 Table 2. Evaluation of the substrate assimilation capabilities in P. pastoris. Model validation An additional step of model validation was performed by comparing the model predicted val- ues with an additional set of experimental data including diverse combinations of glucose, glyc- erol and methanol chemostats [61, 66]. For the glucose chemostats, simulations with different oxygen availabilities, growth rates and CO2 production were successfully predicted with errors lower than 6% for both FAB expressing and non-expressing strains (Fig 4A, 4B and 4C). According to the available experi- mental data [60, 66], when the oxygen availability decreases ethanol and other metabolites are secreted. The present model is also able to predict by-product formation when oxygen-limited conditions are simulated. However, the model predicts a slightly higher production of ethanol and none of the other by-products, such as arabitol or pyruvate. Nevertheless, when ethanol secretion flux is constrained to the experimentally measured value, arabitol secretion is also predicted (Fig 4C). In addition to arabitol, pyruvate is also secreted in the in silico predictions when both ethanol and arabitol are constrained to the experimental values. This discrepancy of the model to directly predict arabitol or pyruvate secretion if no additional constraint is imposed points to additional regulatory constraints other than those strictly stoichiometric. In addition, different cofactor utilization by combinations of isoenzymes [143, 144], and their impact on NAD(P)+/NAD(P)H regeneration, could be one of the key factors for the produc- tion of those alternative products [145, 146]. Constraining the model with additional 13C-label- ling data [147] as well as studying the impact of cofactor perturbation and analyzing these 12 / 24 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs Table 2. Evaluation of the substrate assimilation capabilities in P. pastoris. PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 Integration, Upgrade and Validation of the P. pastoris' GEMs Fig 3. Summary of reaction essentiality results for glucose simulations grouped into major pathways. FBA was performed by optimizing biomass production and sequentially constraining to 0 each reaction in the corresponding simulations. The resulting growth rate was compared with the wild type one. Metabolic reactions were classified in three categories according to the relative growth rate obtained: Essential (E), partially-essential (PE) and non-essential (NE). X axis represent the fraction of each type of reactions in each category of E (in red), PE (in blue) and NE (in green). Reactions are distributed in 8 major subsystems (Y axis). Numbers between brackets indicate number of reactions in each group. Equivalent figures for oxygen limiting conditions and glycerol: methanol simulations can be found in S1 Fig. Fig 3. Summary of reaction essentiality results for glucose simulations grouped into major pathways. FBA was performed by optimizing biomass production and sequentially constraining to 0 each reaction in the corresponding simulations. The resulting growth rate was compared with the wild type one. Metabolic reactions were classified in three categories according to the relative growth rate obtained: Essential (E), partially-essential (PE) and non-essential (NE). X axis represent the fraction of each type of reactions in each category of E (in red), PE (in blue) and NE (in green). Reactions are distributed in 8 major subsystems (Y axis). Numbers between brackets indicate number of reactions in each group. Equivalent figures for oxygen limiting conditions and glycerol: methanol simulations can be found in S1 Fig. doi:10.1371/journal.pone.0148031.g003 cofactor demands for cell growth, as done in other organisms [148] would be interesting approaches to consider in future studies. For the second dataset, (glycerol:methanol mixtures), specific growth rate, together with specific O2 consumption and CO2 production rates were predicted within a 11% of deviation, as shown Fig 4E, 4F and 4G. Similarly to the above described glucose tests, arabitol was only produced when ethanol was constrained to the experimental values; otherwise, ethanol is the preferred product of the stoichiometric model. As in the previous glucose case these results point to another possible level of regulation for arabitol production not included in the model, as without it ethanol production appears as the most efficient way to regenerate NAD+ for maximum biomass production. In order to compare the accuracy of our model with the previous existing models a set of simulations were performed for glucose and glycerol:methanol cultivations (S5 File). The same constrains were set to all the models and growth rate, CO2 production and O2 consumption (only in the glycerol:methanol simulations) were compared to the experimental values [61, 66]. As shown in S5 File, iMT1026 can predict the evaluated macroscopic cultivation parameters more accurately, i.e. with smaller deviations from experimental data. Moreover, our model is also able to describe byproduct secretion under respirofermentative conditions. Model validation +, carbon assimilation and growth; -, no carbon assimilation; v, variable growth/non-growth experiments 13 / 24 Conclusions In summary, a consensus GEM of the yeast P. pastoris integrating the three preexisting models has been obtained. Importantly, the new GEM, iMT1026, is more complete and includes a comprehensive revision and upgrading of several metabolic processes (e.g. fatty acid and sphingolipid metabolism, protein glycosylation and energy metabolism) based on new infor- mation emerged from recent literature. Furthermore, the new GEM has been validated using different sets of experimental data corresponding to a wider range of physiological states than previous GEMs. From our point of view this GEM improves the capabilities in terms of accu- racy of predictions/simulations in relation to previous models. Overall, we provide an improved tool to the P. pastoris community for the physiological analysis and understanding PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 14 / 24 Supporting Information S1 Fig. Reaction essentiality analysis. Summary of reaction essentiality results grouped into major pathways. (PDF) S1 File. iMT1026 model. Pichia pastoris GEM model in xlsx format for use in the RAVEN toolbox. (XLSX) S2 File. Biomass composition. Details of biomass composition for cultivations on glucose and glycerol:methanol mixtures. (XLSX) S3 File. Recombinant protein composition. DNA, RNA and amino acid compositions for the FAB and ROL recombinant proteins expressed in P. pastoris and tested in the model. (XLSX) S4 File. Reaction changes. List of modified, added and excluded reactions in iMT1026. (XLSX) S5 File. iMT1026 model in SBML format. Pichia pastoris GEM model in SBML format gener- ated with the RAVEN toolbox. (XML) S6 File. GEMs performance comparison. Analysis of the prediction capabilities and devia- tions from experimental data in simulations performed with iMT1026 and the previous mod- els. (PDF) S1 Fig. Reaction essentiality analysis. Summary of reaction essentiality results grouped into major pathways. (PDF) S1 File. iMT1026 model. Pichia pastoris GEM model in xlsx format for use in the RAVEN toolbox. (XLSX) S2 File. Biomass composition. Details of biomass composition for cultivations on glucose and glycerol:methanol mixtures. (XLSX) S2 File. Biomass composition. Details of biomass composition for cultivations on glucose and glycerol:methanol mixtures. (XLSX) S3 File. Recombinant protein composition. DNA, RNA and amino acid compositions for the FAB and ROL recombinant proteins expressed in P. pastoris and tested in the model. (XLSX) S3 File. Recombinant protein composition. DNA, RNA and amino acid compositions for the FAB and ROL recombinant proteins expressed in P. pastoris and tested in the model. (XLSX) S3 File. Recombinant protein composition. DNA, RNA and amino acid compositions for the FAB and ROL recombinant proteins expressed in P. pastoris and tested in the model. (XLSX) S4 File. Reaction changes. List of modified, added and excluded reactions in iMT1026. (XLSX) S4 File. Reaction changes. List of modified, added and excluded reactions in iMT1026. (XLSX) S5 File. iMT1026 model in SBML format. Pichia pastoris GEM model in SBML format gener- ated with the RAVEN toolbox. (XML) S5 File. iMT1026 model in SBML format. Pichia pastoris GEM model in SBML format gener- ated with the RAVEN toolbox. (XML) S6 File. GEMs performance comparison. Analysis of the prediction capabilities and devia- tions from experimental data in simulations performed with iMT1026 and the previous mod- els. (PDF) Integration, Upgrade and Validation of the P. pastoris' GEMs Fig 4. Results of the model validation. Graphs with (A) growth rate (B) CO2 and (C) D-arabitol production predictions when simulating glucose ch at different oxygen conditions, with and without recombinant protein production [60, 66] with glucose, O2 and ethanol fluxes constrained to the expe values. (D) Growth rate (E) CO2 production and (F) O2 consumption predictions when simulating different glycerol:methanol chemostats [25, 61]. W black bars correspond to experimental and predicted data respectively.* Not determined in [25]. doi:10.1371/journal.pone.0148031.g004 Integration, Upgrade and Validation of the P. pasto Fig 4. Results of the model validation. Graphs with (A) growth rate (B) CO2 and (C) D-arabitol production predictions when simulating glucose chemostats at different oxygen conditions, with and without recombinant protein production [60, 66] with glucose, O2 and ethanol fluxes constrained to the experimental values. (D) Growth rate (E) CO2 production and (F) O2 consumption predictions when simulating different glycerol:methanol chemostats [25, 61]. White and black bars correspond to experimental and predicted data respectively.* Not determined in [25]. doi:10.1371/journal.pone.0148031.g004 doi:10.1371/journal.pone.0148031.g004 PLOS ONE \| DOI:10.1371/journal.pone.0148031 January 26, 2016 15 / 24 Integration, Upgrade and Validation of the P. pastoris' GEMs of this yeast. It is expected that on-going efforts in the functional (re)annotation of the P. pas- toris genome will allow for further improvements of its GEMs by all the P. pastoris community. From a wider perspective, it also has to be pointed out the importance of curating and manu- ally revising new GEMs of non-model organisms that are based on GEM scaffolds from related model organisms. Despite the comprehensiveness of these scaffolds, an exhaustive analysis of specific metabolic traits of the non-model organism is still essential to construct a GEM describes/predicts its metabolic phenotype accurately. Author Contributions Conceived and designed the experiments: MT JA. Performed the experiments: MT. Analyzed the data: MT JA PF. Contributed reagents/materials/analysis tools: MT JA. Wrote the paper: MT JA PF. Conceived and designed the experiments: MT JA. Performed the experiments: MT. Analyzed the data: MT JA PF. Contributed reagents/materials/analysis tools: MT JA. Wrote the paper: MT JA PF. Acknowledgments The group is a member of 2014-SGR-452 and the Reference Network in Biotechnology (XRB) of Generalitat de Catalunya. The group is a member of 2014-SGR-452 and the Reference Network in Biotechnology (XRB) of Generalitat de Catalunya. References 1. Kim TY, Sohn SB, Kim Y Bin, Kim WJ, Lee SY. 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https://openalex.org/W4386186896	https://zenodo.org/records/8285161/files/LP3.pdf	English	null	THE ENGLISH TEACHER'S PROFILE AND HIS STUDENTS' PERFORMANCE IN ENGLISH LANGUAGE	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,196	Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 Keywords: English Language, Interest, Qualification, Student’s Performance, Teacher’s Profile. Objectives of the Study The objectives of this study include; i. ascertaining if there is any relationship between the teachers’ profile and the performance of his students in English Language. ii. investigating the criteria involved in producing qualified English teachers. ii. investigating the criteria involved in producing qualified English teachers. iii. determining variables involved in improving the academic performance of students in English Language at various examinations. iv. identifying the factors responsible for the improvement of the academic performance of secondary school students in external examinations in English Language and by extension, other subjects. Research Questions i. What are the effects of teacher’s profile on the performance of Secondary school students in external examinations? ii. What are the criteria for producing qualified teachers? ii. What are the criteria for producing qualified teachers? iii. Is there any relationship between the general low performance of students and teacher’s qualification? iv. What are the factors that can improve the academic performance of secondary school students in external examinations in English Language and by extension, other subjects. Introduction It is not an overstatement to say that teachers occupy a very sensitive position in schools. Without the teacher, effective teaching and learning cannot take place. Qualified and dedicated teachers with a high mastery of the teaching of English Language are indispensable tools to students’ excellent performance in the school. The continuous decline in students’ performance in English Language in Secondary Schools has been attributed to many factors such as teachers’ qualification and lack of commitment. Many people who teach English Language have no business in the noble profession, either that they are not interested in teaching the subject or they don’t have much knowledge about the subject. Abstract This is a position paper on the effect of the teacher’s profile on the interest, learning and performance of his students in English Language. By teacher’s profile here, we look at the teacher’s certificate, experience and attitude towards the teaching of English Language. Secondary sources of data were considered and logical conclusions were made based on the literature of notable educators in the field of English. It was discovered that there is a high relationship between the teacher’s profile and the students’ interest and performance in the subject, the higher the teachers’ profile, the higher the interest rate and performance of the students. Variables looked out for under profile include the teacher’s qualification, experience, mastery of subject matter and attitudes towards work, students and their parents. Based on the findings, it was recommended that competent and qualified teachers should be employed to teach English Language and that continuous in-service training, seminars and workshops should be organized to update the teachers’ knowledge periodically. Welfare packages should also be put in place to encourage teachers on the field. Keywords: English Language, Interest, Qualification, Student’s Performance, Teacher’s Profile. A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 36 August 26, 2023 36 August 26, 2023 A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 36 36 August 26, 2023 August 26, 2023 Who is a teacher? A teacher is that person who helps others to acquire knowledge, competence or value via the practice of teaching. He could also be seen as a person who builds up, instructs, trains and guides his students for healthy growth and stable adult life. A teacher’s job transcends that of teaching to that of moulding young lives, guiding youths, motivating students and the general character training (Majasan 1995, Ofojebe and Ezugoh 2010). The role of a teacher is often formal ongoing, and carried out in a school or other places of learning. Aromolaran (2019) defined an effective teacher as an individual who has undergone training and is certified to impart knowledge, skills and positive attitude in the learner. He is expected to have possessed adequate experience in pedagogy and esoteric knowledge to guide the learner to attain achievable goals. He is believed to have been certified by the Teachers’ Registration Council of Nigeria (TRCN). Usman (2012) asserts that a qualified teacher is one who holds a teaching certificate and/or is licensed by the state, owns at least a bachelor’s degree from a four-year institution and well qualified in his/her area of specialization. The teacher occupies an important place in any classroom context if the desired learning outcome is to be attained. There is no doubt that the personality of the teacher affects his students, overtly or covertly. The teacher’s ideology, carriage and other special traits are all being monitored by the students. And being a role model, the likely hood of the students following his footsteps abound. Farrant (1980) believed that the good teacher should possess some characteristics that are worthy of emulation. He should be humorous, energetic, humble, kind and friendly. Onwuegbu (1979), in analyzing the qualities of a good teacher states that a good teacher is not necessarily one who is fluent with words or one who dishes out knowledge. Rather, a good teacher, according to him, must possess the following qualities; i. He must know his subject. ii. He must possess a registered certificate to teach. iii. He must express himself fluently with the official language of instruction – English. iii. He must express himself fluently with the official language of instruction – English. A certified teacher is one who has earned credentials from an authoritative source such as the government, a higher institution or a private source. Methodology The main objective of this paper is to ascertain if there is any relationship between the teachers’ profile and the performance of his students in English Language. To do this, different relevant literatures will be reviewed on the subject and conclusion be subsequently made. 37 37 August 26, 2023 August 26, 2023 A Publication of College of Languages and Communication Arts Education, Lagos State University of Education Some of the objectives of an effective teacher are: • to encourage students achieve success by building a virile teacher-student re • to encourage students achieve success by building a virile teacher-student relationship. • to help students acquire skills, knowledge and attitude g y g p • to help students acquire skills, knowledge and attitude • to instill valuable learning such as skills, competence and positive attitudes to students. • to ensure effective communication takes place during teaching/learning process • to create a conducive classroom environment devoid of hostility and fear. • to teach students from simple to complex, known to unknown and from concrete to abstract. • to instill valuable learning such as skills, competence and positive attitudes to students. • to ensure effective communication takes place during teaching/learning process • to create a conducive classroom environment devoid of hostility and fear. • to teach students from simple to complex, known to unknown and from concrete to abstract. • to teach students from simple to complex, known to unknown and from concrete to abstract. • to help students make discovery and solve their social and academic problems. • to act as a parent to the students in and outside the school • to identify individual differences in students and help them overcome peculiar challenges • to identify individual differences in students and help them overcome peculiar challenges Who is a teacher? This teacher qualification gives the teacher authorization to teach and grade in pre-school, primary or secondary schools. Ogho (2011) said that a teaching qualification is one of a number of academic and professional degrees that enables a person to become a registered teacher. Such teaching qualifications include the Post Graduate Diploma in Education (PDE), Bachelor in Education (B.Ed) and Nigeria Certificate in Education (NCE). A study by Adeogun (2011) found that the quality of any education system depends on the quality of the teachers as no system can rise above the quality of its teachers (you cannot give what you don’t have.) A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 38 August 26, 2023 38 August 26, 2023 38 38 August 26, 2023 August 26, 2023 A Publication of College of Languages and Communication Arts Education, Lagos State University of Education The teacher of English as a Second Language The teacher of English as a second language in Nigeria is one who is specially trained in all the ways discussed earlier and also has a high knowledge of English Language and its teaching (that is, the appropriate teaching methods to be used for different topics). Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 Professional skills encompass the techniques, strategies and approaches that would help teachers to grow in the teaching profession and also work towards its growth. This includes soft skills, counselling skills, interpersonal skills, computer skills, information retrieval and management skills and above all, life-long learning skills. An amalgamation of teacher skills, pedagogical theory and professional skills would serve to create the right knowledge, attitude and skills in teachers, thus promoting wholistic development. Duties of an effective teacher T s, information retr ry and professiona rs, thus promoting a is one who is spe of English Languag erent topics). Duties of an effective teacher • He prepares for his lectures as assigned (prepares his notes of lesson and teaching aids ahead of class) • He prepares for his lectures as assigned (prepares his notes of lesson and teaching aids ahead of class) • He evaluates his teachings to know of problem areas that he may need to re-tea • He directs students learning activities and helps the students to satisfy their peculiar needs H i t k l d kill d i ht ttit d th l F hi t d thi h hi lf • He directs students learning activities and helps the students to satisfy their peculiar needs • He imparts knowledge, skills and right attitude on the learner. For him to do this, he himself must have knowledge, skill and experience in the subject matter. • He imparts knowledge, skills and right attitude on the learner. For him to do this, he himself must have knowledge, skill and experience in the subject matter. • He must ensure that the appropriate instructional materials and methods are applied in his teaching. • He must ensure that the appropriate instructional materials and methods are applied in his teaching. • He must teach according to the syllabus and his guided materials. • He enforces discipline among the students. The teacher education covers teaching skills, sound pedagogical theory and professional skills. The teacher education covers teaching skills, sound pedagogical theory and professional skills. Teaching Skills include providing training and practice in the different techniques, approaches and strategies that would help the teacher to plan and impart instruction, provide appropriate instruction and reinforcement and conduct effective assessment. It includes effective classroom management skills, preparation and use of instructional materials and a good communication skill. Pedagogical theory has to do with the philosophical, sociological and psychological considerations that enable the teacher to have a sound basis of practicing teaching skills in the classroom. This theory is stage-specific and it is based on the needs and requirements that characterize a particular stage. A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 39 August 26, 2023 39 August 26, 2023 A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 39 39 August 26, 2023 August 26, 2023 cation Arts Educa UST 26, 2023 I s and approaches t ards its growth. Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 which can only be met by undergoing trainings that can certify him fit to handle the special needs of his students, that of effective communication in and outside the school. For a teacher to be effective, he has to be able to perform the following tasks effect • Organize his/her students to learn through activities • Organize his/her students to learn through activities • Use good questioning techniques to recall facts from comprehension and application of principles. • Utilize audio-visual aids whenever appropriate to generate and sustain the interest of learners. • Organize and hold meaningful discussions with the students. The study of English in the Secondary School The teacher of English as a second Language in the Secondary School is expected to have an in- depth knowledge of the teaching of the following aspects of English as taught in the Secondary school; • Reading Comprehension • Essay Writing • Grammar • Summary Writing The teacher must have a mastery of the four language skills thus, listening, speaking, reading and writing and how to incorporate these in his everyday lesson. That means that, during every lesson, students must listen to the teacher and classmates. They must answer the teacher’s questions and also speak to their classmates using Standard English (Adebanjo 2020). The students must also read and write during the lesson; this is the best way to help students develop their communication skills in English Language. English teachers have long lasting impacts on the lives of their students and the greatest teachers inspire their students towards greatness. Obaigbena (2017) asserts that within a school, if an English teacher is well educated, intellectually alive and takes keen interest in the performance of his job, it reflects in the academic performance of his students in English Language examinations. The qualification of the teacher therefore must be given a pride of place as the teacher cannot give what he does not have. A number of studies carried out indicated the need for qualified and standard teachers’ academic qualification in English language. Such studies assert that English teachers should receive appropriate training in his subject area so that his classroom instruction could be above board. This will bring positive academic performance of English students especially those preparing for their final examinations. Rubba (2015) indicated that teachers of English language have special needs A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 40 40 August 26, 2023 August 26, 2023 Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 and Oranu (2010), teaching is a partnership between the teacher and the student which ultimately leads to permanent change in the behaviour of the student. The nature of teaching art is so complex and integrated that its dimension scope and outcome need to be planned carefully and systematically even before teaching commences. and Oranu (2010), teaching is a partnership between the teacher and the student which ultimately leads to permanent change in the behaviour of the student. The nature of teaching art is so complex and integrated that its dimension scope and outcome need to be planned carefully and systematically even before teaching commences. Considering all that has been mentioned, this paper therefore recommends that teacher’s profile and his teaching experience affect the level of students’ performance, having a degree in English language will help the language teacher in the delivery of the subject. Workshops, seminars and continuous in-service training are necessary for the English teacher in order to avail him with recent teaching methods in the Language. The English Language teacher should be conversant with the Language laboratory and should use it often with his students especially when dealing with oral drills. An individual who is fluent in English is not necessarily a good teacher of the language and may not be able to impart effectively to the students. Most English teachers get so relaxed once they obtain their degree. The same method of teaching employed several decades ago when they were employed as teachers by their employer remains intact. They have not bothered to improve on their skills, neither are they informed of the latest in the teaching of English Language. This should be discouraged as the government and school owners train and retrain the teachers. The government, school owners and organisations such as the British Council and have been of huge support in the training and re-training of the teachers of English Language in Nigeria. Teachers of English should avail themselves of this great opportunity. Several English Language Associations are also out there to help connect teachers to seminars and workshops by English Language. An Association such as ELTAN is a good example. English Language teachers are enjoined to exploit the opportunity by registering as members. It has also been noticed that teachers generally find it difficult to spend their ‘hard earned’ salaries on such things as subscription to Associations, Journals etc. Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 English teachers should look beyond the paltry sum to the value such training adds to them. It goes a long way and is invaluable. Employers of English teachers also could be of help by inviting experts to their schools to train their teachers as a group without them necessarily going out of their comfort zone. By so doing, both sides would have benefited immensely. In addition to the very good degree a teacher of English possesses, there is need for him to be IT compliant. We have English teachers who do not know how to handle android phones, how much more, computer. Conscious, concerted efforts should be made to learn. The world has become a global village indeed. Desirable Work Habits of a Teacher Aromolaran (2014) posited some of the desirable work habits and social behaviors that promote team spirit in the school environment as: Aromolaran (2014) posited some of the desirable work habits and social behaviors that promote team spirit in the school environment as: • being punctual at school and classes. • being punctual at school and classes. • avoidance of gossip, petitions, bickering, backbiting and unnecessary jealousy • possession of good conduct and loyalty to the management • friendliness with parents, guardians, students, super-ordinates, contemporaries and subordinates • dressing properly to the school and other school activities. • ensuring that criticisms are constructive, genuine and concise. • readiness to work extra hours. • readiness to work extra hours. The effective teacher of English must endeavour to do the above and more as directed by the higher authority. The effective teacher of English must endeavour to do the above and more as directed by the higher authority. English language, generally speaking, is encompassing programmes that equip recipients with the necessary knowledge, skills, attitude that will enable him to succeed in whatever business endeavour he may engage in, and invariably, enhancing economic development. English language, generally speaking, is encompassing programmes that equip recipients with the necessary knowledge, skills, attitude that will enable him to succeed in whatever business endeavour he may engage in, and invariably, enhancing economic development. For teaching and learning to be effective, relevant methods and strategies must be adopted by the teacher who is the distributor of knowledge in order to capture and sustain learners’ interest in the subject being taught and learnt. Variable teaching and learning conditions as well as teacher quality are quite imperative for the achievement of teaching and learning objective in English Language. A Publication of College of Languages and Communication Arts Education, Lagos State University of Education Teaching, according to Overbaugh (2013) is the science and art of assisting a person to learn. To him, teaching entails the use of acquired knowledge from natural and behavioural science in order to help appreciate the circumstances and personality of the learner while the art of teaching involves the use of creative and demonstrate skills in aiding the delivery of instruction. For Ogwu 41 41 August 26, 2023 August 26, 2023 Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ISSN: 2971-5067 According to Olanrewaju (2014) students’ low performance in any subject is as a result of the teachers’ ignorance and lackadaisical attitude. Ferguson and Ladd (2011) also opined that teachers’ qualification does not only matter in students’ achievement but are also major variables in improving the students’ learning and achievement. Qualification and experience are important variables in the efficiency of the teacher. Teachers with long years of teaching experience impact more on the students’ performance than teachers with short years of teaching experience. This is due to the fact that a professionally skilled teacher is skilled in the rudiments of teaching, has mastered his subject area and can relate well with students, parents and even colleagues on the field. The following shortfalls were noticed in the study carried out: 1. Most English teachers do not organize practical lessons. English teachers often often than not, use their native language in the class sometimes to explain certain facts. 1. Most English teachers do not organize practical lessons. English teachers often often than not, use their native language in the class sometimes to explain certain facts. 2. Some teachers teaching English language do not possess the necessary qualifications to teach the language. This was prevalent in the private schools where teachers who read Mass Communication and other Arts subjects were seen teaching the language, probably because they speak good English. 3. Fluency in English does not necessarily make an effective English teacher. 3. Fluency in English does not necessarily make an effective English teacher. 4. A qualification in English Language is a necessary prerequisite for teaching the language at the senior secondary school level. 5. There were no English Language laboratories in most of the schools visited. Conclusion A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 42 August 26, 2023 42 August 26, 2023 A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 42 42 August 26, 2023 August 26, 2023 42 42 Recommendations For teachers to live up to expectation, the government and school owners must ensure that appropriate measures are put in place to motivate the teachers. Some of these measures are; • Further training and in-service training should be encouraged • There should be regular promotion and commendation for the teachers • Materials needed for effective teaching should be supplied. • Job security will encourage teachers to put in their best. • Good working environment will encourage teachers to do their best. • Medical\ Welfare packages should be made available to teachers and their w • Medical\ Welfare packages should be made available to teachers and their wards\families • Recreational facilities will boost the teachers’ morale • Recreational facilities will boost the teachers’ morale. • The education of teachers’ children should free to the university level Fringe benefits will go a long way in retaining teachers and increase productivity A Publication of College of Languages and Communication Arts Education, Lagos State University of Education A Publication of College of Languages and Communication Arts Education, Lagos State University of Education 43 43 August 26, 2023 August 26, 2023 Journal of Colleges of Language and Communication Arts Education, Lagos State University of Education. Vol. 2, No. 1. AUGUST 26, 2023 ** ISSN: 2971-5067 These and so many others will enable teachers put in their best so as to be more effective and this inevitably will affect the performance of the students in English language. References Adedayo, (2018). Improve your school atmosphere. School Planning & Management. 37 (10), 18-21 Adebanjo, M.O (2020). Teaching English Language in Nigerian Secondary Schools. (Revised Edition): Gbadura Press. Adekunle M.A (1969). Journal of the Nigerian English Association: Producing better teacher of English Adeogun (2011) The benefits of Montessori Education. Athens: Ohio University Press Anderson, L.J and Block J.L (2012) Management and Organisation of behavior (7th Edition), New Jersey Prentice Hall. 52-85 Aromolaran, E A. (2019). Fundamentals of teaching (with revised questions). Lagos: Extension Publications Limited Chomsky N (2012). Language and the Mind Language and Education: A source book: Milton Keynes: Open University Press Ferrant, J. S, (1980). Principles and Practice of Education: Longman Education Texts. Majasan, J.A.(1995). The Teacher’s Profession: A Manual for Professional Excellence: Google Books: https://books.google.com Ferguson K. & Ladd, F. (2011). Getting ready for reading: A follow-up study of inner city Second Language learners at the end of key stage. British Journal of Educational Psychology. 74(1, 15-36) Ofejebe & Ezugoh, (2010). Teacher’s Motivation and its Influence on Quality Assurance in Nigerian Educational System: ResearchGate: https://www.researchgate.net Ogho, M. (2011). Education, Development and Peace. Oxford: ANC-CIIO Olarewaju , D. (2014). A very short History of the world. Athens: Penguin Books Rubba, O. (2015). The emotional experience of learning and teaching. London: Karnac Books Olarewaju , D. (2014). A very short History of the world. Athens: Penguin Books 44 44 August 26, 2023 August 26, 2023
https://openalex.org/W2128464157	https://europepmc.org/articles/pmc4181615?pdf=render	English	null	Efficient perovskite solar cells based on low-temperature solution-processed (CH3NH3)PbI3 perovskite/CuInS2 planar heterojunctions	Nanoscale research letters	2,014	cc-by	5,819	Abstract In this work, the solution-processed CH3NH3PbI3 perovskite/copper indium disulfide (CuInS2) planar heterojunction solar cells with Al2O3 as a scaffold were fabricated at a temperature as low as 250°C for the first time, in which the indium tin oxide (ITO)-coated glass instead of the fluorine-doped tin oxide (FTO)-coated glass was used as the light-incidence electrode and the solution-processed CuInS2 layer was prepared to replace the commonly used TiO2 layer in previously reported perovskite-based solar cells. The influence of the thickness of the as-prepared CuInS2 film on the performance of the ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3/Ag cells was investigated. The ITO/CuInS2(2)/ Al2O3/(CH3NH3)PbI3/Ag cell showed the best performance and achieved power conversion efficiency up to 5.30%. Keywords: Solution-processed; Solar cells; (CH3NH3)PbI3; Perovskite; CuInS2 10 nm). Therefore, the new solution-based techniques to improve the crystalline structures of inorganic nanocrys- tals are needed. For example, to enhance the carrier trans- port in CuInS2, a method of using a molecular-based precursor solution has been presented [10] to synthesize CuInS2 nanocrystals with a polycrystalline structure at relatively lower temperatures (<250°C) for the solution- processed inorganic solar cells. Besides, the inorganic materials which can be processed with solution-based techniques and generated charge carriers with long diffu- sion lengths in the bulk are sought. NANO EXPRESS Open Access Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 © 2014 Chen et al.; licensee Springer. 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. Materials Indium acetate (In(OAc)3, 99.99%), copper iodode (CuI, 99.999%), thiourea (≥99.0%), 1-propionic acid (≥99.5%), γ-butyrolactone (≥99.0%), aluminum oxide (Al2O3, 20 wt.% in isopropanol), methylamine (40 wt.% in H2O), hydroiodic acid (57 wt.% in water), diethyl ether, and PbI2 (99.999%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the reagents were used without further purification. Indium tin oxide-coated glass slides (ITO, ≤15 Ω/square, Wuhu Token Sci. Co., Ltd., China) were cleaned by successive ultrasonic treatment in de- ionized water, acetone, and isopropyl alcohol and then dried at 100°C for 10 min. Synthesis of CuInS2 nanocrystal film on ITO substrate CuInS2 nanocrystals were synthesized through a spin- coating method, which is similar to that reported by Li et al. [10]. Briefly, CuI (0.11 mmol), In(OAc)3 (0.1 mmol), and thiourea (0.5 mmol) were dissolved in a mixture of 1- butylamine (0.6 mL) and 1-propionic acid (40 μL) under a nitrogen atmosphere in a glovebox (O2 < 0.1 ppm, H2O < 0.1 ppm). The mixture was shaken for 1 min, and after which, the obtained CuInS2 precursor solution was then spin-cast onto the cleaned ITO substrates at 4,000 rpm for 30 s. Then, the obtained films were calcined at 150°C for 10 min and then heated to 250°C and held for 15 min It is known that CuInS2 as a p-type semiconductor is a very promising light-absorbing material for its direct bandgap of 1.5 eV, which is closely matched to the best bandgap (1.45 eV) of the solar cell materials [27]. Re- cently, a method of using a molecular-based precursor solution to synthesize CuInS2 nanocrystals at relatively lower temperatures (250°C) has been presented by Li et al. [10]. More importantly, the valence band level (−5.6 eV) of the CuInS2 is also matched to that (−5.6 or −6.5 eV) [13] of the (CH3NH3)PbI3, which is very benefi- cial to the hole transfer from the (CH3NH3)PbI3 to the CuInS2. Therefore, replacing the TiO2 with CuInS2 is reasonable. In this study, for the first time, the p-type semiconductor material, CuInS2, as both the light har- vester and hole transporter is prepared by the reported method [10] to replace the commonly used n-type TiO2 in the perovskite-based solar cells. Background h f l l Thin-film solar cells have attracted considerable attention because of simplified and low-cost fabrication procedures compared to conventional silicon-based solar cells. The thin-film solar cells based on inorganic photovoltaic mate- rials processed with expensive vacuum-based techniques and/or high-temperature sintering exhibit high efficiency [1-3]. However, the use of these thin-film solar cells is still limited because the manufacturing costs are still relatively high. To lower the cost of device fabrication, the low- temperature solution-based techniques such as spin coat- ing and chemical bath deposition are needed to prepare inorganic photovoltaic materials. The thin-film solar cells based on the solution-processed inorganic nanocrystals such as PbS [4,5], CdTe [6,7], CdSe [8], copper indium disulfide (CuInS2) [9,10], and Cu2ZnSnS4 [11] have been demonstrated, but their maximum solar power conversion efficiency is still low. The main reason for the low effi- ciency is that the low-temperature solution-processed in- organic nanocrystals are typically amorphous or poorly crystalline, leading to poor charge carrier transport be- cause of short carrier diffusion lengths (typically about The recently reported semiconducting perovskite ma- terials such as (CH3NH3)PbX3 (X = Cl, Br, I) could fulfil these requirements. These perovskites have high charge carrier mobilities and long charge carrier lifetime, which means that the light-generated charges have long carrier transport lengths [12]. It has been reported that the ef- fective diffusion lengths are about 100 nm for both elec- trons and holes [13,14]. In addition, these perovskites with a direct bandgap have a broad range of light absorp- tion and high extinction coefficient [15,16]. Due to their super electrical properties and super light-harvesting char- acteristics, the perovskites have been used in a variety of nanostructured solar cells and have achieved high-power conversion efficiencies (>9%) [16-20]. In solid-state sensi- tized solar cells, the CH3NH3PbI3 used as the sensitizer * Correspondence: mrchenchong@163.com; lfm0613@gmail.com 1School of Physics and Electronics, Henan University, Kaifeng 475004, People’s Republic of China Full list of author information is available at the end of the article * Correspondence: mrchenchong@163.com; lfm0613@gmail.com 1School of Physics and Electronics, Henan University, Kaifeng 475004, People’s Republic of China Full list of author information is available at the end of the article Page 2 of 8 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 as 250°C. After the deposition of CuInS2 film, the Al2O3 and (CH3NH3)PbI3 are successively deposited on the CuInS2 film to form the CuInS2/(CH3NH3)PbI3 planar heterojunction. The porous Al2O3 layer acts as a scaf- fold. Background h f l l Finally, an evaporated Ag top electrode was depos- ited on the (CH3NH3)PbI3 at a pressure of 10−6 Torr to complete the device fabrication. The schematics and energy diagram of the prepared solar cells are shown in Figure 1a, b, respectively. The surface morphology, structure characterization, and optical property of the prepared CuInS2/(CH3NH3)PbI3 film are studied. Fur- thermore, the influence of the thickness of the CuInS2 film on the power conversion efficiency of the fabricated CuInS2/(CH3NH3)PbI3 planar heterojunction solar cell is investigated. has led to a high-power conversion efficiency of 15% [17]. The other perovskite-based nanostructured solar cells that commonly incorporated the perovskite as the absorb- ing layer between an n-type electron-transporting layer such as TiO2 and a p-type hole-transporting layer such as 2,2′,7,7′-tetrakis(N, V-di-p-methoxyphenylamino)-9,9′- spirobifluorene (Spiro-OMeTAD) have also demonstrated high efficiencies [15,18,21]. Moreover, the research results reported by Lee et al.[16], Etgar et al.[22], and Ball et al. [23] showed that the perovskites have good charge (electron or hole)-transport properties, resulting in high efficiencies of the solar cells. Nevertheless, in these perovskite-based nanostructured solar cells, the trans- parent TiO2 compact layer between the conducting sub- strate and perovskite materials or the scaffold (Al2O3) generally requires high-temperature sintering at about 500°C [15,17,18,23,24], which limits substrate choice and is incompatible with the low-cost solar technology. Therefore, the low-temperature solution-processed semi- conductor materials that could replace the TiO2 for the perovskite-based nanostructured solar cells are needed. In addition to the preparation method, the electronic energy levels (EELs) of those substitute materials are needed to match the EELs of the perovskite materials for efficient charge transfer. For this purpose, ZnO compact layer and ZnO nanorods are recently prepared by electrodeposition and chemical bath deposition, respectively, to replace the TiO2 by Kumar et al. [25]. It is worth noting that the ma- terials used to replace the TiO2 do not necessarily have to be the n-type semiconductors such as the ZnO because the perovskites can conduct not only positive holes [22,26] but also electrons [16]. Synthesis of methylammonium iodide and (CH3NH3)PbI3 y y 3 3 3 Methylammonium iodide (CH3NH3I) was synthesized by reacting methylamine (aqueous, 40 wt.%) and hydroiodic acid (aqueous, 57 wt.%) in an ice bath for 2 h with stir- ring, as described elsewhere [18]. After that, the solvent was evaporated and the precipitate was washed using di- ethyl ether three times and dried at 60°C for 24 h in a vacuum oven. The resulting product, CH3NH3I, was used without further purification. To obtain a (CH3NH3) PbI3 precursor, the synthesized CH3NH3I was mixed with PbI2 at a 1:1 mol ratio in γ-butyrolactone (40% by weight) at 60°C. Results and discussion The morphology, structures, and chemical composition of the as-prepared CuInS2 were studied with SEM stud- ies accompanied by energy dispersive X-ray spectrom- etry (EDX). Figure 2a shows a typical top-view SEM image of the ITO/CuInS2(1) film. As shown in Figure 2a, the surface of the ITO substrate is covered with the CuInS2 film. The CuInS2 film is composed of CuInS2 nanoparticles, and these CuInS2 nanoparticles appear to be fused together after heating at 250°C. Moreover, some voids can be clearly seen in the ITO/CuInS2 (1) film, which can be explained by the decomposition of volatile surface ligands and precurse materials [10]. For a com- parison, the SEM top image of the ITO/CuInS2(2) is dis- played in Figure 2b. It can be observed that, compared to the ITO/CuInS2(1) film, the number of the voids in the ITO/CuInS2(2) film decreases significantly, indicat- ing that the voids in the ITO/CuInS2(1) have been filled by the CuInS2 precursor after two times of spin coating. In addition, the cross-sectional SEM images of the ITO/ CuInS2(2), ITO/CuInS2(2)/Al2O3, and ITO/CuInS2(2)/ Al2O3/(CH3NH3)PbI3/Ag films are shown in Figure 2c. It clearly shows that the CuInS2(2) film with an average thickness of 400 nm is formed on the ITO glass, and there are no obvious voids in the film. After the depos- ition of Al2O3, the thickness of the ITO/CuInS2(2)/ Al2O3 film increased. After the deposition of (CH3NH3) PbI3, the thickness of the ITO/CuInS2 (2)/Al2O3/ (CH3NH3) PbI3 film further increased to about 650 nm. The more important thing is that the (CH3NH3) PbI3 precursor solution permeated into the porous Al2O3 layer to form the (CH3NH3) PbI3. The corresponding EDX spectrum of ITO/CuInS2 (2) is shown in Figure 2d, which shows the film is mainly composed of copper (Cu), indium (In), and sulfur (S). The chemical compos- itional analysis reveals that the atomic ratio of Cu, In, and S is 25.33%, 24.9%, and 49.77%, respectively, close to 1:1:2, which confirms the formation of CuInS2. Solar cell fabrication First, a Al2O3 layer was introduced on the ITO/CuInS2(n) films by spin coating isopropanol solution containing Al2O3 nanoparticles at 4,000 rpm for 60 s. After that, the films were dried at 150°C for 30 min to obtain ITO/CuInS2 (n)/Al2O3. Then, the prepared ITO/CuInS2(n)/Al2O3 films were spin-coated with the obtained (CH3NH3)PbI3/γ- butyrolactone solution at 3,000 rpm for 60 s and then dried at 100°C for 1 h to form crystalline (CH3NH3)PbI3. The prepared ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films were naturally cooled to room temperature. All the experi- ment was finished in a nitrogen glovebox (O2 < 0.1 ppm, H2O < 0.1 ppm). Finally, a silver back contact layer was deposited by thermal evaporation onto the ITO/CuInS2 (n)/(CH3NH3)PbI3 films from a silver wire (99.999%). Materials Moreover, the indium tin oxide (ITO) glass rather than the commonly used fluorine-doped tin oxide (FTO) glass in previously re- ported perovskite-based solar cells is used as a light- incidence electrode because the CuInS2 film can directly be deposited on the ITO glass at a temperature as low Figure 1 Schematic diagram (a) and energy diagram (b) of the ITO/CuInS2/Al2O3/(CH3NH3)PbI3/Ag device. Figure 1 Schematic diagram (a) and energy diagram (b) of the ITO/CuInS2/Al2O3/(CH3NH3)PbI3/Ag device. Page 3 of 8 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 at this temperature. To change the thickness of the CuInS2 film, a spinning-drying cycle was repeated several times. The ITO/CuInS2 sample after n cycles of CuInS2 deposition was denoted as ITO/CuInS2(n). measurement, the light intensity was determined using a calibrated Si reference diode. For all measurements, the effective illumination area of the cells was 4 mm2. The monochromatic incident photon-to-electron con- version efficiency (IPCE) spectra for the fabricated solar cells were measured using a commercial setup (QTest Station 2000 IPCE Measurement System, Crowntech, Macungie, PA, USA). Characterization The surface morphology and structure of the prepared ITO/CuInS2(n) and ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films were characterized using a scanning electron micro- scope (SEM) (JSM-7001 F, Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) and power X-ray diffractometry (XRD) (DX-2500, Dandong Fangyuan Instrument Co., Ltd., Dandong, China), respectively. It should be noted that, for XRD measurement, the CuInS2 and (CH3NH3)PbI3 films are individually deposited on the cleaned glass without ITO layer to exclude the influence of the sub- strate on the XRD measurement. UV-visible absorption measurements were conducted using a UV–vis spectro- photometer (UV-2550, Shimadzu Corporation, Kyoto, Japan). Current density-voltage (J-V) characteristics of the as-prepared solar cells were measured using a Keithley 2410 SourceMeter (Keithley Instruments, Inc., Cleveland, OH, USA). A solar simulator (Newport Inc., Irvine, CA, USA) was used as the light source to provide AM 1.5 G simulated solar light (100 mW/cm2). Before each To characterize the crystal structure of the CuInS2, a typical XRD pattern of the as-prepared CuInS2(3) film on a clean glass substrate is shown in Figure 3. The well-defined peaks can be referred to a tetragonal CuInS2 (112), (204), (220), (116), and (312) (JCPDS file no. 85–1575), which is in agreement with the reported results [10,28,29]. Page 4 of 8 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 Figure 2 The morphology, structures, and chemical composition of the as-prepared CuInS2. (a) Top view of the ITO/CuInS2(1) film; (b) the top-view SEM image of the ITO/CuInS2(2) film; (c) the cross-sectional images of the ITO/CuInS2(2), ITO/CuInS2(2)/Al2O3, and ITO/CuInS2(2)/Al2O3/ (CH3NH3)PbI3/Ag films; and (d) the EDX spectrum of the ITO/CuInS2(2) film. Figure 2 The morphology, structures, and chemical composition of the as-prepared CuInS2. (a) Top view of the ITO/CuInS2(1) film; (b) the top-view SEM image of the ITO/CuInS2(2) film; (c) the cross-sectional images of the ITO/CuInS2(2), ITO/CuInS2(2)/Al2O3, and ITO/CuInS2(2)/Al2O3/ (CH3NH3)PbI3/Ag films; and (d) the EDX spectrum of the ITO/CuInS2(2) film. synthesized (CH3NH3)PbI3 film, the XRD analysis of the prepared (CH3NH3)PbI3 film was performed and shown in Figure 4b. It can be seen from Figure 4b that the dif- fraction peaks are in good agreement with the tetragonal phase of the (CH3NH3) PbI3 perovskite [17,30]. Figure 4a shows a top-view SEM image of the ITO/ CuInS2(2)/Al2O3/(CH3NH3)PbI3 film. Characterization By comparing this image with that (Figure 2b) of the ITO/CuInS2(2) film, it can be clearly observed that the (CH3NH3)PbI3 film was deposited on the CuInS2. However, the solution- processed (CH3NH3)PbI3 films are not very uniform and coated the CuInS2 film only partially with micrometer- sized (CH3NH3)PbI3 platelets, which is very similar to the observed phenomenon in the (CH3NH3)PbI3-coved compact TiO2 film [21]. Furthermore, to characterize the crystal structure and phase composition of the To study the light absorption properties of the prepared ITO/CuInS2(n) and ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films for application in photovoltaic devices, light absorp- tion studies are carried out. Figure 5 shows the UV–vis absorption spectra of the ITO/CuInS2(1) and ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3 films (n = 1, 2, and 3). As shown in Figure 5, the ITO/CuInS2(1) film has light absorption at wavelengths below 825 nm, which is similar to the reported results [10,31,32]. After the (CH3NH3)PbI3 film was deposited on the ITO/CuInS2(1) film, the ab- sorbance of the spectra of the ITO/CuInS2(1)/Al2O3/ (CH3NH3)PbI3 film increases significantly in the UV re- gion as well as the visible region. For the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3 films after other spin-cast cycles (n = 2 and 3) in our experiments, similar results are also obtained, which can be attributed to the light absorption of the deposited (CH3NH3)PbI3 film. More- over, for the ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films, Figure 5 also illustrates that the light absorbance was enhanced with an increase in spin-cast cycle number n, indicating an increased deposition amount of CuInS2. In addition, the absorption lines of the ITO/CuInS2/ Figure 3 The XRD pattern of the as-prepared CuInS2(3) film. Figure 3 The XRD pattern of the as-prepared CuInS2(3) film Page 5 of 8 Page 5 of 8 Page 5 of 8 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 Figure 4 SEM image and XRD analysis. (a) Top-view SEM image of the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3 film and (b) the XRD analysis of the prepared (CH3NH3)PbI3 film. The J-V characteristics of the ITO/CuInS2(n)/Al2O3/ (CH3NH3)PbI3/Ag solar cells under simulated AM 1.5 G solar irradiation and in the dark are shown in Figure 6. All device parameters under the light illumination, the open-circuit voltage (Voc), the short-circuit photocurrent (Jsc), the fill factor (FF), and the solar power conversion efficiency (η), extracted from the J-V characteristics are summarized in Table 1. Characterization For the ITO/CuInS2(n)/Al2O3/ (CH3NH3)PbI3/Ag cells, with the increase of CuInS2 de- position number n from 1 to 2, the Jsc increased from 8.85 to 9.92 mA/cm2, the Voc increased from 0.74 to 0.76 V, the FF increased from 0.51 to 0.70, and the η increased from 3.31% to 5.30%. These results might be caused by the voids in the CuInS2 film. As shown in Figure 2a, some voids have been found in the ITO/ CuInS2(1) film. When the (CH3NH3)PbI3 precursor so- lution was spin-cast onto the ITO/CuInS2(1)/Al2O3 film, these voids might be filled by the (CH3NH3)PbI3 precursor solution. Therefore, similar to the observed phenomenon in the mesoporous-TiO2/(CH3NH3)PbI3 film, the (CH3NH3) PbI3 probably infiltrated to the bottom of the CuInS2 film and had a contact with the hole collection electrode (i.e., the ITO electrode), which will enhance the probability of recombination between electrons in the (CH3NH3)PbI3 and holes in the ITO in the ITO/CuInS2(1)/Al2O3/(CH3NH3) PbI3/Ag solar cell. In contrast, as shown in Figure 2c, there are few voids in the ITO/CuInS2(2) film, which may ef- fectively reduce the charge recombination at the ITO/ (CH3NH3)PbI3 interface in the ITO/CuInS2(2)/Al2O3/ (CH3NH3)PbI3/Ag solar cell. This explanation is sup- ported by the J-V characteristics of the ITO/CuInS2(n)/ (CH3NH3)PbI3/Ag in the dark (Figure 6) since the charge recombination can be typically represented by the dark current [2,34,35]. It can be observed that the dark current density of the ITO/CuInS2(2)/Al2O3/(CH3NH3) PbI3/Ag cell is lower than that of the ITO/CuInS2(1)/ Figure 4 SEM image and XRD analysis. (a) Top-view SEM image of the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3 film and (b) the XRD analysis of the prepared (CH3NH3)PbI3 film. (CH3NH3)PbI3 films are very similar to those of the re- ported FTO/TiO2/(CH3NH3)PbI3 films [33], which further confirms the formation of (CH3NH3)PbI3 film and shows the potential of the ITO/CuInS2/Al2O3/(CH3NH3)PbI3 films in photovoltaic application. Figure 6 J-V characteristics. J-V characteristics of the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3/Ag solar cells under simulated AM 1.5G solar irradiation and in the dark (n = 1, 2, and 3). Figure 5 UV–vis absorption spectra of the ITO/CuInS2(1) and ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films (n = 1, 2, and 3). Figure 6 J-V characteristics. J-V characteristics of the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3/Ag solar cells under simulated AM 1.5G solar irradiation and in the dark (n = 1, 2, and 3). Figure 6 J-V characteristics. Characterization J-V characteristics of the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3/Ag solar cells under simulated AM 1.5G solar irradiation and in the dark (n = 1, 2, and 3). Figure 6 J-V characteristics. J-V characteristics of the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3/Ag solar cells under simulated AM 1.5G solar irradiation and in the dark (n = 1, 2, and 3). Figure 5 UV–vis absorption spectra of the ITO/CuInS2(1) and ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3 films (n = 1, 2, and 3). Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 Page 6 of 8 Figure 7 IPCE spectra of the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3/ Ag solar cell. Table 1 Summary of device performance under white light illumination with an intensity of 100 mW/cm2 Cells Voc (V) Jsc (mA/cm−2) FF PCE (%) ITO/CuInS2(1)/Al2O3/ (CH3NH3)PbI3/Ag 0.74 8.85 0.51 3.31 ITO/CuInS2(2)/Al2O3/ (CH3NH3)PbI3/Ag 0.76 9.92 0.70 5.30 ITO/CuInS2(3)/Al2O3/ (CH3NH3)PbI3/Ag 0.76 8.98 0.38 2.60 Table 1 Summary of device performance under white light illumination with an intensity of 100 mW/cm2 Al2O3/(CH3NH3)PbI3/Ag cell, which indicates that the charge recombination is reduced in the ITO/CuInS2(2)/ Al2O3/(CH3NH3)PbI3/Ag cell. g However, the device performance of the ITO/CuInS2 (n)/Al2O3/(CH3NH3)PbI3/Ag cell decreased as the CuInS2 deposition number n increased further from 2 to 3. It can be found from Table 1 that, compared to the ITO/CuInS2 (2)/Al2O3/(CH3NH3) PbI3/Ag cell, all device parameters (Jsc, FF, and η) except Voc of the ITO/CuInS2(3)/Al2O3/ (CH3NH3) PbI3/Ag cell decreased. For the ITO/CuInS2 (3)/Al2O3/(CH3NH3)PbI3/Ag cell, the Jsc, FF, and η de- creased to 8.98 mA/cm2, 0.38, and 2.60%, respectively. The main reason for the decreased device performance may be the increased thickness of CuInS2 film. As shown in Figure 1, in the ITO/CuInS2/Al2O3/(CH3NH3)PbI3/Ag cell, the CuInS2 mainly conducted holes. Therefore, in- creasing the thickness of CuInS2 film would increase the hole-transfer resistance and lead to an increase in overall series resistance (Rs) in the cells, which would inevitably lead to the degradation of Jsc and FF. The Rs can be calcu- lated from the inverse slope of the illuminated J-V charac- teristics at J = 0. The Rs values for the ITO/CuInS2 (2)/Al2O3/(CH3NH3)PbI3/Ag and ITO/CuInS2(3)/Al2O3/ (CH3NH3)PbI3/Ag cells are 6.4 and 29.6 Ω/cm2, respect- ively, which were calculated from the illuminated J-V char- acteristics (shown in Figure 6). Obviously, compared to the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3/Ag cell, the Rs of ITO/CuInS2(3)/Al2O3/(CH3NH3)PbI3/Ag cell increased due to the increased thickness of the CuInS2 film. Characterization Further- more, a too thick CuInS2 film may dramatically reduce the amount of light absorbed by the (CH3NH3)PbI3 film, which results in a sizeable reduction in the number of the photo-generated electrons in the (CH3NH3)PbI3 film and therefore reduces the Jsc and FF. Figure 7 IPCE spectra of the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3/ Ag solar cell. spectra for the perovskite solar cells without a CuInS2 layer [19,36,37]. It should be noted that, for these reported perovskite solar cells, the IPCE values are nearly zero in the long wavelength region (820 to 1,000 nm). However, in our case, IPCE of over 9% is observed at an entire wave- length range from 820 to 1,000 nm, resulting from the photocurrent originating from the CuInS2 layer. There- fore, the CuInS2 layer can improve the IPCE values of the solar cells in the long wavelength region. Our experimental results demonstrated that, for the ITO/CuInS2(n)/Al2O3/(CH3NH3)PbI3/Ag cells, the ITO/ CuInS2(2)/Al2O3/(CH3NH3)PbI3/Ag cell showed the high- est solar power conversion efficiency of 5.30%. However, it should be noted that the highest power conversion effi- ciency presented here was just taken from the solar cells with a simplified architecture. The solar cell architecture can be further optimized. For example, a hole-selective layer can be inserted between the ITO and the CuInS2 layers to reduce the charge recombination at the ITO/ CuInS2 interface. Similarly, inserting an electron-selective layer between the (CH3NH3)PbI3 layer and the Ag elec- trode may also suppress the charge recombination at the (CH3NH3)PbI3/Ag interface. 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Conclusions Figure 7 shows the IPCE spectra of the ITO/CuInS2(2)/ Al2O3/(CH3NH3)PbI3/Ag solar cell. It can be observed that the solar cell shows a spectral response in the almost entire wavelength region from 370 to 1,000 nm. The IPCE of over 31% is observed at a wavelength range from 370 to 750 nm. Furthermore, for the IPCE value, a sharp decrease in the wavelength region from 750 to 820 nm is observed. The threshold wavelength of 820 nm is related to the bandgap of about 1.5 eV for (CH3NH3)PbI3 [36]. These re- sults are in agreement with the previously reported IPCE In summary, the solution-processed (CH3NH3)PbI3 per- ovskite/CuInS2 planar heterojunction solar cells with a Al2O3 scaffold have been successfully fabricated, in which the CuInS2 films as both the light harvester and hole transporter were prepared at a relatively low temperature (250°C) via a simple solution-based chem- ical approach to replace the commonly used n-type TiO2 layer. The influence of the thickness of CuInS2 film on the performance of the fabricated ITO/CuInS2/Al2O3/ Page 7 of 8 Page 7 of 8 Chen et al. Nanoscale Research Letters 2014, 9:457 http://www.nanoscalereslett.com/content/9/1/457 (CH3NH3)PbI3/Ag solar cells was investigated. Our ex- perimental results demonstrated that an optimum power conversion efficiency of up to 5.30% can be achieved by the ITO/CuInS2(2)/Al2O3/(CH3NH3)PbI3/Ag cell. Opti- mizing the device architecture may further improve the performance of the ITO/CuInS2(n)/Al2O3/(CH3NH3) PbI3/Ag solar cells. The present research findings offer a new approach to achieve low-cost and high-efficiency solar cells. 11. Todorov T, Sugimoto H, Gunawan O, Gokmen T, Mitzi DB: High-efficiency devices with pure solution-processed Cu2ZnSn(S, Se) (4) absorbers. IEEE J Photovolt 2014, 4:483–485. 12. Hodes G: Perovskite-based solar cells. Science 2013, 342:317–318. 12. Hodes G: Perovskite-based solar cells. Science 201 13. 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Adv Mater 2013, 25:3727–3732. doi:10.1186/1556-276X-9-457 Cite this article as: Chen et al.: Efficient perovskite solar cells based on low-temperature solution-processed (CH3NH3)PbI3 perovskite/CuInS2 planar heterojunctions. Nanoscale Research Letters 2014 9:457. 36. Kim HS, Lee JW, Yantara N, Boix PP, Kulkarni SA, Mhaisalkar S, Gräetzel M, Park NG: High efficiency solid-state sensitized solar cell-based on submicrometer rutile TiO2 nanorod and CH3NH3PbI3 perovskite sensitizer. Nano Lett 2013, 13:2412–2417. 37. Jeng JY, Chiang YF, Lee MH, Peng SR, Guo TF, Chen P, Wen TC: CH3NH3PbI3 perovskite/fullerene planar-heterojunction hybrid solar cells. Adv Mater 2013, 25:3727–3732. doi:10.1186/1556-276X-9-457 Cite this article as: Chen et al.: Efficient perovskite solar cells based on low-temperature solution-processed (CH3NH3)PbI3 perovskite/CuInS2 planar heterojunctions. Nanoscale Research Letters 2014 9:457. 36. Kim HS, Lee JW, Yantara N, Boix PP, Kulkarni SA, Mhaisalkar S, Gräetzel M, Park NG: High efficiency solid-state sensitized solar cell-based on submicrometer rutile TiO2 nanorod and CH3NH3PbI3 perovskite sensitizer. Nano Lett 2013, 13:2412–2417. 37. Jeng JY, Chiang YF, Lee MH, Peng SR, Guo TF, Chen P, Wen TC: CH3NH3PbI3 perovskite/fullerene planar-heterojunction hybrid solar cells. Adv Mater 2013, 25:3727–3732. doi:10.1186/1556-276X-9-457 Cite this article as: Chen et al.: Efficient perovskite solar cells based on low-temperature solution-processed (CH3NH3)PbI3 perovskite/CuInS2 planar heterojunctions. Nanoscale Research Letters 2014 9:457. doi:10.1186/1556-276X-9-457 Cite this article as: Chen et al.: Efficient perovskite solar cells based on low-temperature solution-processed (CH3NH3)PbI3 perovskite/CuInS2 planar heterojunctions. Nanoscale Research Letters 2014 9:457. 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https://openalex.org/W2012643223	https://jiasociety.biomedcentral.com/track/pdf/10.1186/1758-2652-11-S1-P133	English	null	Evaluation of kidney toxicity in HIV patients with tenofovir-based regimen: the role of boosted protease inhibitor in real clinical setting	Journal of the International AIDS Society	2,008	cc-by	408	Conclusion In conclusion, our study demonstrated that also in real- life conditions, patients receiving TDF in combination with PI/r-based regimens had greater declines in renal function than did TDF NNRTI. Open A Poster presentation Evaluation of kidney toxicity in HIV patients with tenofovir-based regimen: the role of boosted protease inhibitor in real clinical setting F Ortu, P Piano, P Serra, R Meleddu, N Corso and PE Manconi F Ortu, P Piano, P Serra, R Meleddu, N Corso and PE Mancon Address: Azienda Ospedaliero Universitaria di Cagliari, Monserrato, Italy * Corresponding author from Ninth International Congress on Drug Therapy in HIV Infection Glasgow, UK. 9–13 November 2008 Published: 10 November 2008 Journal of the International AIDS Society 2008, 11(Suppl 1):P133 doi:10.1186/1758-2652-11-S1-P133 This abstract is available from: http://www.jiasociety.org/content/11/S1/P133 © 2008 Ortu et al; licensee BioMed Central Ltd. Purpose of the study p y Tenofovir (TFV) disoproxil fumarate (TDF) is an oral pro- drug of the nucleotide reverse transcriptase inhibitor TFV. Following oral administration, TDF undergoes rapid con- version to TFV in plasma. TFV is eliminated from systemic circulation renally through a combination of glomerular filtration and active tubular secretion. Active tubular secretion of TFV has been inferred from the observation that TFV renal clearance exceeds that of creatinine clear- ance in patients. Renal drug-drug interactions may occur between therapeutics that are substrates for the same tubular transport pathways or that inhibit the pathway of a drug subject to renal excretion: both of them, ritonavir (RTV) and tenofovir, share the same transport pathways (MRP4) [Ray et al. Antim Ag Chem 2006 (Oct); 50: 3297– 3304]. The objective of the present study was to investi- gate in a real clinical setting the relationship between PI/r co-administration with TDF and changes in estimated renal function. The mean baseline renal function was within the normal range and was similar between treatment groups. In uni- variate analysis, decreases in C-G estimates of CrCl were not significantly different among the two groups during the first 12 months of therapy. Journal of the International AIDS Society BioMed Central Open Access Page 1 of 1 (page number not for citation purposes) Methods Our study involved 187 patients in TFV-based regimen: 49 in NNRTI and 138 with PI/r. In a follow-up of one year, we evaluated renal function by calculated glomerular fil- trate using the Cockcroft-Gault (C-G) equation and the unabbreviated Modification of Diet in Renal Disease (MDRD) equation. Page 1 of 1 (page number not for citation purposes)
https://openalex.org/W4313320291	https://www.nature.com/articles/srep35425.pdf	English	null	Functionalized Stress Component onto Bio-template as a Pathway of Cytocompatibility	null	2,022	cc-by	10,645	Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 This in-vitro study introduces residual stress as a third dimension of cell stimulus to modulate the interaction between cells and bio-template, without the addition of either chemical or physical stimuli onto the bio-template surface. Ultrashort Pulsed Laser (USPL) irradiation of silicon-based bio-template causes recrystallization of silicon, which mismatches the original crystal orientation of the virgin silicon. Consequently, subsurface Induced Residual Stress (IRS) is generated. The IRS components demonstrated a strong cytocompatibility, whereas the peripheral of IRS, which is the interface between the IRS component and the virgin silicon surface, a significant directional cell alignment was observed. Fibroblast cells shown to be more sensitive to the stress component than Hela cancer cells. It revealed that cytocompatibility in terms of cell migration and directional cell alignment is directly proportional to the level of the IRS component. Higher stress level results in more cell alignment and border migration width. There is a stress threshold below which the stress component completely loses the functionality. These results pointed to a functionalized bio-template with tunable cytocompatibility. This study may lead to a new tool for the designing and engineering of bio-template. There is an ample amount of literature surrounding the needs for modulation of cell behavior for bio-applications, which indicates that study of cell interaction with biomaterials/bio-templates is of great importance for con- trolling cell behaviour, growth, and migration. With advancement in Nano and Micro-scale technologies, a vari- ety of approaches have been developed to engineer in-vitro cell interaction, including topographical patterning, changing surface chemistry, mechanical loading (stiffness), and the combinations of these methods1. Micro and nano topographic patterns is a popular tool for directional migration of cells. This method mechanically guide the cell migration by restricting cell mobility in channels and grooves1–6. Lithography, for instance, is one of the methods to create micro channels with an acceptable feature resolution7. The major challenge of this approach is that the fabricated topographic pattern do not represent the true extra cell matrix in nature. Cells in a real tissue are subject to an environment of three-dimensional space, multiple soluble factors, and cell-cell interactions1,8,9. Some effort have been made to partially address this issue. For example, Zhou et al. has demonstrated a two-level topographic pattern made by an integrated lithography to cue cells8. Modification of surface chemistry is another approach of bio-template. Premnath et al. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Functionalized Stress Component onto Bio-template as a Pathway of Cytocompatibility received: 13 June 2016 accepted: 29 September 2016 Published: 19 October 2016 1Micro/Nanofabrication Laboratory, Department of Aerospace Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, M5B 2K3, Canada. 2Ultrashort laser nanomanufacturing research facility, Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada. 3Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada. Correspondence and requests for materials should be addressed to K.V. (email: venkat@ryerson.ca) Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 Aside from biological in favor of using silicon as a bio-template, slight changes in crystal orientation of a silicon substrate leads to the build-up of a residual stress component. This unique property can be attributed to the mono crystallinity of silicon r, making it a favourable material for use as a bio-template. In order to accurately tune the functionalization of stress components on the IRS, the bio-template was exposed to the irra- diation from an Ultrashort Pulsed Laser (USPL). The combination of shockwave formations and intense thermal expansion generated by the laser irradiation, induces crystal distortion. The resulting mismatch of distorted crys- tal orientation and virgin crystals causes residual stress. The functionality of the stress component was controlled by varying repetition rate (rep.rate) of the laser. The repetition rate determines the level of stress of the functional- ized stress component, and it in turn tunes the degree of biocompatibility of the bio-template. This functionalized property elicits precise control on cell behaviour, such as directional cell migration and self-cytocompatibility. Fibroblast and cancerous (HeLa) cells seeded respectively onto IRS zones and the responses were observed after 24 hours of incubation. Both cell lines showed a significant collective migration tendency towards virgin areas on the bio-template. In contrast to the cell migration trend, cell mobility was confined by the functionalized stress component. In this case, the migration distance can be regulated by the level of stress within the functionalized stress component. The Fibroblast (NIH3T3) cells is found to be distinctively more sensitive to the functionalized stress component. Precisely speaking, the threshold of cytocompatibility is comparatively lower in Fibroblast (NIH3T3) cells. Moreover, directional cell alignment was observed on the proximity of IRS. The results showed that, for the first time, significant promise of creating self-cytocompatibility properties in silicon bio-component without resorting to chemical stimuli and/or topographical structures. Figure 1 is a schematic drawing shows the response of cells to IRS. The cell interactions, topographical, elemental, compositional, and crystallographic properties of the IRS bio-template were characterized by the Scanning Electron Microscopy (SEM), Florescent Microscope, X-ray Diffraction (XRD), Energy Dispersive X-ray (EDX), and Electron Backscatter Diffraction (EBSD), respectively. Finally, to identify the threshold of cytocompatibility, the minimum level of stress to observe cytocompatbility, was evaluated with Micro-Raman spectrum shift. knowledge, this the first time subsurface stress has been used on silicon bio-template for the modification of cytocampatibility. Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 A monocrystalline silicon wafer has been chosen as an experimental substrate because sil- icon crystals are sensitive to mechanical and thermal excitation. Silicon is increasingly being considered for bio-applications20–22. Aside from biological in favor of using silicon as a bio-template, slight changes in crystal orientation of a silicon substrate leads to the build-up of a residual stress component. This unique property can be attributed to the mono crystallinity of silicon r, making it a favourable material for use as a bio-template. In order to accurately tune the functionalization of stress components on the IRS, the bio-template was exposed to the irra- diation from an Ultrashort Pulsed Laser (USPL). The combination of shockwave formations and intense thermal expansion generated by the laser irradiation, induces crystal distortion. The resulting mismatch of distorted crys- tal orientation and virgin crystals causes residual stress. The functionality of the stress component was controlled by varying repetition rate (rep.rate) of the laser. The repetition rate determines the level of stress of the functional- ized stress component, and it in turn tunes the degree of biocompatibility of the bio-template. This functionalized property elicits precise control on cell behaviour, such as directional cell migration and self-cytocompatibility. Fibroblast and cancerous (HeLa) cells seeded respectively onto IRS zones and the responses were observed after 24 hours of incubation. Both cell lines showed a significant collective migration tendency towards virgin areas on the bio-template. In contrast to the cell migration trend, cell mobility was confined by the functionalized stress component. In this case, the migration distance can be regulated by the level of stress within the functionalized stress component. The Fibroblast (NIH3T3) cells is found to be distinctively more sensitive to the functionalized stress component. Precisely speaking, the threshold of cytocompatibility is comparatively lower in Fibroblast (NIH3T3) cells. Moreover, directional cell alignment was observed on the proximity of IRS. The results showed that, for the first time, significant promise of creating self-cytocompatibility properties in silicon bio-component without resorting to chemical stimuli and/or topographical structures. Figure 1 is a schematic drawing shows the response of cells to IRS. The cell interactions, topographical, elemental, compositional, and crystallographic properties of the IRS bio-template were characterized by the Scanning Electron Microscopy (SEM), Florescent Microscope, X-ray Diffraction (XRD), Energy Dispersive X-ray (EDX), and Electron Backscatter Diffraction (EBSD), respectively. Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 Finally, to identify the threshold of cytocompatibility, the minimum level of stress to observe cytocompatbility, was evaluated with Micro-Raman spectrum shift. Meysam Keshavarz1, Bo Tan1 & Krishnan Venkatakrishnan2,3 has recently performed an in-depth study of programing cancer cells through phase-functionalized silicon-based biomaterials10. It revealed that changing the surface chemistry of the silicon substrate by formation of oxides contributes to the control of directional migration, cytoskeleton shape, and cell population10–12. This finding has provided valuable insight into cellular responses to changes of material-phase stimuli. However, the functionality of this method relies on the changing of surface chemistry. Recent studies have shown that mechanical loading (stress and strain) can be implemented as a cue for cell sign- aling and migration13–17. However, the application of this method is restricted to polymer-based bio-templates17. It cannot be applied to silicon-based bio-templates which are widely used as biocompatible templates, due to the brittle nature of silicon18,19. In this research work, we introduced a novel concept of using laser induced residual stress (IRS) on a monocrystalline silicon substrate as a stimuli for cell modulation. This method does not employ chemicals or topographic structures. It stimulates cell migration and alignment through subsurface stress. To the best of our 1Micro/Nanofabrication Laboratory, Department of Aerospace Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, M5B 2K3, Canada. 2Ultrashort laser nanomanufacturing research facility, Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada. 3Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada. Correspondence and requests for materials should be addressed to K.V. (email: venkat@ryerson.ca) Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 1 1 www.nature.com/scientificreports/ Figure 1. Schematic illustration shows the response of cells to IRS. Two types of encounters on the proximity of IRS are: cell breakage and cell protrusion. SEM images of HeLa and Fibroblast (NIH3T3) represent collective cell migration towards stress-free area on the bio-template. Figure 1. Schematic illustration shows the response of cells to IRS. Two types of encounters on the proximity of IRS are: cell breakage and cell protrusion. SEM images of HeLa and Fibroblast (NIH3T3) represent collective cell migration towards stress-free area on the bio-template. knowledge, this the first time subsurface stress has been used on silicon bio-template for the modification of cytocampatibility. A monocrystalline silicon wafer has been chosen as an experimental substrate because sil- icon crystals are sensitive to mechanical and thermal excitation. Silicon is increasingly being considered for bio-applications20–22. Results and Discussion Ablation threshold for a single laser pulse is given by, where Pavg is the Laser average power [W] and Frep [KHz]. Ablation threshold for a single laser pulse is given by, = .       T K F F ln (3) t th (3) where Ft is the laser fluence, Fth is the ablation threshold, and K is related both to the absorption depth of the light in the bulk material, and the thermal diffusion length (2√D × τ where D is the thermal diffusivity and τ is the laser pulse duration).l Figure 2A shows the variation of laser fluence versus the ablation threshold in terms of energy generated per unit area at distinct repetition rates and 214 (fs) laser pulse width. In this plot the effect of pulse-to-pulse interval was not taken into consideration. However, multi-photon absorption simulation using COMSOL Multiphysics unveiled that the accumulative influence of laser pulses increases by decreasing pulse-to-pulse interval, that is, laser rep.rate. When the bio-template is irradiated with a USPL the surface temperature of substrate exponen- tially rises by increasing laser rep.rate. The difference in temperature between face and subsurface of the silicon bio-template, caused by rapid heat dissipation, creates intricate crystal distortions. This changes the crystal orien- tation of the IRS zone from inherent (100) to (211) and a different order of crystallinity. Another advantage of uti- lizing tunable USPL is the ability to precisely modulate the IRS, avoiding collateral damage created by exceeding the ablation threshold of an irradiated bio-template. In particular, amplifying the repetition rate from 4 MHz to 26 MHz causes extensive accumulation of heat on the exposure zone - owing to maximizing laser matter interac- tion time, more thermal and shockwave energy is transferred to the substrate. Therefore, incremental heat gener- ation intensifies thermal stress, resulting in formation of the laser induced residual stress zone. Earlier mentioned laser parameters such as fluence and rep.rate also have a similar effect in amplifying the laser shockwave. pl pf p y g In an attempt to understand the heat accumulation generated by the interaction between an ultrafast laser and a bio-template, COMSOL Multiphysics was exploited to simulate the average heat flux disturbed during multiphoton absorption. Results and Discussion Results and Discussion Fabrication and characterization of IRS onto bio-template. Interaction of ultra-short pulsed laser with matter falls into two steps: a localized thermal accumulation and shockwave caused by multi-photon absorp- tion, and a blast of plasma formation23,24. Thereby the sequential interaction of USPL trains with the substrate is triggered to increase the localized functional stress component along with the laser path25,26. Laser irradiation of silicon-based bio-templates using different laser repetition rates has prompted a gradient of residual stress at the peripheral of IRS27,28. Amplitude of this laser-induced stressed zone is proportional to the repetition rate of the laser. However for a given pulse width, the gradient of laser fluence decreases with increasing laser pulse repeti- tion rate from 4 MHz to 26 MHz; here, the transmitted energy, in terms of heat and pressure of the shockwave, is intensified. To expound upon the relationship of laser ablation threshold and laser fluence with repetition rate, the following equations were considered;l Laser fluence is given by, Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 2 www.nature.com/scientificreports/ Figure 2. Plot A, shows variation of laser fluence versus ablation threshold of Silicon bio-template a distinct laser pulse width of (214 fs) and repetition rates ranging from 4 to 26 MHz. Plot B, Temperature variation with rep.rate, shows temperature rise exponentially by increasing laser rep.rate. Plot C, shows the temperature vs. heat dissipation time. D to G show the temperature gradient simulated by COMSOL Multiphysics. (All the data were taken after 10 consecutive pulses). Figure 2. Plot A, shows variation of laser fluence versus ablation threshold of Silicon bio-template a distinct laser pulse width of (214 fs) and repetition rates ranging from 4 to 26 MHz. Plot B, Temperature variation with rep.rate, shows temperature rise exponentially by increasing laser rep.rate. Plot C, shows the temperature vs. heat dissipation time. D to G show the temperature gradient simulated by COMSOL Multiphysics. (All the data were taken after 10 consecutive pulses). = E A Ft (1) eff (1) where E is laser pulse energy [J] and Aeff is effective focal spot area [cm2]. Laser Pulse energy is given by, where E is laser pulse energy [J] and Aeff is effective focal spot area [cm2]. Laser Pulse energy is given by, = P F E (2) avg rep (2) where Pavg is the Laser average power [W] and Frep [KHz]. = ∗ q emissivity an x y t 4( , , ) o (8) Here, emissivity was taken as 0.8. Within COMSOL, the bottom surface of the silicon wafer geometry was thermally insulated and the initial temperature of the wafer was defined at 293.15 K. A free tetrahedral mesh was used on the entirety of the geometry with a maximum element edge length of 1.8E-6 meters. Upon completion of the computations, the wafer thickness was scaled up to five times to allow improved observation of the depth of the heat gradient within the thickness. For the following study, a classic heat transfer model was applied in order to describe the phenomenon of accumulative temperature rise caused by amplifying the repetition rate from 4 MHz to 26 MHz. However, as cal- culated and plotted on Fig. 2A, laser fluence is expected to decrease with increasing repetition rates. Also, short- ening pulse to pulse interval at higher repetition rates, increases the initial temperature for the subsequent pulse. Therefore, heat accumulation is expedited by multiplying the number of pulses, as shown on Fig. 2B. As seen in Fig. 2C, heat dissipation time is longer than pulse interval at a higher rep.rate and thus, thermal energy would be expected to build up by increasing the rep.rate. In contrast, at a lower rep.rate, accumulated thermal energy is insufficient, since the heat induced by a single pulse has dissipated by the time the next pulse arrives. Figure 2D to G show the heat generated by multiphoton absorption at different rep.rates, the cross sectional view displays the heat dissipation depth and expansion of IRS at higher rep.rates. The pulse-to-pulse duration is a function of the repetition rate of a laser and therefore, has a significant influence on laser matter interaction - the substrate in the focal point gains energy and is thermally loaded by sequential pulses. Therefore, different repetition rates (4, 8, 12 and 26 MHz) at constant a 214 fs pulse-width have been implemented to irradiate the bio-template, through which the functionalized stress component can be induced. Moreover, to characterize the formation of distinct crystal orientations XRD and EBSD have been carried out.hh y The crystalline orientation map of the IRS zone is shown in Fig. 3. The mapping area shown in Fig. 3A is 200 μm2, with a mapping step of 0.2 μm where an index of over 85% was achieved. The different colors of the orientation map correspond to the different crystallographic orientations of scanned area. = ∗ q emissivity an x y t 4( , , ) (8 o = ∗ q emissivity an x y t 4( , , ) o Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 Results and Discussion As mentioned, the simulation was created on COMSOL Multiphysics software and the built-in Heat Transfer in Solids Module was used to solve the following heat transfer equation: Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 3 www.nature.com/scientificreports/ ρ ρ ∂ ∂ + ⋅∇ = ∇⋅ ∇ + u C T t C T k T Q ( ) (4) p p ρ ρ ∂ ∂ + ⋅∇ = ∇⋅ ∇ + u C T t C T k T Q ( ) p p (4) where k is the thermal conductivity of silicon, ρ is the material density, and Cp is the heat capacity of silicon sub- strate at constant pressure. The pulse width and rep.rate of the USPL were varied for each computational run of the simulation by adjusting the parameters for each new computation. To represent the silicon wafer, a cylindrical geometry was created with a thickness of 1 μm and with a radius identical to the spot size (5 μm) in order to facil- itate more precise meshing. A Gaussian pulse (I) was used to mimic the distribution of intensity within a laser beam, the highest intensity being in the center with decreasing intensity towards the circumference. A standard deviation of r_spot/3, where r_spot is the radius of the circular spot, was entered to create increasingly higher temperatures in the centre of the ablation spot. This allowed the simulation of a more realistic temperature dis- tribution. Results and Discussion Within the heat flux equations of COMSOL, the laser power (p_laser) was multiplied to the Gaussian functions to produce a gradient intensity (hf) within the laser spot area: = ∗ ∗ hf x y p gp x gp y ( , ) 1( ) 1( ) (5) laser = ∗ ∗ hf x y p gp x gp y ( , ) 1( ) 1( ) laser (5) The rep.rate and pulse width were incorporated into a single equation to create a rectangular (rect2) pulse train (an3) with parameters that could be manipulated: = ∗ an t x y rect t hf x y 3( , , ) 2( ) ( , ) (6) = ∗ an t x y rect t hf x y 3( , , ) 2( ) ( , ) (6) A heat flux function (an4) was then formulated: = ∗ an t x y hf x y an t 4( , , ) ( , ) 3( ) (7) = ∗ an t x y hf x y an t 4( , , ) ( , ) 3( ) (7) Finally, within the Heat Transfer in Solids Module, the above equation was entered as a general inward heat flux to produce: = ∗ q emissivity an x y t 4( , , ) (8) o = ∗ q emissivity an x y t 4( , , ) o The (001) crystallographic plane was set as the reference direction. Hence, comparison of collected information from backscattered Kikuchi dif- fraction patterns at each EBSD mapping point revealed a deviation from the reference orientation. The EBSD mapping result shows that the IRS zone is blue in color, whereas crystal orientation on the unirradiated zone is red in color (according to the EBSD relative Euler orientation map Fig. 3C) which indicates a deviation from (100) to (211). The color variation of the orientation map shown on Fig. 3B corresponds to the different crystal- lographic orientations. In this case, (211) is the dominant crystal plane. The corresponding orientation for each color is represented on Fig. 3C. This EBSD outcome is in line with the resulting XRD pattern.hl p gh g p The influence of laser irradiation on the crystal orientation of bio-templates was acquired by utilizing X-ray diffraction, as plotted on Fig. 4. Diffraction patterns of the IRS bio-templates at different laser repetition rates were compared with a single crystal silicon substrate as a reference. D-spacing calculation of diffraction pattern of reference substrate shows a characteristic peak at 2θ (69.2), which stands for (100). While after exposure to an USPL, distinct peaks such as (111), (211) and (220) were emerged. An increment in intensity of a peak represents (211) crystal orientation observed by increasing repetition rate of the laser pulse from 4 to 26 MHz. However, this peak has relatively lower intensity compared to a dominant (100) peak due to the fact that only a thin facial layer of a bio-template undergoes crystal orientation changes. The XRD result agrees with the EBSD outcome, both of methods demonstrating changes of crystal orientation on IRS zones. Further characterization has been done using EDX to examine the possibility of surface oxidation or surface compositional changes. The XRD analysis Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 4 www.nature.com/scientificreports/ tificreports/ Figure 3. (A) Back scatter scanning area. (B) EBSD mapping of the IRS zone. (C) EBSD relative Euler orientation map. (The color is related to the crystallographic direction that is parallel to the surface). Figure 4. XRD patterns of laser irradiated bio-templates at 4, 8, 12, and 26 MHz. Enlarged pattern shows emerging peaks, representing of (111), (211) and (221) crystal orientations. Figure 3. (A) Back scatter scanning area. (B) EBSD mapping of the IRS zone. (C) EBSD relative Euler orientation map. = ∗ q emissivity an x y t 4( , , ) o (The color is related to the crystallographic direction that is parallel to the surface). Figure 3. (A) Back scatter scanning area. (B) EBSD mapping of the IRS zone. (C) EBSD relative Euler orientation map. (The color is related to the crystallographic direction that is parallel to the surface). Figure 4. XRD patterns of laser irradiated bio-templates at 4, 8, 12, and 26 MHz. Enlarged pattern shows emerging peaks, representing of (111), (211) and (221) crystal orientations. Figure 4. XRD patterns of laser irradiated bio-templates at 4, 8, 12, and 26 MHz. Enlarged pattern shows emerging peaks, representing of (111), (211) and (221) crystal orientations. Figure 5. EXD spectrum through LIRS IRS and plain bio-template. (A) SEM image of take spectrum. (B) EDX Spectrum identifying the presence of Oxygen. Figure 5. EXD spectrum through LIRS IRS and plain bio-template. (A) SEM image of take spectrum. (B) EDX Spectrum identifying the presence of Oxygen. revealed that the compositional aspect of all samples remained intact. Surface compositional characterization of IRS at different laser repetition rates was examined by EDX. Figure 5 shows an EDX spectral. Elemental anal- ysis of the spectrum revealed that there is no compositional difference in comparison to a plain silicon wafer. This indicates that multiphoton substrate interaction to induce functional stress component using the previously mentioned parameters, does not cause compositional changes. However, alternations in crystal orientation have clearly been observed in XRD patterns and through EBSD mapping. Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 5 www.nature.com/scientificreports/ Figure 6. (A) Raman spectra of IRS at 26 MHz. (B) Raman shifts at 4, 8, 12, and 26 MHz. Figure 6. (A) Raman spectra of IRS at 26 MHz. (B) Raman shifts at 4, 8, 12, and 26 MHz. To analyze the relationship between laser irradiation energy and functionalized stress components, IRS was characterized by Raman spectroscopy in terms of wavelength shift. In general, as a result of compressive stress, Raman frequency increases while tensile stress results in downward shift of Raman frequency29–32. Figure 6A shows Raman spectra of a bio-template irradiated at 26 MHz. As shown in Fig. 6A, there is a slight difference in measured peak position when moving outward within the IRS zone. Raman shift variation measured at each 10 μm (up to 50 μm) from the proximity of IRS at different rep.rates, are illustrated in Fig. 6B. = ∗ q emissivity an x y t 4( , , ) o The characteristic Raman scattering frequency of the silicon-based bio-template used in this study was ω = 521 cm–1 however, this value shifted as a result of USPL-induced stress. Mapping of Raman shift differences through the IRS zone leads us to determine the thresholds of HeLa and Fibroblast (NIH3T3) cell lines in sensing the minimum required functionalized stress component by which a cell-free zone can be created as a result of collective cell migration. Slight changes in measured frequency, in this case for 0.5 cm−1 peak shift in silicon, represents a stress sensitivity of about 4 KPa32. The relation between Raman peak shift and stress was assumed to be simply linear, and the effect of all stain tensor components are not considered31,32. Components of strain in crystalline materials affect the frequency of the Raman phonons and as such, the position of the Raman peak. For instance, for uniaxial or biaxial stress in the (100) plane of silicon, this relation is: σ = − ∆ω − Uniaxial (MPa) 435 (cm ) (9) 1 σ = − ∆ω − Uniaxial (MPa) 435 (cm ) 1 (9) σ + σ = − ∆ω − Biaxial (MPa) 435 (cm ) (10) xx yy 1 σ + σ = − ∆ω − Biaxial (MPa) 435 (cm ) xx yy 1 (10) Laser fluence that can be precisely tuned through the adjustment of laser parameters, such as rep.rate, laser power, to accurately determine the tensile and compressive stress. The results from cell culture show that the degree of functionalization of the induced stress component depends on the level of stress. A general observation is that a considerable amount of compressive stress was induced at rep.rates higher than 8 MHz, as showed in Fig. 7. Comparison of collective cell migration distances from the IRS, demonstrated that compressive stress has a profound impact on cell response. Cell interaction with IRS bio-template. Collective cell migration. We employed a culture medium that contained 3× 106 cells/mL; HeLa and Fibroblast (NIH3T3) cell lines were separately seeded and incubated for 24 hrs. Interestingly, collective cell migration from the IRS zone towards the plain surface was observed on flu- orescent and SEM images. As shown on images in Fig. 8 both Fibroblasts (NIH3T3) (Fib) and cancerous cells (Hela) responded to a functionalized stress component as a migration stimulus. = ∗ q emissivity an x y t 4( , , ) o Thus the sensitivity of cells to the residual stress required to cue them has been secured as a threshold of cytocompatibility. Moreover, as evidenced in Fig. 8, higher sensitivity of cells to the compressive stress compared to the tensile stress is significantly noticea- ble. As presented in Fig. 8 (cell migration distance) by intensifying the repetition rate, compressive stress increases and hence the cells migration distance was broadened. It is implied that, as a result of increasing the repetition rate, the intensity of residual stress has magnified in linear regression with the average migration distance of cells from IRS zones. Therefore, distinct boundary on proximity of the IRS zone has created a cell-free zone. Given the cell-free zone indirectly rule out the possibility of topographical changes in terms of surface 20 ness, as the pro- found influence of surface roughness on proliferative behavior of cell by which increment of roughness enhances the cell growth and proliferation is well known33. The width of this cell-free zone is directly proportional to the induced residual stress and hence laser repetition rate. It can be seen that induced residual stress on bio-templates can be functionalized in order to regulate cells behavior.ht g The measured Raman shift indicates that compressive stress is dominant at higher laser repetition rates (Fig. 7). In addition, transition of tensile to compressive stress at 4 and 8 MHz has been determined to be a thresh- old of cytocompatibility, by which a minimum width of 30 μm for Fibroblast (NIH3T3)and 22.3 μm for HeLa have been formed through collective cell migration. Higher sensitivity of Fibroblast (NIH3T3) cells was noticed Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 6 www.nature.com/scientificreports/ Figure 7. Shows magnitude of stress component as function of repetition rate. Figure 7. Shows magnitude of stress component as function of repetition rate. Figure 7. Shows magnitude of stress component as function of repetition rate. Figure 8. Collective cell migration of HeLa and Fibroblast (NIH3T3) in response to functionalized stress component induced on bio-template at different rep.rate are shown on SEM images. The histogram on the right illustrates distance of cell migration corresponding to the magnitude of functionalized stress component. Error bars show SEM; two independent experiments were repeated with n = 3 in each experiment. Statistical significance is shown with p < 0.05, p < 0.01. Figure 8. = ∗ q emissivity an x y t 4( , , ) o Collective cell migration of HeLa and Fibroblast (NIH3T3) in response to functionalized stress component induced on bio-template at different rep.rate are shown on SEM images. The histogram on the right illustrates distance of cell migration corresponding to the magnitude of functionalized stress component. Error bars show SEM; two independent experiments were repeated with n = 3 in each experiment. Statistical significance is shown with p < 0.05, *p < 0.01. compare to HeLa cells. Due to the higher mobility and smaller size of the cytoplasm of HeLa cells, their reduced sensitivity to the IRS can be speculated. Additionally, cytoskeleton breakage on cells moving away from the IRS and protrusion on cells who encounters IRS were observed. Cell response to IRS was characterized in terms of cell directionality and persistence distance. The polar diagrams (Fig. 9) show directionality of collective cells on four different conditions within a 100 μm proximity of central the IRS zone. Given polar diagrams on Fig. 9, a significant difference between cell angles on Fig. 9A,D has been observed, each image compares a tendency of cell alignment on the proximity of the IRS with the control area far from the IRS. To calculate directionality of the cell migration, symmetry of IRS zone was taken into account. The boundary of cell interaction with IRS was defined as (0, 0) of the virtual origin, an absolute value of cell angle considered as directional value. At higher functional- ized stress component induced onto the bio-template by shorter laser pulse separation time (high rep.rate), cells tend to be oriented more closely along the X vector of the virtual coordinate than for a longer pulse separation compare to HeLa cells. Due to the higher mobility and smaller size of the cytoplasm of HeLa cells, their reduced sensitivity to the IRS can be speculated. Additionally, cytoskeleton breakage on cells moving away from the IRS and protrusion on cells who encounters IRS were observed. Cell response to IRS was characterized in terms of cell directionality and persistence distance. The polar diagrams (Fig. 9) show directionality of collective cells on four different conditions within a 100 μm proximity of central the IRS zone. Given polar diagrams on Fig. 9, a significant difference between cell angles on Fig. 9A,D has been observed, each image compares a tendency of cell alignment on the proximity of the IRS with the control area far from the IRS. = ∗ q emissivity an x y t 4( , , ) o To calculate directionality of the cell migration, symmetry of IRS zone was taken into account. The boundary of cell interaction with IRS was defined as (0, 0) of the virtual origin, an absolute value of cell angle considered as directional value. At higher functional- ized stress component induced onto the bio-template by shorter laser pulse separation time (high rep.rate), cells tend to be oriented more closely along the X vector of the virtual coordinate than for a longer pulse separation Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 7 www.nature.com/scientificreports/ Figure 9. Polar diagrams show, decrease of cell angle by increasing the magnitude of functionalized stress component. (A–D) The fluorescent images of Fibroblast (NIH3T3) cell lines seeded on the IRS bio-template with different magnitudes of functionalized stress component, 217.5, 50, −870 and −1305 MPa respectively. Comparison of images (A,D) shows that by increasing the functionalized stress component, diversity of cell angle decreases. Figure 9. Polar diagrams show, decrease of cell angle by increasing the magnitude of functionalized stress component. (A–D) The fluorescent images of Fibroblast (NIH3T3) cell lines seeded on the IRS bio-template with different magnitudes of functionalized stress component, 217.5, 50, −870 and −1305 MPa respectively. Comparison of images (A,D) shows that by increasing the functionalized stress component, diversity of cell angle decreases. Figure 9. Polar diagrams show, decrease of cell angle by increasing the magnitude of functionalized stress component. (A–D) The fluorescent images of Fibroblast (NIH3T3) cell lines seeded on the IRS bio-template with different magnitudes of functionalized stress component, 217.5, 50, −870 and −1305 MPa respectively. Comparison of images (A,D) shows that by increasing the functionalized stress component, diversity of cell angle decreases. Figure 10. (A) Schematic illustration of the distribution of functionalized stress component and cyto-breakage as a result of cell-IRS interaction. (B,C) SEM images shows cyto-breakage of Fibroblast (NIH3T3) cells. (D,E) same phenomena has observed on HeLa cells facing IRS as well. (Arrows and dashed line indicate the side of breakage and the boundary of IRS zone respectively). Figure 10. (A) Schematic illustration of the distribution of functionalized stress component and cyto-breakage as a result of cell-IRS interaction. (B,C) SEM images shows cyto-breakage of Fibroblast (NIH3T3) cells. (D,E) same phenomena has observed on HeLa cells facing IRS as well. (Arrows and dashed line indicate the side of breakage and the boundary of IRS zone respectively). time (short rep.rate). Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 = ∗ q emissivity an x y t 4( , , ) o Cell angles gradually increase towards the unirradiated zone on which cells on this zone do not show preferential direction. In other words, cells tend to become parallel at the proximity of IRS. The same propensity was observed on HeLa cell lines. Preferable migration towards plain areas was pronounced on cells at outer edges of peripheral laser induced zones. This attribute allowed us to manipulate the directionality and morphology of the cells. It can be concluded that cell morphology is an effect of cell migration and self-adoption of a cell’s cytoskeleton that implies better understanding of the effects of controlling cell behaviour. Although the morphology of cells away from the IRS zone (control area) showed a tendency toward a polygonal shape. Cytocompatibility to induced residual stress (inter-cellular response). Quantitative information about cells-matter interaction can be inferred by investigation of cell morphology34,35. Therefore, interaction of an individual cell with functionalized stress component was examined. Two distinct phenomena of cyto-breakage and cell protru- sion were observed on cells in the proximity of the IRS zone. Upon formation of filopodia after seeding the cells on the bio-template, they perpetually sense their environs to further formation of lamellipodia and ultimately direct cell movement. Cell retraction by which cyto-breakage occurred was reaction to failed adhesion and pre- mature formation of filopodia facing the IRS. The SEM images of HeLa and Fibroblast (NIH3T3) cells, shown in Fig. 10, indicate that although filopodia have been developed on the both sides of cells, cyro-breakages have taken place on the side of the cell facing to the IRS zone. This is evidence that cell migration through retraction and expansion is prohibited by a functional stress component. On the other hand, during 24 hours of incubation, it is likely that cells migrate towards IRS zone. In this case, cells tend to protrude on the side facing the IRS zone as shown in Fig. 11. Additionally, as statistical survey in Fig. 11E,F evidenced that cell protrusion is more dominant on HeLa cell line compared to Fibroblast (NIH3T3) cell line, in which cyto-breakages was common. Lamellipodia are the actual motor which pulls the cell forward during the process of cell migration. Protruding of lamellipo- dium, indicates no preference of a cell to attach to the IRS zone as SEM images showed on Fig. 11A–D. Therefore, 8 Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 www.nature.com/scientificreports/ Figure 11. = ∗ q emissivity an x y t 4( , , ) o SEM images show cells protrusion on the side facing IRS. (A,B) Fibroblast (NIH3T3) cell line, and (C,D) Hela cell lines. The statistical diagrams (E,F) compares the number of cell protrusions and cyto- breakages on 4, 8, 12 and 26 MHz for Fibroblast (NIH3T3) and HeLa. Error bars show SEM; two independent experiments were repeated with n = 3 in each experiment. Statistical significance is shown with p < 0.05, *p < 0.01. (Arrows are indication the lamellipodium protrusion on the SEM images and dashed line indicate the boundary of the IRS zone). Figure 11. SEM images show cells protrusion on the side facing IRS. (A,B) Fibroblast (NIH3T3) cell line, and (C,D) Hela cell lines. The statistical diagrams (E,F) compares the number of cell protrusions and cyto- breakages on 4, 8, 12 and 26 MHz for Fibroblast (NIH3T3) and HeLa. Error bars show SEM; two independent experiments were repeated with n = 3 in each experiment. Statistical significance is shown with p < 0.05, *p < 0.01. (Arrows are indication the lamellipodium protrusion on the SEM images and dashed line indicate the boundary of the IRS zone). disinterest of lamellipodia to the IRS zone causes cell protrusions. On the other hand, there is a strong belief that the cell crawling in the process of migration is initiated by the development of stable filopodium-substrate adhesion, followed by development of lamellipodium36–38. Hence cell protrusion is dependent on collaborative interaction of filopodia and lamellipodia39. However lamellipodia function as key structure on cell migration, filopodia extensions are essential for guidance and cellular architecture40. Investigation of cell protrusion were made possible by high resolution SEM imaging and tilting technique of substrate up to 45°. Filopodial extensions. Filopodia serve as probing sensors at the frontier of cells that are extended beyond the leading edge of lamellipodia. This attribution leads them to have a significant function in cell migration as well as cell environmental sensing37. The number of filopodial extensions are the evidence of cell preference to adhere and grow41. Quantitative image analysis, revealed that the side of cells facing the peripheral of the IRS zone has relatively no or a reduced number of filopodial extensions compared to the opposite side of cell that faces untreated substrate (control area) as depicted in Fig. 12E. This immature development of filopodial extensions was observed on both cell lines of HeLa and Filapodia as shown on Fig. 12. = ∗ q emissivity an x y t 4( , , ) o In addition, the formation of new filopodia has proven to be strongly dependant on pre-existing filopodia. Taking into account the attributes of filopodial, the two main functions of environmental sensing and driving force generated for cell migration together with filopodial-originated actin bundles, best describe the role of these needle-like structures in direct- ing cell migration42,43. Filopodia are the starting point for essential adhesion and movement. Therefore, the final cellular position, actin bundles, and formation of new filopodia are affected by interaction of these structures with IRS. Stress fibres and cell migration. Stress fibers play an important role in providing numerous functions such as cellular contractility, cell adhesion, and migration44. Upon attachment of a cell to the bio-template, the pulling force required for traction is provided by adhesion. Thus, cell transition processes are made possible by contrac- tion of the cell body to the new site. Vacillant cells such as Fibroblast (NIH3T3) showed oriented ventral stress fibers perpendicular to the vector of migration. Moreover, a higher concentration of actin bundles formed on the locomotion face of cells are observed. This is caused by stronger adhesion at the migration side compared the rear. Cells in the proximity of IRS have shown, actin bundle concentration on the opposite side of cell breakage, while stress fibers are stretched perpendicular to the direction of migration. (Fig. 13). Taken together, here we reported a novel method of remote stimulation of cell migration by laser-induced residual stress on silicon-based bio-template. It provided a precise manipulation of cell-bio-template interaction, unlike topographical and/or chemical surface modification, cell response to IRS relies on environmental probing of cells. Conclusion h h In this research work we created an IRS component on a bio-template, which regulates filopodia formation, adhe- sion, and collective cell migration. Fluorescent and SEM imaging revealed that at the periphery of IRS cell mor- phology and direction are strongly dependent on the level of stress and the formation and expansion of fiopodia Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 9 www.nature.com/scientificreports/ Figure 12. (B,D) SEM images show the Filopodial extension on the side of the cells opposite to the IRS zone. (A,C) Illustrate the Fibroblast (NIH3T3) cells and HeLa cells respectively on the control samples. Bar graph on the left side (E) comparing the number of filopodia of HeLa and Fibroblat seeded on the IRS bio-template and respective control samples. (Hundred cells were counted for each cell group of samples; error bars denote SD. for each group). Figure 12. (B,D) SEM images show the Filopodial extension on the side of the cells opposite to the IRS zone. (A,C) Illustrate the Fibroblast (NIH3T3) cells and HeLa cells respectively on the control samples. Bar graph on the left side (E) comparing the number of filopodia of HeLa and Fibroblat seeded on the IRS bio-template and respective control samples. (Hundred cells were counted for each cell group of samples; error bars denote SD. for each group). Figure 13. High magnification Fluorescent images (single cell analysis) of Fibroblast (NIH3T3) and HeLa cell lines. (A,F) Show single Fibroblast (NIH3T3) and HeLa cell interacting with IRS, arrows indicate both sides of a cell. (B,D) Are example of Fibroblast (NIH3T3) cells seeded on the laser irradiated bio-template at 26 and 4 MHz, compared to (C,E) where extension of filopodia and lamellipodia are directional. (G,I) Show the HeLa cells seeded on the laser irradiated bio-template at 4 and 26 MHz respectively (arrows indicate the cell breakage on side of cell facing the IRS). (H,J) Are HeLa cells on the control area. (Optical images showing actin cytoskeleton (green) and nuclei (blue) of cells after 24 hours of culture on IRS bio-template). Figure 13. High magnification Fluorescent images (single cell analysis) of Fibroblast (NIH3T3) and HeLa cell lines. (A,F) Show single Fibroblast (NIH3T3) and HeLa cell interacting with IRS, arrows indicate both sides of a cell. (B,D) Are example of Fibroblast (NIH3T3) cells seeded on the laser irradiated bio-template at 26 and 4 MHz, compared to (C,E) where extension of filopodia and lamellipodia are directional. Materials and Methods Materials and laser irradia Materials and laser irradiation. High-quality polished single crystal N-type silicon wafers with crystal- lographic orientation of (100) and 625 μm ±25 μm thickness were supplied by University Wafer. After dicing the wafers into 2 cm2 square-shape samples, they were ultrasonically cleaned in a 50 degree Celsius acetone bath for 15 minutes, followed by rinsing and drying. In order to induce residual stress, these substrates were exposed to a diode-pumped, Yb-doped femtosecond laser beam (Clark-MXR, Inc.; IMPULSE Series ultrashort pulse laser). This laser system is capable of producing central wavelengths of 1040 nm and a laser pulse width varying from 214 fs to 1428 fs. As well as, the laser pulse repetition rate is tuneable between 4 MHz (Low rep.rate) and 26 MHz (High rep.rate). The maximum operating average power is 16 W. Utilizing an ultrashort pulsed laser (USPL) (femtosecond range) is advantageous since the absorption of multiphoton excitation energy is confined to the localized laser beam spot within the focal point. This results in minimal collateral damage caused by laser-matter interaction. To precisely functionalize the stress component on the IRS, a pulse repetition rate of 4, 8, 12 and 26 MHz, and scanning velocity of 10 mm s−1 were implemented. Laser pulse width and average power were set up and remained constant at 214 fs and 16 W respectively. The laser spot-size at the focal point was adjusted to 10 μm diameter after manipulation by a pair of galvanometers through optics lenses. Prior to seeding the cells, the sample was irradiated at the focal point by a computerized Galvano scanner to precisely implement an IRS biomimetic architecture. Cell culture and seeding. In order to remove all debris and impurities, samples were ultrasonically cleaned, following the procedure as previously mentioned. Laser treated bio-templates were rinsed with Phosphate Buffered Saline (PBS) (1X strength compose of: 138 mM NaCl, 2.7 mM KCl, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4) before seeding the cells then dried and sterilized under UV light for 20 minutes. Fibroblasts cell lines (NIH3T3) and human cervical cancer cell lines (HeLa, ATCC, American Type Culture Collection, and ATCC No. CCL-2) were employed in cell experiments to ascertain comparative cell controllability of mammalian and cancer cell lines. Fibroblast (NIH3T3) cells were grown in a DMEM medium containing 10% heat activated fetal bovine serum with 1% penicillin-streptomycin antibiotics (Pen-strep). Materials and Methods Materials and laser irradia HeLa cells were grown in a DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% Pen-strep. Subsequently, the cells were separately cultured on the substrates that are placed in petri dishes with seeding density of 750,000 cells/cm2 of substrate surface area. The petri dishes were placed in an incubator for 24 hours at 37 °C temperature. Statistics. All cell assay were carried out at least three times and the data points are averages unless other- wise mentioned. The error bars indicate as mean ± standard deviations (SD). Statistical significance were com- pleted using one-way analysis of variance (ANOVA), with p < 0.05, **p < 0.01 suggesting significant difference. Additionally, the control sample were compared for each significant level shown in (Figs 8 and 11). Cell imaging and morphology. The surface morphology of the samples, seeded with cells, were observed using a (Hitachi, SU-1500) scanning electron microscope (SEM). For this regard, after the prescribed time period, spent medium were aspirated. This was followed by the fixing of samples in 2% glutaraldehyde in 0.1 M pH 7.3 sodium cacodylate buffer for an hour. Next, the samples were immersed in 0.1 M sodium cacodylate buffer with 0.2 M pH 7.3 sucrose for 20 minutes. Preceded by dehydration as an increasing concentrations of alcohol for 20 minutes. The samples were then dried to a critical point. At the conclusion of the experiment, cells were ready to be directly observed using Scanning Electron Microscopy (SEM). Images were acquired at 100× to 10,000× magnification with an accelerating voltage of 10 kV, and a probe current of 50 to 60 μA. Fluorescence microscopy. To conduct fluorescent visualization the samples are first fixed in methanol-free paraformaldehyde followed by incubation in skim milk to prevent non-specific binding. Actin and cytoskele- ton are stained by incubation of the samples with phalloidin-Alexa Fluor 488 (Life Technologies) followed by DAPI (4′,6′-diamidino-2-phenylindole, Life Technologies) to stain the nucleus. The samples are studied using an epi-fluorescent Nikon E-400 microscope (Nikon, Canada). Electron backscattered diffraction (EBSD). A fundamental difference between EBSD and XRD is that X-ray penetrates through the entire thickness of the sample with a 10 mm beam size, whereas EBSD receives backscattered electrons from the top 10–50 nm due to limited backscattering depth of the electrons [22]. The local change in the crystal orientation due to laser strike associated with rapid cooling and shock waves can be deter- mined using EBSD. Conclusion h h (G,I) Show the HeLa cells seeded on the laser irradiated bio-template at 4 and 26 MHz respectively (arrows indicate the cell breakage on side of cell facing the IRS). (H,J) Are HeLa cells on the control area. (Optical images showing actin cytoskeleton (green) and nuclei (blue) of cells after 24 hours of culture on IRS bio-template). Figure 13. High magnification Fluorescent images (single cell analysis) of Fibroblast (NIH3T3) and HeLa cell lines. (A,F) Show single Fibroblast (NIH3T3) and HeLa cell interacting with IRS, arrows indicate both sides of a cell. (B,D) Are example of Fibroblast (NIH3T3) cells seeded on the laser irradiated bio-template at 26 and 4 MHz, compared to (C,E) where extension of filopodia and lamellipodia are directional. (G,I) Show the HeLa cells seeded on the laser irradiated bio-template at 4 and 26 MHz respectively (arrows indicate the cell breakage on side of cell facing the IRS). (H,J) Are HeLa cells on the control area. (Optical images showing actin cytoskeleton (green) and nuclei (blue) of cells after 24 hours of culture on IRS bio-template). Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 10 www.nature.com/scientificreports/ are directly altered by IRS component. It was found that varying laser parameters have a significant influence on the magnitude of stress and consequently the distance of cell migration can be controlled by carefully tuning the laser parameters. Cell culture study indicates that in order for a cell to respond, the induced residual stress must reaches a minimal level, which is defined as threshold of cytocompatibility. The findings of this study opens a new front on cell regulation and cell patterning using silicon based bio-templates. Materials and Methods Materials and laser irradia Crystal orientation was measured with EBSD systems, Oxford Instruments INCA interfaced to a Hitachi SU3500 SEM with a LaB6 electron gun. These systems can automatically obtain a crystal orientation map by scanning a rectangular area on the surface of the specimen, which is tilted 70° from the horizontal. X-ray diffraction (XRD). The elemental composition and crystal structure of the substrate was charac- terized using Rigaku MiniFlex 600 Benchtop X-ray diffraction (XRD) under Cu-Kα radiation (λ = 0.154 nm). Additionally, XRD analysis of the IRS revealed a pronounced (211) peak that emerged when the laser rep.rate was increased. These spectra are in well agreement with EBSD results in Fig. 5. Secondary phases were not detected in the XRD spectra of IRS samples, which shows there is no compositional deviation from an untreated silicon wafer. To further corroborate with the XRD analysis, Raman and EDX spectra were acquired. Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 11 www.nature.com/scientificreports/ Energy-dispersive X-ray spectroscopy (EDX). Further surface analytical techniques were employed to identify possible compositional changes on the IRS zone and compression within the untreated silicon surface. The advantage of employing SEM/EDX lies in shallow penetration depth of the interactive electron beam in con- trast to XRD. Thus, surface elemental analysis can be achieved. Micro-Raman spectroscopy (μRS). Micro-Raman spectroscopy is capable of characterizing types of stresses (Tensile/Compressive) within a small measurement spot size, below 5 μm in diameter, and a submi- cron spatial resolution30. Although Micro-Raman spectroscopy was mostly being used in chemical composition studies as a complementary technique to other methods, deducing crystallinity information is advantageous to this technique as a non-destructive method to measure residual stress29. Raman spectra were obtained using a Bruker SENTERRA, Dispersive Raman Microscope having a central wavelength of 532 nm as an excitation source. The Raman shift measurements were taken perpendicularly along the IRS of several samples at 10 μm intervals. 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Acknowledgementsh g The authors would like to thank Malvika Iyer for her assistance with simulation and Daryna Skybina for her assistance with statistical analyses. This research was funded by Natural Science and Engineering Research Council of Canada (NSERC Discovery Grant 126042, 119087). g The authors would like to thank Malvika Iyer for her assistance with simulation and Daryna Skybina for her assistance with statistical analyses. This research was funded by Natural Science and Engineering Research Council of Canada (NSERC Discovery Grant 126042, 119087). Author Contributions M.K., K.V. and B.T. worked together in designing the project. M.K. performed the experiments and wrote the manuscript. K.V. and B.T. assisted in results, discussion and editing the manuscript. M.K., K.V. and B.T. worked together in designing the project. M.K. performed the experiments and wrote manuscript. K.V. and B.T. assisted in results, discussion and editing the manuscript. Additional Informationihi Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 Additional Informationih Competing financial interests: The authors declare no competing financial interests. Competing financial interests: Competing financial interests: The authors declare no competing financial interests. How to cite this article: Keshavarz, M. et al. Functionalized Stress Component onto Bio-template as a Pathway f Cytocompatibility. Sci. Rep. 6, 35425; doi: 10.1038/srep35425 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports \| 6:35425 \| DOI: 10.1038/srep35425 13
https://openalex.org/W1516052975	https://revistaseletronicas.pucrs.br/ojs/index.php/civitas/article/download/3757/4584	Portuguese	null	Novas configurações na produção do conhecimento: a dinâmica das modernas biotecnologias na periferia do sistema mundial de ciência e tecnologia	Civitas - Revista de Ciências Sociais	2,009	cc-by	7,251	* Mestre em Políticas Sociais pela Universidade Estadual do Norte-Fluminense (Uenf) e doutor em Sociologia pela Universidade Federal do Rio Grande do Sul (Ufrgs), em Porto Alegre, Brasil. <fabriciomneves@yahoo.com.br> A dinâmica das modernas biotecnologias na periferia do sistema mundial de ciência e tecnologia New trends in knowledge production The dynamics of modern biotechnology in the outskirts of the global science and technology Civitas Porto Alegre v. 9 n. 2 p. 307-323 maio-ago. 2009 New trends in knowledge production The dynamics of modern biotechnology in the outskirts of the global science and technology The results allow us to propose two models of scientific production, namely, the mode 2 and triple helix. We compare both models with each other, evaluating its possible explanations for the Brazilian case. Key words: Innovation; Science; Technology; Mode 2; Triple helice New trends in knowledge production The dynamics of modern biotechnology in the outskirts of the global science and technology Fabrício Monteiro Neves* Resumo: Este artigo propõe estudar a forma como a produção do conhecimento científico está organizada atualmente valendo-se de pesquisa de campo realizada em 2004, no município de Campos dos Goytacazes, RJ, com grupos de pesquisa em biotecnologia da cana-de-açúcar localizados em duas universidades públicas, a saber, a Universidade Federal Rural do Rio de Janeiro (Ufrj) e a Universidade Estadual do Norte Fluminense (Uenf). Nós também usamos estudo exploratório realizado em 2006 em outros locus científico e tecnológico: o Centro de Biotecnologia da Universidade Federal do Rio Grande do Sul (Ufrgs) e em laboratório e companhia biotecnológica, na Pontifícia Universidade Católica do Rio Grande do Sul (Pucrs), respectivamente, Laboratório de Genética e Biologia Molecular e 4G pesquisa e desenvolvimento. Os resultados são relacionados às proposições de dois modelos de produção científica, quais sejam, o modo 2 (mode 2) e a hélice tripla (triple helix), comparando-os entre si a partir da análise dos resultados, buscando avaliar sua possibilidades explicativas para o caso brasileiro. Palavras-chave: Inovação; Ciência; Tecnologia; Modo 2; Hélice tripla Abstract: This article aims at studying the way scientific knowledge production is currently organized. For this purpose, in 2004, we conducted a field research at Campos dos Goytacazes city, State of Rio de Janeiro, with research groups in sugar cane biotechnology from two public universities, namely the Federal Rural University of Rio de Janeiro (Ufrj) and the State University of Norte Fluminense (Uenf). In this research, we also used an exploratory study conducted in 2006 at other loci of science and technology: the Biotechnology Center of the Federal University of Rio Grande * Mestre em Políticas Sociais pela Universidade Estadual do Norte-Fluminense (Uenf) e doutor em Sociologia pela Universidade Federal do Rio Grande do Sul (Ufrgs), em Porto Alegre, Brasil. <fabriciomneves@yahoo.com.br> 308 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 do Sul (Ufrgs) and at the Pontifical Catholic University of Rio Grande do Sul (Pucrs), the Laboratory of Genetics and Molecular Biology and 4G research and development laboratory. The results allow us to propose two models of scientific production, namely, the mode 2 and triple helix. We compare both models with each other, evaluating its possible explanations for the Brazilian case. do Sul (Ufrgs) and at the Pontifical Catholic University of Rio Grande do Sul (Pucrs), the Laboratory of Genetics and Molecular Biology and 4G research and development laboratory. Sociedade moderna e formas de organização da ciência Atualmente há razões suficientes para se crer na importância alcançada pelo âmbito tecnocientífico da sociedade. Expressões como “Sociedade do risco” (Beck, 2002; Douglas e Wildavsky, 1983), “sociedade do conhecimento” (Maciel, 2001, Knorr-Cetina, 1999) e “capitalismo do conhecimento” (May, 2005) têm centralizado a descrição da sociedade contemporânea em riscos científicos e tecnológicos e no conhecimento científico, concomitantemente. Destas maneiras de compreensão do fenômeno social sucedem modelos analíticos que buscam dar conta desta centralidade do conhecimento científico e tecnológico para a reprodução da sociedade. Shinn (2002) tem enfatizado o surgimento desses modelos a respeito da centralidade do conhecimento científico na sociedade moderna, relacionando-o principalmente à crise energética da década de 1970 e à desaceleração econômica sequente. Nesta época de crise, governos e empresários propuseram saídas relacionadas a políticas científicas e tecnológicas, engendrando novas expectativas industriais e sociais, incidindo na forma de produção do conhecimento científico. Dois estudos lograram um lugar de destaque na percepção de que uma transformação na produção do conhecimento estava se processando, a saber, a publicação de Michael Gibbons et al. (1994), intitulada A nova produção do conhecimento, e um conjunto de estudos fragmentados de Loet Leydesdorff e Henry Etzkowitz consagrado como “hélice tripla”, publicados esporadicamente a partir de 1995. Atualmente há razões suficientes para se crer na importância alcançada pelo âmbito tecnocientífico da sociedade. Expressões como “Sociedade do risco” (Beck, 2002; Douglas e Wildavsky, 1983), “sociedade do conhecimento” (Maciel, 2001, Knorr-Cetina, 1999) e “capitalismo do conhecimento” (May, 2005) têm centralizado a descrição da sociedade contemporânea em riscos científicos e tecnológicos e no conhecimento científico, concomitantemente. De acordo com os autores de A nova produção do conhecimento, há tempos vem se desenvolvendo um processo de mudança na produção de conhecimento em sociedades contemporâneas. Isto quer dizer que a maneira mais familiar de fazer pesquisa, modo 1, está sendo substituída por um novo modo, mesmo que ainda difuso, sem contornos evidentes, a saber, o modo 2. Àquele correspondiam operações processadas no interior de limites disciplinares, com fronteiras claramente definidas, operando dentro dos limites acadêmicos e das descobertas científicas, ao passo que este se define dentro 309 F. Neves – Novas configurações na produção do conhecimento do contexto de sua aplicação, o que envolve uma dinâmica mais complexa de vertentes disciplinares díspares, refletindo uma estrutura transdisciplinar, de interesses distintos, de alcance amplo, o que leva os autores à constatação de ser a natureza deste conhecimento mais socialmente distribuído. Sociedade moderna e formas de organização da ciência O que quer dizer “aumento da importância do foro híbrido – grupos constituídos através de relações de especialistas e leigos – na configuração do conhecimento (Gibbons et al., 1994, p. 156). Assim, no modo 1 a solução de problemas se limita ao contexto da pesquisa básica e acadêmica e não necessariamente se aplica os resultados, enquanto que no seguinte modo há pretensão de aplicação já na proposta, e isto envolve acomodação de interesses de vários atores, que não são somente aqueles que atuam no mercado. Mesmo as ciências aplicadas no estilo do modo 1, como as enge- nharias, se defrontam com um contexto mais complexo quando são praticadas em modo 2, o que envolve a superação da mera incidência acadêmica, tendo que responder agora à demandas de outras ordens. Decorrem deste cenário ampliações de várias ordens: de lugares potenciais onde se produz conhecimento (universidades, institutos, centros de pesquisa, agências governamentais, incubadoras, força-tarefa, consultorias, entre outros), à articulação e diferenciação simultânea desses lugares através de várias formas de redes de comunicação, reconfigurando subcampos de pesquisa. Assim, “o modo 2 cria um novo ambiente no qual o conhecimento flui mais facilmente através de fronteiras disciplinares, recursos humanos mais móveis e organização de pesquisa mais flexível e aberta” (Gibbons et al., 1994, p. 20). O consenso em modo 2 é condicionado pelo próprio contexto da aplicação, ou seja, ele se dá conforme a interação de diferentes habilidades e especialidades, em uma estrutura transdisciplinar. Este consenso não diz respeito somente a métodos e teorias, mas envolve a perspectiva de não- especialistas, apresentando acomodação de interesses quanto à agenda de pesquisa, à solução de problemas, à qualidade da pesquisa. O modo 2 então, ao incorporar uma série de perspectivas advindas de esferas diferentes e de distintas disciplinas, recai em uma estrutura produtora de conhecimento mais responsável. Prestam-se contas não mais a restritas comunidades de pesquisadores, esta nova produção do conhecimento envolve um amplo espectro de interesses, de firmas a laboratórios, de associação de consumidores a governos, das empresas às agências reguladoras, e neste sentido produz avaliações mais estritas e reflexivas de “boa pesquisa”. Como resultado o controle de qualidade não é avaliado através do modelo tradicional de “revisão por pares”, este passa a se resumir a uma perspectiva dentro de um amplo 310 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. Sociedade moderna e formas de organização da ciência 2009 espectro de atores envolvidos na avaliação, cada qual com seus critérios de julgamento (Hemlin e Rasmussem, 2006; Fujigaki e Leydesdorff, 2000). Isto envolve um jogo com o futuro, já que no contexto de aplicação as consequências futuras em cada âmbito são levadas em conta, “‘a solução, se encontrada, será competitiva no mercado?’, ‘qual será o custo efetivo?’, ‘será socialmente aceitável?’” (Gibbons et al., 1994, p. 8). Ainda que o cenário de produção do conhecimento seja o mesmo, o modelo de hélice tripla, de Leydesdorff e Etzkowitz, desenvolve uma interpretação diferente da do modo 2. O modelo de hélice tripla aponta continuidades no papel que instituições participantes na produção de conhecimento cristalizaram historicamente. O foco da análise não é tanto o papel que elas desempenham, mas a relação entre basicamente três dessas instituições, a saber: a universidade, a indústria e o governo. Pode-se pensar em três modelos a partir da relação institucional básica universidade-indústria-governo na formação de sistemas de inovação (Etzkowitz e Leydesdorff, 2000). Uma hélice tripla 1, definida pelo abarcamento e direcionamento da relação com academia e indústria pelo estado, verificada na União Soviética, no Leste Europeu e em alguns países da América Latina. Seria o caso brasileiro sob governo militar (Morel, 1979). Uma hélice tripla 2, de matriz político-institucional de laissez-faire, tipicamente americana, em que as três esferas estão separadas em funções definidas. E uma hélice tripla 3, com sobreposição entre esferas, cada uma assumindo o papel da outra, criando organizações híbridas nas interfaces, através de redes de comunicação. O que importa ressaltar é que a relevância de cada esfera é salvaguardada no processo, conservando papéis tradicionais, ao mesmo tempo em que, na dinâmica com os outros âmbitos, cria novas formas de produção de conhecimento, formas emergentes como incubadoras e laboratórios industriais, cuja dinâmica não pode ser reduzida à dinâmica de nenhuma das esferas envolvidas. Etzkowitz (2003) resume as premissas da hélice tripla em proposições sintéticas que apresentam o debate até ali. Afirma que os arranjos e redes entre as esferas institucionais da hélice tripla são as fontes da inovação atualmente. A inovação é um fenômeno mais amplo que envolve arranjos emergentes sobre as esferas isoladas. Mas, retroativamente, cada esfera muda seu processo operacional, mantendo a função específica, na medida em que aumenta a interação entre pesquisa acadêmica, avanço industrial e políticas governamentais de desenvolvimento econômico. Sociedade moderna e formas de organização da ciência As inovações organizacionais emergem desse processamento em rede, tornando-se tão importantes quanto o patrimônio físico envolvido na pesquisa. Surgem incubadoras e parques tecnológicos com uma dinâmica inovadora capaz de promover intercâmbios 311 F. Neves – Novas configurações na produção do conhecimento de experiências, colaboração, e, até mesmo, formação de novas disciplinas (Etzkowitz, 2003). Neste sentido, a inovação não segue um modelo linear tradicional: através dessas redes comunicativas o processo de produção do conhecimento é permanentemente assistido pelos participantes, o que muda inclusive o controle de qualidade, os recursos implicados e a avaliação de resultados. Faz parte desta dinâmica, consequentemente, a “capitalização do conhecimento” concomitantemente a “cognitização do capital” (Etzkowitz, 2003 p. 297). Isto aponta para privatização da pesquisa, como sugerem Mirowski e Van Horn (2005) para o caso da indústria farmacêutica pós-anos 1980. Novos mecanismos emergem para a produção de conhecimento, como as incubadoras de riscos empresariais, e outros já existentes ampliam seus mecanismos, como os sistemas de patentes e as regras sobre direito de propriedade (Chesnay, 1996). As formas de capital se complexificam, e elas são intercambiáveis na mesma esfera e entre elas (Bourdieu, 1983). Nesta nova dinâmica estão imbricados capital financeiro, social, intelectual, e novas formas estão surgindo na medida em que emergem novos âmbitos de criação do conhecimento (Etzkowitz, 2003). A globalização intensifica o intercâmbio de informação e as colaborações globais emergem sem barreiras territoriais ou linguísticas, com acordos sobre patentes e propriedade intelectual decorrentes dos processos inovadores produzidos em conjunto. As estratégias de desenvolvimento de países e regiões passam a se basear na promoção de nichos de conhecimento e de desenvolvimento tecnológicos. As universidades passam a funcionar como centros estratégicos na produção de desenvol- vimento regional, atraindo empresas para perto com suporte governamental (Etzkowitz, 2003). Decorre deste processo de produção de conhecimento uma mudança substancial no que diz respeito aos processos de inovação. Há um deslocamento do que se convencionou chamar de “fronteira sem fim” a partir do relatório Bush (Bush, 1999), ou seja, a imagem de uma ciência em que a pesquisa básica era traduzida em uso em longo prazo, para uma “transição sem fim”, em que a pesquisa básica se liga à aplicada através de uma série de processos intermediários, não necessariamente de forma linear (Etzkowitz e Leydesdorff, 2000, p. 110). Sociedade moderna e formas de organização da ciência Isto muda drasticamente a autoimagem da ciência, da autonomia científica aos interesses externos, do direcionamento de recursos através do mecanismo de “revisão por pares”, mas também dos próprios valores internos, que, segundo Hess (1997, p. 58) responderia, desta vez, por essa nova dinâmica da pesquisa: os valores e normas atuais estariam então emergindo nas interfaces de uma hélice tripla. 312 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 1 É o caso do estudo de Sutz (2000), que aborda a relação entre universidade, indústria e estado na América Latina, ressaltando as dificuldades na relação entre indústria e universidade, mas apontando dinâmicas inovadoras. Modelos para a periferia? O que estes modelos teriam a oferecer para as análises da produção científica em países periféricos, científica e tecnologicamente? Eles seriam adequados para tanto? No livro de Gibbons et al. (1994) é feita uma análise do caso brasileiro, onde se apontam os mecanismos que falharam na promoção de uma produção de conhecimento em modo 2, sendo típico a produção em modo 1, com a comunidade científica reduzindo sua prática a sua própria dinâmica, resistindo às demandas externas (Gibbons et al., 1994, p. 134). Neste estudo, o Brasil é comparado a países asiáticos que, segundo os autores, tiveram sucesso na promoção de uma economia de livre mercado e foram capazes de promover parcerias público-privadas para a promoção de avanços tecnocientíficos. No Brasil, ciência e tecnologia foram conduzidas somente por iniciativas estatais e no final da década de 1970, com o endividamento crescente, o estado não suportou os mesmos níveis de financiamento. Mediante a defesa de seu mercado, o Brasil postergou a busca por investimentos estrangeiros, à medida que os países asiáticos desenvolveram seu próprio mercado concomitantemente a um aumento de sua participação na competição global. Em relação à hélice tripla, a conferência ocorrida no Rio de Janeiro, em 2000, poderia ser um indicativo de suas possibilidades analíticas em países em desenvolvimento. Mas um outro indicativo são os estudos empíricos utilizando este modelo que emergiram nos últimos tempos.1 Porém, existem ressalvas quanto ao uso destes modelos para outras realidades, como a brasileira. Trigueiro (2001) entende o caso brasileiro a partir da articulação de várias “hélices”: organizações não-governamentais, estado, comunidades científicas nacionais, empresários e universidades, entre outros. Estas hélices se rearranjam na elaboração de um complexo modelo de relações. Algumas iniciativas de estado tiveram lugar na década de 1990, buscando aprimorar o sistema de gestão de ciência e tecnologia, impulsionado pela nova dinâmica na produção, baseada na capacidade de inovação científica e tecnológica, e em um cenário de hiper-competitividade industrial. Os esforços foram feitos para incorporar setores industriais nos arranjos de produção de conhecimento. No plano de políticas de inovação industrial, os anos 1990 engendraram uma diferenciação processual no estado brasileiro, buscando maximizar o potencial da indústria nacional através de políticas científicas e tecnológicas e política industrial de inovação. A última se concretizará 313 F. 2 Foram muitos os programas governamentais para a área. Destacam-se o Programa Integrado de Genética (1975), o Programa Integrado de Engenharia Genética (1978) e o Programa Nacional de Biotecnologia (1981). Foram incentivadas redes de colaboração científica e tecnológica entre empresas e universidades através de parques tecnológicos. Modelos para a periferia? Neves – Novas configurações na produção do conhecimento através da criação de programas específicos voltados para a área, como, por exemplo, o PCI – Programa de Competitividade Industrial; o PBQP – Programa Brasileiro de Qualidade e Produtividade; o Pacti – Programa de Apoio à Capacitação Tecnológica da Indústria; e o Pace – Programa de Apoio ao Comércio Exterior. O modelo anterior de ciência e tecnologia baseado no gasto militar e na pesquisa básica foi sendo substituído, devido em parte ao fim da guerra fria que reestruturou as ações militares alterando a ideologia da “defesa nacional”, por um novo modelo, mais voltado para a competitividade do sistema industrial, isto impelido pelas condições internacionais a que estavam submetidas as empresas locais. A falta de amplitude e de modernidade da matriz industrial teria que ser superada pela criação de um núcleo de setores motores de inovação tecnológica: informática, biotecnologia, novos materiais, químicas fina etc. Em relação à política nacional de biotecnologia, o tema está na agenda política brasileira há pelo menos três décadas. Embora a base técnico-científica necessária ao acompanhamento das pesquisas internacionais de ponta na área fosse limitada, foram vislumbradas potencialidades no que tangia a agricultura e saúde, o que poderia inclusive atrair a indústria nacional para o desenvolvimento de uma base endógena de pesquisa e desenvolvimento (Azevedo et al., 2002). Havia os velhos problemas relacionados à baixa iniciativa empresarial e à falta de quadros científicos especializados em áreas biológicas necessárias à implementação da pesquisa genética. Neste contexto, as pesquisas adquiriram organização própria, refletindo o arranjo e o desenvolvimento das esferas. O incentivo governamental naquele momento não foi suficiente para que a biotecnologia deixasse de ter fins estritamente acadêmicos.2 Atualmente, têm aparecido iniciativas que apontam para a emergência de novos arranjos de produção de conhecimento, desta vez com uma participação mais destacável, embora ainda tímida, do empresariado nacional (Trigueiro, 2001). Como se mostrará à frente, os arranjos e a complexidade do sistema de ciência e tecnologia brasileiro não permite que se reduza sua dinâmica a um modelo de desenvolvimento específico. Seu operar combina setores de ponta na produção de conhecimento, com características globalizadas no que tange a suas publicações, patentes e colaborações; arranjando-se nas interfaces entre universidade, estado e indústria; operando suas pesquisas em uma Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 314 matriz transdisciplinar; e inovando processos e produtos no próprio contexto de aplicação. Modelos para a periferia? Ao mesmo tempo em que se opera reproduzindo uma ciência tradicional, disciplinar, voltada para a reprodução da comunicação científica unicamente, com poucas possibilidades de redes internacionais e pouca capacidade inovadora. A complexidade de formas que emergem no sistema científico e tecnológico brasileiro desafia as possibilidades analíticas dos modelos apresentados. Uma miríade emergente de formas Os estudos a seguir foram feitos em um espaço de dois anos entre eles, utilizando entrevista semi-estruturada e pesquisa bibliográfica, para coleta de dados, e análise de conteúdo. Os grupos localizados no estado do Rio de Janeiro foram entrevistados em fins de 2003 e início de 2004, enquanto os grupos e a empresa localizados no estado do Rio Grande do Sul foram abordados em 2006. A despeito das diferenças, entende-se que ambos os grupos estavam sujeitos às mesmas contingências organizacionais, às mesmas linhas de financiamentos, aos mesmos programas, podendo oscilar o montante de recursos. Deste modo, pode-se dizer que tais grupos experimentaram um contexto institucional parecido, ainda que as diferenças regionais tenham consequências consideráveis, mas que não serão discutidas aqui. Os grupos de biotecnologia pesquisados no Rio de Janeiro estão sediados em Campos dos Goytacazes, região norte-fluminense, em duas universidades públicas, Universidade Federal Rural do Rio de Janeiro (Ufrrj) através de seu campus avançado (Dr. Leonel Miranda) e seu Programa de Melhoramento Genético da Cana-de-Açúcar (formado em 1973); e a Universidade Estadual do Norte-Fluminense (Uenf) e seus grupos, basicamente surgidos após 2000, a saber, o núcleo de sequenciamento de DNA vegetal e o grupo de bactérias endofídicas. No Rio Grande do Sul foi feita pesquisa exploratória em um instituto público de pesquisa, localizado na Universidade Federal do Rio Grande do Sul (Ufrgs), o Centro de Biotecnologia do Rio Grande do Sul (criado em 1981); em um laboratório de biotecnologia residindo em um parque tecnológico gerenciado pela Pontifícia Universidade Católica do Rio Grande do Sul (Pucrs), o Laboratório de Genética e Biologia Molecular; e, finalmente, em uma empresa incubada residente no mesmo parque, a 4G Pesquisa & Desenvolvimento Ltda. Tanto laboratório quanto empresa foram criados no início desta década. 315 F. Neves – Novas configurações na produção do conhecimento Entre os grupos podem-se diferenciar formas distintas de produzir conhecimento, e isto fica mais claro quando se pergunta pela finalidade da pesquisa. Neste sentido, perguntar “para quem?” condiciona o “como?”. Com isto se quer dizer que a supracitada relação entre contexto e práticas de pesquisa apresenta uma correlação positiva. Contexto aqui é o lado da demanda, os interesses, as “perturbações externas”, e isso não necessariamente está fora da pesquisa científica; no sistema científico há uma demanda por novos conhecimentos, assim como no sistema econômico, por meio das empresas. A configuração da pesquisa vai basicamente responder por estes interesses construídos em sistemas distintos. 3 O Planalsucar foi o Programa Nacional de Melhoramento Genético da Cana-de-açúcar criado junto ao IAA. O plano criou quatro estações experimentais, em Carpina (PE), Rio Largo (AL), Campos dos Goytacazes (RJ) e Araras (SP), para o desenvolvimento de projetos tecnológicos para o setor sulcroalcooleiro. 4 A sigla significa Ridesa Brasil 80, e diz respeito à variedade de cana liberada pela Ridesa, Rede Interuniversitária de Desenvolvimento do Setor Sucroalcooleiro, criada concomitantemente ao Planalsucar e ao IAA. Uma miríade emergente de formas Portanto, dois lados: expectativas acadêmicas e empresariais. Porém, há uma forma emergente que é a articulação de ambas as expectativas e interesses. O Programa de Melhoramento Genético da Cana-de-Açúcar (Grupo 1-RJ) pratica o melhoramento clássico em suas pesquisas, ou seja, seleciona espécimes mais adaptados às condições climáticas, pluviométricas e geográficas de Campos dos Goytacazes. Seus resultados dizem respeito basicamente àquela região, afinal, ao mudar as condições ambientais o grau de adaptação da planta decresce. Portanto, a demanda é majoritariamente local, de usineiros locais, e a pesquisa tem como único objetivo o suprimento desta demanda, que financia a totalidade da pesquisa. Em especial este grupo teve sua estrutura organizacional alterada com o tempo em decorrências de mudanças de governo e ciclos econômicos ligados à produção de álcool e açúcar. Até fins da década de 1980, o Instituto do Açúcar e do Álcool (IAA) tinha um Programa Nacional de Melhoramento da Cana-de-açúcar que financiava o grupo desde 1973. Com a extinção do IAA em 1990, o grupo foi incorporado pelo Campus avançado na Ufrrj, tendo pesquisas interrompidas, quadro de pesquisadores reduzido por aposentadorias e redução orçamentária. De acordo com um dos entrevistados: A pesquisa principal do grupo sempre foi melhoramento genético da cana, melhoramento sempre visando a produtividade, o melhor rendimento agrícola e industrial. (...) Então, quem faz o objetivo da pesquisa é o pessoal que paga por ela, as empresas conveniadas. (...) a tendência que a gente começa a perceber está nas pessoas procurarem variedades mais resistentes à seca, à salinidade e às próprias doenças. Essas variedades são bem produtivas (...) (Entrevista Grupo 1-RJ/1). Mais à frente, se refere à perda de complexidade na pesquisa ligada à perda de especialistas, técnicas e linhas de pesquisa. Houve um caminho inverso, da interdisciplinaridade para uma abordagem disciplinar. 316 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 Antigamente, quando se tinha o Planalsucar3 (a gente não foi da era do Planalsucar), o que a gente lê e vê é que se tinha um recurso muito bom, então se pesquisavam todas as áreas da cana, desde a entomologia, fitopatologia, melhoramento. Então você já tinha todas as áreas cobertas de pesquisa. Hoje a área da cana coberta por pesquisa somente é o melhoramento, por pouco a fitopatologia (Entrevista Grupo 1-RJ/1). 5 O projeto Genoma-cana é o maior projeto de análise de genes expressos em plantas já realizado por uma instituição pública em todo o mundo. Envolve 200 pesquisadores de 60 laboratórios localizados em 8 estados brasileiros: São Paulo, Pernambuco, Bahia, Rio de Janeiro, Paraná, Rio Grande do Norte, Minas Gerais e Alagoas. Estes laboratórios, em conjunto, identificaram 53 mil genes e agora desenvolvem trabalhos para identificar a função de cada um deles. Uma miríade emergente de formas Antigamente, quando se tinha o Planalsucar3 (a gente não foi da era do Planalsucar), o que a gente lê e vê é que se tinha um recurso muito bom, então se pesquisavam todas as áreas da cana, desde a entomologia, fitopatologia, melhoramento. Então você já tinha todas as áreas cobertas de pesquisa. Hoje a área da cana coberta por pesquisa somente é o melhoramento, por pouco a fitopatologia (Entrevista Grupo 1-RJ/1). Esta pesquisa não tem nenhum resultado convertido em publicações científicas, seu impacto é local e relativo à produtividade dos empresários que a financiam. Não há financiamento estatal que não seja o pagamento de salários de pesquisadores e técnicos e a manutenção do centro de pesquisa, bem precária no momento da investigação. A pesquisa é totalmente desenvolvida no contexto da aplicação, e depende fundamentalmente deste. Não tem possibilidades de grandes inovações e, assim, o patenteamento de espécies melhoradas, se houvesse esta possibilidade, não seria interessante, já que o interesse é puramente local. O controle social produzido por este tipo de contexto de aplicação é intrínseco à produtividade da espécie liberada, já que os avaliadores são fundamentalmente os produtores de cana-de-açúcar. Mesmo conclusões sobre desempenho na produtividade de determinadas espécies dependerão do parecer dos produtores, e muitas vezes a avaliação é contraditória: O agricultor está interessado em plantas que acumulam sacarose mais rápido, que sejam resistentes à pragas e doenças. Então eu não posso dizer que a RB 804 é a melhor, o que adianta dizer isto para o agricultor se para o agricultor a melhor é a 92, que produz mais açúcar? O agricultor ganha pela quantidade de sacarose que a planta produz. Então eu tenho que adequar as minhas pesquisas às demandas do agricultor (Entrevista Grupo 1-RJ/2). No caso dos grupos da Uenf ocorre fenômeno oposto, e este se enquadra mais fielmente às configurações tradicionais de pesquisa no Brasil, ou seja, sua incidência é puramente no sistema científico, não tendo implicações tecnológicas, pelo menos a curto prazo. Tanto o núcleo de sequenciamento de DNA vegetal (Grupo 2-RJ) quanto o grupo de bactérias endofídicas (Grupo 3-RJ), realizam pesquisas com técnicas mais modernas que aquelas do grupo 317 F. Neves – Novas configurações na produção do conhecimento acima, porém, até o momento da pesquisa (2004) não haviam tido nenhum resultado aplicado. Uma miríade emergente de formas Há interações em rede com outros grupos de pesquisa, como entre o Grupo 2-RJ e a Universidade Federal do Rio de Janeiro, que envolvem orientações e uso compartilhado de instrumentos, e também com a rede de laboratórios do Genoma-Cana5 financiados pela Fundação de Apoio à Pesquisa do Estado de São Paulo (Fapesp). Além da importante fase de conhecimento da estrutura genômica da cana-de-açúcar, o grupo busca também conhecer a função de cada gene, fase que poderá revelar novas vias de interesse biotecnológico. Estes grupos são totalmente financiados pelo estado e a única ligação com a indústria é indireta e não envolve transferência de tecnologia. Nossa interação direta com os produtores regionais não é feita em contato com eles e sim através da Universidade Rural, onde este grupo do campus Leonel Miranda tem contato com os produtores da região, e para isso atualmente o que estamos fazendo é analisar as variedades de cana mais resistentes para descobrir quais são os genes que se expressam nestas variedades, mais resistentes à seca e à salinidade, em contraposição àquelas variedades mais sensíveis. E assim descobrir quais são os genes responsáveis pela resistência (Entrevista Grupo 2-RJ/1). Através da identificação da estrutura e função dos genes de espécies avaliadas como mais produtivas para a região, avaliação que passa pelo parecer técnico dos pesquisadores do Grupo 1-RJ, além dos próprios produtores, a pesquisa acadêmica pode apresentar suas possibilidades tecnológicas aventadas pela pesquisa genética. Surge então uma rede que envolve pesquisa básica e aplicada, não necessariamente em uma relação causal. A rede se mantém principalmente pelo compartilhamento de instrumentos de pesquisa, pela complexificação das técnicas utilizadas e pela estratégia de chegar aos principais usuários. Estes modelos de pesquisas apresentam uma dinâmica que pode ser observada segundo um modelo que articule as três esferas principais da hélice tripla. Isto é bem ilustrado na rede formada entre grupos 1, 2 e 3, porém estas esferas estão operando de maneira indireta nos resultados. O grupo 1 não tem financiamento estatal, não faz pesquisa básica, e é formado por um corpo técnico disciplinar, mas tem financiamento privado; já os grupos 2 e 3 318 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 invertem a relação: contam com financiamento estatal, fazem pesquisa básica e transdisciplinar, mas nenhum apoio privado. 6 Esta linha está incluída no projeto nacional “Genolyptus: Rede Brasileira de Pesquisa do Genoma de Eucalyptus”, com financiamento estatal (Fundo Setorial “Verde-Amarelo”, Ministério de Ciência e Tecnologia - MCT) e industrial (Aracruz Celulose S. A., por exemplo). Uma miríade emergente de formas A rede formada une ao mesmo tempo, financiamento privado e estatal, pesquisa básica e aplicada, ciência e tecnologia, cria núcleos transdisciplinares, mas de maneira indireta. Neste sentido, parece haver uma diferenciação funcional relacionada ao papel desempenhado por cada esfera no Brasil. Estes casos indicam a existência de limites operacionais entre participação privada e estatal na pesquisa científica. À pesquisa privada se relaciona aplicação e disciplinaridade, com avaliação ex- terna; à pesquisa pública, investigação básica e transdisciplinaridade, e avalia- ção interna (peer review). A dinâmica em rede extrapola estes limites operacio- nais, criando uma dinâmica que maximiza as possibilidades que cada esfera expõe na pesquisa, podendo criar maiores chances de inovação tecnológica. Nos grupos do Rio Grande do Sul, a dinâmica produtora de conhecimento apresenta novos processos, novas formas e relação universidade-indústria- estado diferenciada. Os centros pesquisados têm, em sua forma de produção de conhecimento e inovação, as três esferas já articuladas, criando um mecanismo não-linear entre pesquisa básica e aplicada, pesquisa e desenvolvimento de novos produtos e processos. Estes centros têm incubadoras de empresas, escritório de transferência de tecnologia, laboratórios produtores de insumos e de diagnósticos moleculares para outros laboratórios e empresas de produção animal. Toda esta estrutura propicia a ligação entre pesquisa, desenvolvimento e inovação e demandas externas, criando redes de produtores-consumidores e produtores-produtores das mais variadas formas. O Centro de Biotecnologia do Rio Grande do Sul, com 25 anos de existência, apresenta 14 grupos de pesquisa no Diretório dos Grupos de Pesquisa do CNPq, com várias linhas prestando serviços para laboratórios acadêmicos, laboratórios clínicos e indústrias, como Laboratório de Imunologia Aplicada à Sanidade Animal (Grupo 1-RS), que oferece anticorpos sobre encomenda. Suas linhas de pesquisa envolvem ainda biologia molecular e biotecnologia de plantas, fixação de nitrogênio e biotecnologia de fungos de importância biotecnológica, entre outras. De forma geral, estas pesquisas são financiadas com recursos públicos em sua maioria, mas algumas linhas, como a “Genômica vegetal”, conta com colaboradores de diversas instituições nacionais e de empresas, com o objetivo de sequenciamento e análise de genes expressos e de genomas vegetais.6 319 F. Neves – Novas configurações na produção do conhecimento Há o projeto do Genoma do eucalipto nacional, que tem a par- ticipação das empresas, em 10 ou 20%. 7 Genotox - Laboratório de Genotoxicidade, Tecfarm (Enzi-Far) e Pirisa (Bio-Pirinset). Uma miríade emergente de formas Então as empresas devem entrar com 1, 2, 3 milhões de reais; é um projeto que envolve muitas universidades e praticamente todas as empresas de papel e celulose; não conheço nenhum projeto completo financiado por uma empresa (Entrevista Grupo 1-RS/1). O centro conta com uma incubadora de empresas (IE-Cbiot) onde, no momento, residem três empresas incubadas7 desenvolvendo atividades em genética toxicológica, monitoramento ambiental, enzimas para a formulação de detergentes biológicos biodegradáveis e inseticida não-tóxico, para o controle de mosquitos e borrachudos em fase larval. Duas dessas empresas são ramificações de empresas que já atuavam no mercado, a saber, Tecfarm Tecnologia Química e Farmacêutica Ltda. (Tecfarm – incubada) e Pirisa Pietro Industrial Ltda. (Pirisa – incubada). Duas empresas, após período de incubação, passaram a ter sede própria e desenvolverem suas atividades a partir da experiência com a universidade, são elas a Ikro Biotec, que produz reagentes para testes de coagulação só disponíveis no mercado externo, e Simbios Biotecnologia, que desenvolve diagnósticos moleculares para laboratórios de análises clínicas e patológicas. O controle destas tecnologias foi possível graças à interação universidade-empresa, e desta interação surgiram algumas patentes divididas entre as esferas. Estes dois lados do centro, quais sejam, aquele ligado às aplicações industriais presente na incubadora e aquele que se localiza no marco acadêmico com pouca ligação industrial, parecem reproduzir o continuum não nivelado da dinâmica da inovação no Brasil. Eles envolvem processos característicos que lhe são próprios. Do lado da incubadora se tem aplicações industriais, produtos e serviços e avaliação externa (empresa e mercado), do lado dos grupos, quase nenhuma interação com empresas, seguindo a lógica da publicação, ficando a avaliação condicionada estritamente aos pares. O caso do Laboratório de Genética e Biologia Molecular (Grupo 2-RS) e da empresa encubada 4G Pesquisa & Desenvolvimento Ltda, localizado no parque tecnológico da Pontifícia Universidade Católica do Rio Grande do Sul (Tecnopuc) difere ainda sobremaneira dos demais. Há uma maior conjunção entre interesses acadêmicos e empresariais, onde as duas lógicas, praticamente apartadas nos casos acima, se encontram, inclusive no que se relaciona à criação de papéis emergentes, como o do cientista-empresário. A organização do parque tecnológico apresenta estruturas criadas com o objetivo de superar a incidência acadêmica. Têm-se laboratórios acadêmicos, 320 Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 incubadoras, agência de gestão tecnológica e escritório de transferência de tecnologia, articulados na produção de inovação. Uma miríade emergente de formas incubadoras, agência de gestão tecnológica e escritório de transferência de tecnologia, articulados na produção de inovação. O Tecnopuc tem um escritório de patentes. O Tecnopuc é organizado da seguinte maneira: tem uma agência que se chama Agência de Gestão Tecnológica. Esta agência tem uma sede e dentro da Agência tem um escritório de patentes, só cuida de patentes e propriedade intelectual. Você tem a facilidade porque este tipo de estrutura cuida de tudo: contratos, burocracia, eles têm os advogados etc... então é um negócio que facilita enormemente o trabalho (Entrevista Grupo 2-RS/1). A relação entre laboratório e empresa é indissociável, com temáticas relacionadas à capacidade da pesquisa do primeiro com as potencialidades técnicas do segundo. Inclusive, laboratório e empresa são vizinhos geograficamente, com cientistas e empresários circulando livremente entre os dois espaços. Isto aqui é uma experiência nova no desenvolvimento de biotecnologia no Brasil. Que é o seguinte: se cria um grupo de pesquisa forte, na área acadêmica, para você gerar conhecimento nas áreas de biotecnologia, e, ao mesmo tempo, se cria uma empresa para todo conhecimento gerado ser convertido em tecnologia. Isto tem dado certo (Entrevista 2-RS/1). O sistema de financiamento é mais complexo, envolvendo recursos da universidade, da empresa, do estado e ainda de empresas não incubadas. Embora ainda a maior parte dos recursos venha de bolsas de instituições federais (CNPq e Capes), alguns empreendimentos já ficam a cargo tanto de recursos da empresa ou da universidade, quanto dos contratos assinados com empresas conveniadas. Os recursos da empresa vêm através da venda de produtos criados e patenteados internamente e recentemente foi assinado um contrato entre a Pucrs, o grupo farmacêutico paulista Farmasa e a empresa 4G. É o primeiro convênio no Brasil entre universidade e empresas para a criação de medicamentos com tecnologia totalmente nacional, desde a elaboração até a produção em grande escala. O acordo prevê a construção de um novo centro de pesquisa e desenvolvimento (P&D) a ser construído no Parque Tecnológico, com financiamento aprovado pela Financiadora de Estudos e Projetos (Finep). Esta forma de organização da pesquisa já envolve um número maior de avaliadores, de distintas perspectivas e um ajuste fino destas perspectivas em torno do quê pesquisar, como pesquisar e para quem pesquisar. Isto envolve a empresa incubada, o laboratório, a universidade, os empresários, os órgãos F. Neves – Novas configurações na produção do conhecimento 321 governamentais, entre outros. Uma miríade emergente de formas Toda esta dinâmica de produção de conhecimento envolvendo universidade, estado e indústria – se se aceita que esta relação produz uma série de processos emergentes inéditos – acaba retornando para cada esfera e esta agora tem que reagir a esta nova forma, re-significando seus processos internos característicos. Por exemplo, em relação às publicações científicas, se observa que a submissão a determinadas revistas segue objetivos que estão além do prestígio acadêmico ou da acumulação do capital científico. O sentido de publicar, nesta forma de produzir conhecimento, é alterado como consequência da dinâmica das três esferas. Um dia eu recebi um e-mail, o cara perguntando que tinha lido em revistas tais, um artigo tal, aí me perguntou: você tem o clone? (...) Eu não fiz o contato, eles é que fizeram o contato. Mais uma vez é a importância da pesquisa científica no desenvolvimento de produtos. Sem a pesquisa científica nós não teríamos publicado estes papers, e o cara não teria visto, se eu partisse direto para produzir e não publicasse, ele não iria me conhecer (Entrevista Grupo 2-RS/1). Neste caso se pode observar que o estreitamento de laços entre universidade, indústria e estado produz um processamento em feedback entre as esferas de produção de ciência e tecnologia e a forma emergente inaugurada. Relaciona avaliação por pares e avaliação externa, uma indissociabilidade entre pesquisa básica e aplicada, ciência e tecnologia, criando um lócus mais propício à inovação. As consequências dessa nova forma de operar a produção do conhecimento científico, no plano das esferas envolvidas, são absorvidas de acordo com a estrutura característica de cada uma, que se altera e é alterada concomitantemente, em um processo denominado “transição sem fim”. Conclusão Destas observações acima decorrem algumas conclusões que dizem respeito aos modelos analíticos importados, mas também às formas de se construir modelos analíticos alternativos. O quadro empírico que acima se apresenta extrapola os limites conceituais do modo 2 e da hélice tripla. Não que estes modelos sejam equivocados, pelo contrário, algumas proposições correspondem a processos observados, mas talvez a configuração institucional da periferia do sistema exija modelos mais complexos de observação. Os casos desaprovam o enquadramento dos dados empíricos dentro de modelos duais, como uma passagem de modo 1 para modo 2, ou ainda um modelo de inovação que pressuponha necessariamente a existência de relação entre universidade, industria e estado, como o modelo de hélice tripla. Pesquisas disciplinares Civitas, Porto Alegre, v. 9, n. 2, p. 307-323, maio-ago. 2009 322 podem ser feitas no contexto da aplicação, serem avaliadas por pares e ao mesmo tempo pelos consumidores, como ilustra o caso do grupo 1-RJ. Pesquisas transdisciplinares podem envolver um sistema de avaliação simples, de tipo acadêmico (peer review) como é o caso dos grupos da Uenf. Porém, com a formação da rede de pesquisa entre estes grupos, isto se mistura, criando um âmbito emergente de inovação. O caso da Ufrgs ilustra uma dificuldade maior de análise. Nele, combinam-se interesses acadêmicos e empresariais, avaliação por pares e atores externos, ciência básica e aplicada, ou seja, depende de quais grupos e âmbitos se observam (centro de pesquisa ou incubadora). Em alguns grupos se articulam interesses empresariais e acadêmicos, como naquele que é vinculado ao mapeamento do genoma do eucalipto, em outros a incidência é acadêmica. O caso da incubadora já ilustraria uma dinâmica de outra ordem, modo 2 ou hélice tripla, que se apresenta também na incubadora da PUC e na empresa 4G. Nestes casos a interação é instantânea e eles se autoalimentam, na escolha de temáticas de pesquisas específicas e no uso de técnicas, por exemplo. Há uma imbricação de lógica empresarial com acadêmica, repercutindo na criação de um papel social escasso no Brasil, a saber, a do cientista-empresário. As distintas formas de produção de conhecimento se reproduzem e se articulam constrangidas por esferas funcionais como o estado e a economia. À autonomia processual da ciência, de métodos e teorias, se contrapõe sua existência contextual, que lhe impõe limites financeiros, legais, culturais, entre outros. Conclusão Assim, discutir produção do conhecimento envolve a discussão da forma como a própria sociedade se organiza em contextos distintos. Atualmente, uma miríade de outros atores é envolvida na produção de conhecimento científico e tecnológico, e é esta complexificação na forma tecnocientífica contemporânea que tem desafiado modelos analíticos. AZEVEDO, Nara et al. Pesquisa científica e inovação tecnológica: a via brasileira da biotecnologia. Dados, Rio de Janeiro, v. 45, n. 1, p. 139-176, 2002. Referências AZEVEDO, Nara et al. Pesquisa científica e inovação tecnológica: a via brasileira da biotecnologia. Dados, Rio de Janeiro, v. 45, n. 1, p. 139-176, 2002. BECK, Ulrich. The terrorist threat: world risk society revisited. Theory, Culture & Society, London, v. 19, n. 4, p. 39-55, 2002. BOURDIEU, Pierre. O campo científico. In: ORTIZ, Renato (Org.). Pierre Bourdieu: Sociologia. São Paulo: Ática, 1983. BUSH, Vanevar. Ciência, la frontera sin fin. Redes, Buenos Aires, v. 14, n. 1, p. 89-137, nov. 1999. CHESNAY, François. A mundialização do capital. São Paulo: Xamã, 1996. 323 F. Neves – Novas configurações na produção do conhecimento DOUGLAS, Mary; WILDAVSKY, Aaron. Risk and culture: an essay on the selection of technological end environmental dangers. Berkeley: University of California Press, 1983. ETZKOWITZ, Henry; LEYDESDORFF, Loet. The dynamics of innovation: from national Systems and ‘mode 2’ to a triple helix of university-industry-government relations. Research Policy, New York, v. 29, p. 109-123, 2000. ETZKOWITZ, Henry. Innovation in innovation: the triple helix of university-industry- government relations. Social Science Information, London, v. 42, n. 3, p. 293-337, 2003. FUJIGAKI, Yuko; LEYDESDORFF, Loet. Quality control and validation boundaries in a triple helix of university-industry-government: ‘mode 2’ and the future of university research. Social Science Information, London, v. 39, n. 4, p. 636-655, 2000. GIBBONS, Michael et al. The new production of knowledge: the dynamics of science and research in contemporary societies. London: Sage, 1994. 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The Triple Helix and New Production of Knowledge: Prepackaged Thinking on Science and Technology. Social Studies of Science, London, v. 4, n. Referências 32, p. 599-614, 2002. SUTZ, Judith. The university-industry-government relations in Latin America. Research Policy, v. 29, p. 279-290, 2000. TRIGUEIRO, Michelangelo G. S. A comunidade científica, o estado e as universidades do atual estágio de desenvolvimento científico tecnológico. Sociologias, Porto Alegre, v. 6, p. 30-51, jul.-dez. 2001. Recebido em: 28 jul. 2008 Aprovado em: 27 ago. 2009 Recebido em: 28 jul. 2008 Aprovado em: 27 ago. 2009
https://openalex.org/W1802943043	https://hal.science/hal-01341431/document	English	null	Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon	Veterinary research	2,015	cc-by	7,453	Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon Maria Aamelfot, Alastair Mcbeath, Debes H. Christiansen, Iveta Matejusova, Knut Falk To cite this version: Maria Aamelfot, Alastair Mcbeath, Debes H. Christiansen, Iveta Matejusova, Knut Falk. Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon. Veterinary Research, 2015, 46 (1), pp.120. 10.1186/s13567-015-0265-1. hal-01341431 Maria Aamelfot, Alastair Mcbeath, Debes H. Christiansen, Iveta Matejusova, Knut Falk Maria Aamelfot, Alastair Mcbeath, Debes H. Christiansen, Iveta Matejusova, Knut Falk To cite this version: Maria Aamelfot, Alastair Mcbeath, Debes H. Christiansen, Iveta Matejusova, Knut Falk. Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon. Veterinary Research, 2015, 46 (1), pp.120. 10.1186/s13567-015-0265-1. hal-01341431 © 2015 Aamelfot et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon aria Aamelfot1†, Alastair McBeath2†, Debes H. Christiansen3, Iveta Matejusova2 and Knut Falk Abstract All viruses infecting fish must cross the surface mucosal barrier to successfully enter a host. Infectious salmon anae- mia virus (ISAV), the causative agent of the economically important infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L., has been shown to use the gills as its entry point. However, other entry ports have not been investigated despite the expression of virus receptors on the surface of epithelial cells in the skin, the gastrointestinal (GI) tract and the conjunctiva. Here we investigate the ISAV mucosal infection in Atlantic salmon after experimental immersion (bath) challenge and in farmed fish collected from a confirmed outbreak of ISA in Norway. We show for the first time evidence of early replication in several mucosal surfaces in addition to the gills, including the pectoral fin, skin and GI tract suggesting several potential entry points for the virus. Initially, the infection is localized and primarily infecting epithelial cells, however at later stages it becomes systemic, infecting the endothelial cells lining the circula- tory system. Viruses of low and high virulence used in the challenge revealed possible variation in virus progression during infection at the mucosal surfaces. for many infectious agents [5] as they are constantly exposed to the water. Previously we showed that a low virulent ISAV (LVI) infected and replicated more rapidly than a highly virulent virus (HVI) in the gills following immersion challenge, suggesting that the mechanism of entry and early infection phase of ISAV may vary between different isolates [6, 7]. Both endothelial cells and gill epi- thelial cells may be infected with the virus [8]. In addition, primary gill epithelial cell cultures support ISAV rep- lication [9]. Whilst ISAV infection of endothelial cells is generalized and associated with high mortality [8], infec- tion with low- or putatively non-virulent variants of ISAV (HPR0) has only been detected in epithelial gill cells by IHC (unpublished results) leading to localized and tran- sient infection not associated with the ISA disease [10]. HAL Id: hal-01341431 https://hal.science/hal-01341431v1 Submitted on 4 Jul 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Aamelfot et al. Vet Res (2015) 46:120 DOI 10.1186/s13567-015-0265-1 Open Access Correspondence: knut.falk@vetinst.no †Maria Aamelfot and Alastair McBeath contributed equally to this work 1 Norwegian Veterinary Institute, Oslo, Norway Full list of author information is available at the end of the article Introduction I f i l Infectious salmon anaemia (ISA), caused by the ISA virus (ISAV) [1] is an important disease of farmed Atlan- tic salmon, Salmo salar L., and listed as notifiable by the OIE. First reported in Norway in 1984, the prevalence peaked in the 90s, resulting in the implementation of strict biosecurity measures to combat the disease [2]. ISA has also caused significant problems in Scotland, the Faroe Islands, Chile, and on the east coast of Canada and USA. Outbreaks occur as single cases, or localised epidemics. Without any intervention, accumulated fish mortality may reach 80% over several months [3]. No treatment exists and the efficacy of available vaccines is disputed. Anaemia, haemorrhaging in the eyes and skin, pale gills, ascites, dark liver and swollen spleen are com- mon signs in diseased fish suggesting circulatory failure as a major mechanism of pathogenesis (reviewed in [4]). To our knowledge, other entry ports for ISAV such as the skin, eye and GI tract have not been investigated. In mammals or poultry, influenza A virus can use the eye, nose and GI tract as port of entry and primary replication site in addition to the respiratory tract [11, 12], and the virus receptor has been detected in conjunctival cells in humans [13]. The skin of mammals is an effective barrier against viral infections. However, in fish, the outermost epidermal cells remain viable and non-keratinized [14], All aquatic viruses, including ISAV, must cross mucosal barriers [e.g. skin, gills or gastrointestinal (GI) tract] to enter the host. The gills are believed to be the entry port Correspondence: knut.falk@vetinst.no †Maria Aamelfot and Alastair McBeath contributed equally to this work 1 Norwegian Veterinary Institute, Oslo, Norway Full list of author information is available at the end of the article Page 2 of 11 Aamelfot et al. Vet Res (2015) 46:120 stained with haematoxylin and eosin, followed by routine histological examination. All organs samples were exam- ined for the presence of ISAV by immunohistochemistry (IHC) as previously described [18] using rabbit antibodies to recombinant ISAV nucleoprotein (NP) [19] and scored using our previously described IHC ISAV scoring system [6]. Immunofluorescent labelling (IFAT) was performed on acetone fixed cryo-sections as previously described [8, 20] using a monoclonal antibody to ISAV haemagglutinin esterase (HE) [20] and Alexa Fluor® 488 conjugated anti- mouse IgG (Life technologies) for detection. Mucosal infection of experimentally challenged fish Skin swabs All skin swabs were positive for virus and replication was demonstrated by a viral load increase in the days immediately following infection (Figure 1A; Table 1). The swabs yielded a mean ELF Ct value of 20.7 (range 18.01– 25.16) suggesting the samples contained cells (most likely epithelial cells covering the scales) in addition to mucus. Despite considerable variation in the quantities of virus Resultsh The number of challenged fish positive for LVI and HVI respectively at each sampling point by different detection methods in all tested organs is shown in Table 1. In addition, five moribund fish were sampled from a marine site in Norway with a confirmed outbreak of ISA. Autopsy included recording of macroscopic pathological changes. Gill, pectoral fin, pelvic fin, skin from the side- line, hind-gut, mid-gut and conjunctiva (around the eye) were collected in formalin and 1 mL RNAlater. Heart, liver, kidney and spleen were collected in formalin for histological examination. Organ sampling during immersion challenge and ISA outbreak Details regarding the virus production, infection chal- lenge and organ sampling procedure are described else- where [6]. Briefly, fish were challenged by immersion with either 104 TCID50 of highly virulent ISAV Glesvær/2/90 (HVI) or low virulent Can/F679/99 (LVI). Four fish were sampled at 6 h post infection (pi) and on days 1–8, 10, 12, 14, 19 and 23 pi from each challenge group, respectively. Cotton swabs were used to sample skin mucus from the base of the left pectoral fin and placed individually in RLT buffer (Qiagen). Pectoral fin (including skin at the fin base), gill, eye, mid-gut and hind gut were collected in 10% phosphate buffered formalin; pectoral fin (no skin), gill and hind-gut were also stored in 1 mL RNAlater (Inv- itrogen). Gills were snap-frozen in isopentane (Sigma) chilled in liquid nitrogen. The experimental trial was approved by the Norwegian Animal Research Authority. In addition, five moribund fish were sampled from a marine site in Norway with a confirmed outbreak of ISA. Autopsy included recording of macroscopic pathological changes. Gill, pectoral fin, pelvic fin, skin from the side- line, hind-gut, mid-gut and conjunctiva (around the eye) were collected in formalin and 1 mL RNAlater. Heart, liver, kidney and spleen were collected in formalin for histological examination. Details regarding the virus production, infection chal- lenge and organ sampling procedure are described else- where [6]. Briefly, fish were challenged by immersion with either 104 TCID50 of highly virulent ISAV Glesvær/2/90 (HVI) or low virulent Can/F679/99 (LVI). Four fish were sampled at 6 h post infection (pi) and on days 1–8, 10, 12, 14, 19 and 23 pi from each challenge group, respectively. Cotton swabs were used to sample skin mucus from the base of the left pectoral fin and placed individually in RLT buffer (Qiagen). Pectoral fin (including skin at the fin base), gill, eye, mid-gut and hind gut were collected in 10% phosphate buffered formalin; pectoral fin (no skin), gill and hind-gut were also stored in 1 mL RNAlater (Inv- itrogen). Gills were snap-frozen in isopentane (Sigma) chilled in liquid nitrogen. The experimental trial was approved by the Norwegian Animal Research Authority.ii Introduction I f i l Sections were mounted in SlowFade® Gold (Molecular Probes), and were examined with a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss, Germany) using a 63× oil immersion objective and scored [6]. covered and protected by a mucus layer. In fact, fin and skin have both been implicated as important entry points for other viruses in fish including viral haemorrhagic septicaemia (VHSV), infectious haematopoietic necrosis virus (IHNV) and koi herpes virus (KHV) [15–17]. The presence of the ISAV cellular receptor in epithelial cells of the GI tract, skin and conjunctiva was previously con- firmed [8], suggesting they may also be entry routes for ISAV. The present study investigated the significance of mucosal infection for ISA pathogenesis by revealing alternative entry routes and sites of early viral replication other than the gills. An experimental immersion chal- lenge with two ISAV isolates of different virulence [6] as well as fish collected during a disease outbreak in farmed Atlantic salmon in Norway (2014), were used. Under- standing the early primary replication phase at mucosal surfaces and responses may open new possibilities for disease prevention and vaccine development, i.e. vaccines aimed at stimulating the mucosal immune mechanisms. Real‑time PCR Total RNA was extracted using either the QIAsym- phony® RNA robotic system (Qiagen) (challenge mate- rial; 5 mg hind gut and gill, 20 mg pectoral fin and skin/ mucus swabs) or the RNeasy tissue kit (Qiagen) (ISA out- break samples; 5 mg) according to manufacturer’s proto- cols. For hind gut and all ISA outbreak samples, cDNA was synthesized using the TaqMan® Reverse Transcrip- tion Reagents kit (Life Technologies, UK) with oligo- d(T)16 as described previously [21] and real-time PCR (qPCR) analysis was performed on a Lightcycler LC96 (Roche) as described in [6] using assays targeting ELF and ISAV segment 8 [7]. For skin swabs and pectoral fin, one-step real-time RT-qPCR (Quantitect Probe RT-PCR kit, Qiagen) was performed as outlined in McBeath et al. [7]. Transcription of immune genes type I and II IFN, Mx and γIP were analysed on a Lightcycler LC96 (Roche) for all tissues and statistical analysis was performed as previ- ously described [7]. RNA species specific RT and qPCR to specifically examine replication (cRNA) and transcrip- tion (mRNA) was performed according to McBeath et al. [7, 22]. Histology, immunohistochemistry and immunofluorescent labellinghifi Number of fish positive for ISAV in gills, hind gut, skin/pectoral fin (fin only for RT-qPCR), eye, and skin/mucus swabs with RT-qPCR or IHC/IFAT in fish infected with low and highly virulent ISAV na not applicable a The “negative” sample was much smaller than the others and produced an ELF Cp 9 cycles higher than expected b Only 3 fish tested c Only 2 fish tested Day 6 h 1 2 3 4 5 6 7 8 10 12 14 19 23 Gills 4 4 4 4 4 4 4 4 4 4 4 4 4 4 qPCR Low 0/0 0/0 0/4 0/4 2/4 3/1 4/1 4/1 2/2 3/2 4/3 3/na 4/na 1/na IHC/IFAT 4 4 4 4 4 4 4 4 4 4 4 4 4 4 qPCR High 0/0 0/0 0/0 0/1 0/3 1/0 3/2 2/2 2/3 4/4 4/4 4/na 4/na 4/na IHC/IFAT Hind gut 0 2 3 3a 4 4 4 4 4 4 4 4 4 4 qPCR Low 0 0 0 0 1 2 0 3 1 4 3 1 2 0 IHC 0 1 0 4 4 4 4 4 4 4 4 4 4 3b qPCR High 0 0 0 2 0 0 1 1 1 3 4 3 3 3 IHC Skin and/or fin 4 4 4 2c 4 4 4 4 4 4 4 4 4 na qPCR Low 0 0 0 0 3 4 4 4 0 4 4 4 4 2 IHC 3 3 4 4 4 4 3 4 4 4 4 4 4 na qPCR High 0 0 0 1 3 4 4 4 0 3 4 4 4 4 IHC Eye (n = 2) 0 0 0 0 0 0 2 0 1 1 2 2 0 0 IHC Low 0 0 0 0 0 0 1 1 0 2 2 2 0 0 IHC High Mucus 4 4 4 4 4 4 4 4 4 4 4 4 4 na qPCR Low 4 4 4 4 4 4 4 4 4 4 4 4 4 na qPCR High Table 1 Number of fish positive for ISAV in gills, hind gut, skin/pectoral fin, eye, and skin/mucus. Hind gut detected in the individual swabs (as indicated by wide confidence intervals and dispersal of data, including sev- eral apparently “super-infected” HVI fish sampled after day 4 pi) a statistically significant difference in dynamics of LVI and HVI was observed (Figure 1A). LVI increased rapidly during the first 4 days pi, with a tenfold increase of virus between 6 h and day 1 pi, indicating early replica- tion, followed by a gradual decrease until the termination of the challenge. Contrary to this, HVI increased slowly in skin swabs throughout the challenge (Figure 1A). The RNA species specific assay detected both mRNA and cRNA in all LVI infected fish on days 2 and 3, but not in HVI infected fish at these timepoints (data not shown). detected in the individual swabs (as indicated by wide confidence intervals and dispersal of data, including sev- eral apparently “super-infected” HVI fish sampled after day 4 pi) a statistically significant difference in dynamics of LVI and HVI was observed (Figure 1A). LVI increased rapidly during the first 4 days pi, with a tenfold increase of virus between 6 h and day 1 pi, indicating early replica- tion, followed by a gradual decrease until the termination of the challenge. Contrary to this, HVI increased slowly in skin swabs throughout the challenge (Figure 1A). The RNA species specific assay detected both mRNA and cRNA in all LVI infected fish on days 2 and 3, but not in HVI infected fish at these timepoints (data not shown). g Virus was detected in the hind gut by RT-qPCR on day 1 in both HVI and LVI fish (1 and 2 fish respectively, Table 1) but with very high Ct values (>35), indicating low viral load. All fish were positive from day 3 with LVI level increasing faster than HVI up to day 8 (Figure 1C). Both cRNA and mRNA were first detected in hind gut of one LVI fish on day 4 and of 3 fish on day 5 (data not shown). In addition, the measuring of four select immune response related genes indicated little difference in the type I response (IFNα and Mx; Additional file 1). How- ever, the expression of type II IFN and γIP was sug- gested to be earlier in response to LVI compared to HVI between days 2 and 8 pi (Additional file 1). Histology, immunohistochemistry and immunofluorescent labellinghifi The formalin fixed samples were embedded in paraffin and processed using standard histological procedures, Page 3 of 11 Page 3 of 11 Aamelfot et al. Vet Res (2015) 46:120 Table 1 Number of fish positive for ISAV in gills, hind gut, skin/pectoral fin, eye, and skin/mucus. Cell tropism of experimentally infected fish Gills Virus was detected by RT-qPCR in all pectoral fin samples except for one HVI fish at each of 6 h and day 1 pi (Table 1). The statistically significant faster increase of LVI (as dem- onstrated in skin swabs) was also observed in the pectoral fin, further indicating rapid viral entry and replication of this low virulent strain in the epithelial layer (Figure 1B). This was supported by RNA species-specific analysis, although mRNA and cRNA levels only reached detectable levels on day 3 pi for LVI and HVI in 2 and 1 fish respec- tively. On subsequent days, more LVI- than HVI-infected fish were positive for both RNA types, indicating more rapid replication and transcription (data not shown). In general, epithelial ISAV IFAT labelling in cryo-sections (Figure 2) was observed with the amount of positive cells increasing from sparse to moderate between days 2 and 5 pi and scored from 1 to 2 (of maximum 3). Figure 3 shows the number of challenged fish positive for ISAV in the gill cryo-sections and shows the shift from immune staining of epithelial to endothelial cells from day 5 to 6. Epithe- lial IHC staining was observed between days 4 and 8 pi in the gills from the challenged fish (score 1) (Figure 2). After day 6 by IFAT and day 8 by IHC no epithelial cells Aamelfot et al. Vet Res (2015) 46:120 Page 4 of 11 Figure 1 ISAV segment 8 profiles measured by qPCR. Statistical modelling of ISAV segment 8 total RNA load profiles measured by real-time RT-qPCR of high virulent (dashed line and triangles) and low virulent (solid line and circles) strains from skin swabs (A), pectoral fin (B) and hind gut (C) post-immersion infection on day 0. Dotted lines indicate 95% confidence intervals. Figure 1 ISAV segment 8 profiles measured by qPCR. Statistical modelling of ISAV segment 8 total RNA load profiles measured by real-time RT-qPCR of high virulent (dashed line and triangles) and low virulent (solid line and circles) strains from skin swabs (A), pectoral fin (B) and hind gut (C) post-immersion infection on day 0. Dotted lines indicate 95% confidence intervals. were positive for ISAV; however endothelial labelling was observed by both methods. HVI (day 7 pi) indicating that LVI established a systemic endothelial infection earlier than HVI, possibly due to the more pronounced primary replication phase in mucosal gill epithelial cells. Cell tropism of experimentally infected fish Gills By IFAT, LVI was detected in epithelial gill cells 1 day earlier (day 2 pi) compared to HVI (day 3 pi) indicating a rapid early establishment of infection for both viruses, but in particular LVI. The number of ISAV positive epi- thelial cells was low to moderate in the LVI infected fish and scored from 1 to 2 whilst low in the HVI infected fish, all with the score 1. Labelling of endothelial gill cells was observed 1 day earlier in LVI (day 6 pi) compared to Discussion Here we demonstrate for the first time that ISAV was taken up and replicated in mucosal surfaces other than gills, such as the pectoral fin, skin and GI tract. We also present evidence of a shift in cell tropism, from the initial infection of epithelial cells to endothelial cells as the dis- ease progressed, suggesting a primary replication phase in mucosal epithelial cells. Finally, we present observa- tions suggesting a role for the secondary vascular system (SVS) in ISA pathogenesis. Mucosal surfaces are significant barriers to viral infec- tion. In the present study, following an immersion infec- tion, detection of ISAV in the skin swabs after only 6 h pi indicated an early accumulation of viral particles in the mucus and skin. Interestingly however, the signifi- cant temporal increase in viral RNA after 6 h pi indicated ISAV was indeed infecting cells contained in the mucus and actively replicating as confirmed by RNA specific assay. Taken together, this strongly suggests ISAV uses the skin and fin epithelial cells as entry points and sites of primary replication. To our knowledge this is the first description of ISAV using this port of entry in Atlantic salmon.hi Figure 3 Number of fish positive for ISAV HE in gill cryo- sections by IFAT. Fish positive on days 1–5 pi displayed positive epithelial cells, while fish positive on days 6–12 pi displayed positive endothelial cells. A transition from epithelial infection (days 2–5 pi) into endothelial infection (days 6–12 pi) occurs between day 5 and 6. from each virus group at day 4 pi. The positive cells [interpreted to be part of the secondary vascular system (SVS)] were found close to the epidermis (Figures 4A– C) and scored from 1.5 to 2.5. From day 4 all fish in both groups had positive endothelial cells in the skin. At day 23 pi positive necrotic epithelial cells (Figure 4D) were found in 1 fish infected with LVI showing that epithe- lial skin cells can become infected by the virus. Macro- scopic skin haemorrhages were observed in the positive fish. The infection pattern in the pectoral fin by RT-qPCR was very similar to that seen in gill [6, 7] indicating that the pectoral fins also play a part in the initial infection. Discussion Even though the present study used only pectoral fin and skin to examine virus load on outer mucosal surfaces, we speculate that the whole mucosal surface plays a role in early infection. This is supported by live fish real time infection studies with IHNV and VHSV in salmonids and KHV in carp where skin and fin play a role in uptake and primary infection [15–17]. Due to variable sensitivities of the differing assays, virus production in both gill and fin was likely occurring at undetectable levels using IHC and IFAT prior to the positive timepoints, as indicated by the increase in virus level by RT-qPCR. The sample prepa- ration procedures may also have caused loss of surface epithelial cells during histological processing, putatively explaining why no positive epithelial cells were found in the pectoral fin/skin by IHC at early timepoints. Unfortu- nately no frozen skin or fin were sampled to confirm that the positive RT-qPCRs were indeed caused by epithelial infection and this therefore remains an important aspect for future studies. Hind gut and eye In the hind gut, the first ISAV observations by IHC were in epithelial cell nuclei at day 3 pi for the HVI and at day 4 for the LVI. Examples of positive cells are shown in Figures 4E and F. Endothelial labelling was observed from day 6. In the eye, only endothelial cells were positive and only after the virus had been detected in internal organs (Table 1). This suggests the positive endothelial cells in the eye were infected through the cardio vascular system and not as a result of primary viral entry. Skin/pectoral fin ISAV positive epithelial cells were not detected by IHC during the first days pi in the skin/pectoral fin (Table 1). However, large amounts of positive endothelial cells were detected from day 3 pi in 1 HVI fish and in 3 fish Page 5 of 11 Aamelfot et al. Vet Res (2015) 46:120 re 2 IFAT and IHC of gills from challenged Atlantic salmon. IFAT (A, B) and IHC (C–F) labelling of gill samples from challenged Atlantic on showing cells positive for ISAV HE and NP respectively. A, B Positive epithelial cells on gill lamellae, day 2 post infection (pi) LVI. C, D Positive elial cells on gill lamellae, day 5 pi LVI. E, F Positive endothelial cells in the gill, day 6 pi HVI. Arrows show positive cells. Figure 2 IFAT and IHC of gills from challenged Atlantic salmon. IFAT (A, B) and IHC (C–F) labelling of gill samples from challenged Atlantic salmon showing cells positive for ISAV HE and NP respectively. A, B Positive epithelial cells on gill lamellae, day 2 post infection (pi) LVI. C, D Positive epithelial cells on gill lamellae, day 5 pi LVI. E, F Positive endothelial cells in the gill, day 6 pi HVI. Arrows show positive cells. Figure 2 IFAT and IHC of gills from challenged Atlantic salmon. IFAT (A, B) and IHC (C–F) labelling of gill samples from challenged Atlantic salmon showing cells positive for ISAV HE and NP respectively. A, B Positive epithelial cells on gill lamellae, day 2 post infection (pi) LVI. C, D Positive epithelial cells on gill lamellae, day 5 pi LVI. E, F Positive endothelial cells in the gill, day 6 pi HVI. Arrows show positive cells. Page 6 of 11 Page 6 of 11 Aamelfot et al. Vet Res (2015) 46:120 0 1 2 3 4 1 2 3 4 5 6 7 8 10 12 LVI HVI Posive epithelial cells Posive endothelial cells Number of posive ﬁsh Days Figure 3 Number of fish positive for ISAV HE in gill cryo- sections by IFAT. Fish positive on days 1–5 pi displayed positive epithelial cells, while fish positive on days 6–12 pi displayed positive endothelial cells. A transition from epithelial infection (days 2–5 pi) into endothelial infection (days 6–12 pi) occurs between day 5 and 6. Naturally infected fishi All five Atlantic salmon from the ISA outbreak were anaemic and showed typical clinical and pathological signs of ISA. Histological examination revealed haemor- rhaging in several organs and hepatic necrosis associated with ISA. IHC labelling of the mucosal organs revealed positive endothelial cells in gills, pectoral fin, skin, mid- gut and hind-gut (Figure 5). Positive epithelial cells were found in conjunctiva only. All fish were highly positive by IHC (score 2.5–3) and RT-qPCR in all organs tested. Notable was the significantly higher viral load in the skin/ side-line samples (Figure 6). Interestingly, the cell tropism and kinetics of the mucosal infection of LVI and HVI appeared to be differ- ent. Our previous study indicated a primary replication phase in the gills for the LVI, that was not observed for the HVI [6, 7]. Here we showed that the LVI specifically infects epithelial cells earlier and with a higher score than the HVI, suggesting epithelial tropism. At early time- points the LVI appears similar to the non-virulent ISAV Page 7 of 11 Aamelfot et al. Vet Res (2015) 46:120 Figure 4 IHC labelling of pectoral fin/skin and gut from challenged Atlantic salmon. IHC labelling of pectoral fin/skin and gut samples from challenged Atlantic salmon showing cells positive for ISAV NP. A Positive endothelial cells (ECs) in vessel in skin, day 3 pi HVI. B, C Positive ECs in ves- sel in skin, day 4 pi HVI. D Skin of LVI on day 23 pi, showing necrotic epithelial cells in skin positive for ISAV. E, F Positive epithelial cells (EPs) in hind gut, day 4 pi LVI. Arrows show positive cells. kin and gut from challenged Atlantic salmon. IHC labelling of pectoral fin/skin and gut samples from positive for ISAV NP. A Positive endothelial cells (ECs) in vessel in skin, day 3 pi HVI. B, C Positive ECs in ves- y 23 pi, showing necrotic epithelial cells in skin positive for ISAV. E, F Positive epithelial cells (EPs) in hind . Figure 4 IHC labelling of pectoral fin/skin and gut from challenged Atlantic salmon. IHC labelling of pectoral fin/skin and gut samples from challenged Atlantic salmon showing cells positive for ISAV NP. A Positive endothelial cells (ECs) in vessel in skin, day 3 pi HVI. B, C Positive ECs in ves- sel in skin, day 4 pi HVI. Naturally infected fishi D Skin of LVI on day 23 pi, showing necrotic epithelial cells in skin positive for ISAV. E, F Positive epithelial cells (EPs) in hind gut day 4 pi LVI Arrows show positive cells i Figure 4 IHC labelling of pectoral fin/skin and gut from challenged Atlantic salmon. IHC labelling of pectoral fin/skin and gut samples from challenged Atlantic salmon showing cells positive for ISAV NP. A Positive endothelial cells (ECs) in vessel in skin, day 3 pi HVI. B, C Positive ECs in ves- sel in skin, day 4 pi HVI. D Skin of LVI on day 23 pi, showing necrotic epithelial cells in skin positive for ISAV. E, F Positive epithelial cells (EPs) in hind gut, day 4 pi LVI. Arrows show positive cells. Aamelfot et al. Vet Res (2015) 46:120 Page 8 of 11 Figure 5 IHC labelling of mucosal tissues from Atlantic salmon from outbreak. IHC labelling of mucosal tissues from Atlantic salmon from confirmed outbreak of ISA showing cells positive for ISAV NP. Gill (A), pectoral fin (B), side-line skin (C), hind gut (D) and mid gut (E) with ISAV posi- tive endothelial cells. Conjunctiva (F) with positive epithelial cells. Arrows show positive cells. Figure 5 IHC labelling of mucosal tissues from Atlantic salmon from outbreak. IHC labelling of mucosal tissues from Atlantic salmon from confirmed outbreak of ISA showing cells positive for ISAV NP. Gill (A), pectoral fin (B), side-line skin (C), hind gut (D) and mid gut (E) with ISAV posi- tive endothelial cells. Conjunctiva (F) with positive epithelial cells. Arrows show positive cells. Aamelfot et al. Vet Res (2015) 46:120 Page 9 of 11 1,E-1 1,E+0 1,E+1 1,E+2 1,E+3 1,E+4 1,E+5 1,E+6 1,E+7 1,E+8 1,E+9 gill pectoral ﬁn pelvic ﬁn skin hind gut midgut Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Mean Figure 6 ISAV segment 8 measured by qPCR. Log of RT-qPCR ISAV segment 8 load in fish 1–5 from outbreak of ISA standardised to ELF and mean of 5 fish with standard error. Detection of HVI in endothelial and, only to a limited extent, in epithelial cells at early stages suggests HVI, to a greater extent than LVI, enters directly through the epi- thelium through transcytosis, thus reaching the endothe- lial cells underneath. Naturally infected fishi This strategy is one of a variety of mechanisms used by viruses to gain entry to the host [26] and was seen studying VHSV on polarized epithelial rainbow trout cultures [27]. Low virulent strains infected the epithelial layer, while the highly virulent strains passed straight through without replication. LVI on the other hand infects the mucosal epithelium. This may give the fish infected with LVI an advantage by priming the local mucosal immune system, potentially triggering an early systemic protective response, before virus overflows the circulation [4].i HPR0, which is largely detected in gill epithelial cells and does not progress into an endothelial infection as demon- strated by IHC examinations (unpublished results), even though it has been detected in kidneys in some cases by qPCR [10, 23]. HVI does not appear to be as active in the epithelial cells, however at the later stages it establishes an extensive endothelial infection leading to higher viral load and higher mortality [6].hi HPR0, which is largely detected in gill epithelial cells and does not progress into an endothelial infection as demon- strated by IHC examinations (unpublished results), even though it has been detected in kidneys in some cases by qPCR [10, 23]. HVI does not appear to be as active in the epithelial cells, however at the later stages it establishes an extensive endothelial infection leading to higher viral load and higher mortality [6].hi The vascular system in fish is divided into two compo- nents, the primary vascular system (PVS) containing the red blood cell (RBC) circulation, and a secondary vascu- lar system (SVS) consisting of thin-walled capillaries of varying sizes, not containing RBCs under resting condi- tions. The SVS is a large-volume low-flow system con- nected to the PVS through anastomoses, and has only been found in mucosal surfaces in direct contact with surrounding water, e.g. gills, skin, fins, and oral mucosa. The volume of SVS was reported to be 1.5 times the PVS in rainbow trout, Oncorhynchus mykiis (Walbaum) [24]. Given that endothelial infection in the skin, inter- preted to be part of the SVS, was found preceding IHC endothelial detection in any other organ indicates the cells in the skin were not infected through the cardio- vascular system. Thus, we speculate that HVI may have a primary replication phase in the SVS. Naturally infected fishi Furthermore, the significantly higher viral loads in samples from the side- line skin in outbreak fish could also indicate a role of the SVS in ISA pathogenesis. The functional role of the SVS is poorly understood, although ion/water balance, oxy- gen uptake, acid–base regulation, and communication between the systemic and mucosal protective systems have been suggested [25]. The further exploration of the SVS in ISA pathogenesis is a topic for future studies. Statistically modelled profiles of LVI and HVI in the hind gut were similar to those observed in heart [6]. As previously suggested for the heart, the earlier increase of LVI in the hind gut may be attributed to the more rapid infection of epithelial cells which systemically alerted the immune system. This can be seen in two of the immune markers tested (Additional file 1). The RT-qPCR posi- tives on days 1 and 2 pi may be a result of ingested virus however further work is required to understand the role of the GI tract for ISAV uptake and infection in Atlantic salmon. Virus shedding was not a focal point of this study how- ever the continuous circulation of new water combined with an increase in the amount of ISAV in the skin swabs from the challenged fish, indicate that the skin may take part in shedding. A high level of virus particularly in the skin of the outbreak fish supports this. Positive epi- thelial cells in the conjunctiva of the outbreak fish may Page 10 of 11 Page 10 of 11 Aamelfot et al. Vet Res (2015) 46:120 References 1. Dannevig BH, Falk K, Namork E (1995) Isolation of the causal virus of infec- tious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J Gen Virol 76:1353–1359 y 2. Håstein T, Hill BJ, Winton JR (1999) Successful aquatic animal disease emergency programmes. Rev Sci Tech 18:214–227 3. Thorud K, Djupvik HO (1988) Infectious anaemia in Atlantic salmon (Salmo salar L). Bull Eur Assoc Fish Pathol 8:109–111 4. Aamelfot M, Dale OB, Falk K (2014) Infectious salmon anaemia—patho- genesis and tropism. J Fish Dis 37:291–307 5. Roberts RJ (2001) Fish pathology. W B Saunders, London 6. McBeath A, Aamelfot M, Christiansen DH, Matejusova I, Markussen T, Kaldhusdal M, Dale OB, Weli SC, Falk K (2015) Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L. J Fish Dis 38:3–15 Acknowledgements suggest its involvement in dispersal of virus at a later stage of infection however this should be investigated further. Individuals vary substantially in their responses to infection and this influences the host’s ability to limit or clear the infection. More susceptible hosts are likely to shed more virus than a less susceptible host. A study using IHNV and VHSV in rainbow trout showed that even under substantially controlled conditions, between- individual variation in shedding was prevalent [28]. The considerable variation in the quantities of virus detected in the individual skin swabs, in particular for the HVI, could simply be due to unequal swabbing during sam- pling however may indicate that not all fish shed compa- rable levels of virus. The authors wish to thank Brit Sofie Nordvik, Randi Terland and Sylvie Benestad at the Norwegian Veterinary Institute, Oslo, Norway, Annabelle Duff and Malcolm Hall at Marine Scotland Science, Aberdeen, Scotland, and Kristín Baldvinsdóttir and Ann Siri Borg Hentze at the Food and Veterinary Authority, Tórshavn, Faroe Islands for laboratory assistance. Thanks also to the staff at the VESO Vikan research station and to Rune Hafsmo at Marine Harvest. The research leading to these results has received funding from the Norwegian Research Council, HAVBRUK project No. 207024, the European Community’s Seventh Framework Programme (FP7, 2007–2013), the Faroese Research Council and the Research Infrastructures action, Grant agreement no. FP7- 228394 (NADIR). Council and the Research Infrastructures action, Grant agreement no. FP7- 228394 (NADIR). Abbreviations ECs: endothelial cells; EPs: epithelial cells; GI: gastrointestinal; HE: haemaggluti- nin esterase; HPR0: putatively non-virulent variants of ISAV; HVI: highly virulent ISAV; IFAT: immunofluorescent labelling; IHC: immunohistochemistry; IHNV: infectious haematopoietic necrosis virus; ISA: infectious salmon anaemia; ISAV: infectious salmon anaemia virus; KHV: koi herpes virus; LVI: low virulent ISAV; NP: nucleoprotein; pi: post infection; PVS: primary vascular system; SVS: secondary vascular system; VHSV: viral haemorrhagic septicaemia virus. 10. Christiansen DH, Ostergaard PS, Snow M, Dale OB, Falk K (2011) A low-pathogenic variant of infectious salmon anemia virus (ISAV-HPR0) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L) in the Faroe Islands. J Gen Virol 92:909–918 11. Belser JA, Wadford DA, Xu JG, Katz JM, Tumpey TM (2009) Ocular Infection of Mice with influenza A (H7) viruses: a site of primary replication and spread to the respiratory tract. J Virol 83:7075–7084 Authors’ contributions MA carried out all histology, IHC, IFAT analysis and co-drafted the manuscript. AM and DHC carried out all molecular analysis. AM performed the statistical analysis and co-drafted the manuscript. KF designed and organised the exper- imental challenge and prepared virus. MA, AM, DHC, IM and KF participated in the design and execution of the experimental aquarium challenge and in data analysis. All authors significantly contributed to the revision and editing of the manuscript and approved the final version. All authors read and approved the final manuscript. 12. Reperant LA, van de Bildt MW, van Amerongen G, Leijten LM, Watson S, Palser A, Kellam P, Eissens AC, Frijlink HW, Osterhaus AD, Kuiken T (2012) Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats. J Virol 86:1158–1165 13. Kumlin U, Olofsson S, Dimock K, Arnberg N (2008) Sialic acid tissue distribution and influenza virus tropism. Influenza Other Respir Viruses 2:147–154 14. Genten F (2009) Atlas of Fish Histology Science Publishers, Enfield, New Hampshire, USA Additional file 7. McBeath AJ, Ho YM, Aamelfot M, Hall M, Christiansen DH, Markussen T, Falk K, Matejusova I (2014) Low virulent infectious salmon anaemia virus (ISAV) replicates and initiates the immune response earlier than a highly virulent virus in Atlantic salmon gills. Vet Res 45:83 Additional file 1. Statistical modelling of 4 immune marker expres- sion profiles. Statistical modelling of 4 immune marker expression profiles, type I IFN (A), Mx (B), type II IFN (C) and γIP (D) in hind gut of fish infected with high virulent (dashed line and triangles) or low virulent (solid line and circles) strains measured by real-time RT-qPCR. Dotted lines indicate 95% confidence intervals. Additional file 1. Statistical modelling of 4 immune marker expres- sion profiles. Statistical modelling of 4 immune marker expression profiles, type I IFN (A), Mx (B), type II IFN (C) and γIP (D) in hind gut of fish infected with high virulent (dashed line and triangles) or low virulent (solid line and circles) strains measured by real-time RT-qPCR. Dotted lines indicate 95% confidence intervals. 8. Aamelfot M, Dale OB, Weli SC, Koppang EO, Falk K (2012) Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus. J Virol 86:10571–10578 9. Weli SC, Aamelfot M, Dale OB, Koppang EO, Falk K (2013) Infectious salmon anaemia virus infection of Atlantic salmon gill epithelial cells. Virol J 10:5 Received: 3 July 2015 Accepted: 1 October 2015 In conclusion, we show for the first time that ISAV uses the skin and pectoral fin as entry ports and primary replication sites in addition to the gill. As the infection progresses, the type of cells infected with the virus shifts from epithelial to endothelial, resulting in a shift from a localized to a generalized infection. LVI and HVI appear to have a different mechanism or pathway of infecting host cells with the LVI having a greater affinity for epi- thelial cells in the gill, skin and fin while the HVI appears to directly infect endothelial cells leading to more pro- nounced systemic infection. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 3 July 2015 Accepted: 1 October 2015 Received: 3 July 2015 Accepted: 1 October 2015 Author details 1 15. Costes B, Raj VS, Michel B, Fournier G, Thirion M, Gillet L, Mast J, Lieffrig F, Bremont M, Vanderplasschen A (2009) The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin. J Virol 83:2819–2830 1 Norwegian Veterinary Institute, Oslo, Norway. 2 Marine Scotland Science, Marine Laboratory, Aberdeen, Scotland, UK. 3 Food and Veterinary Authority, Tórshavn, Faroe Islands. 1 Norwegian Veterinary Institute, Oslo, Norway. 2 Marine Scotland Science, Marine Laboratory, Aberdeen, Scotland, UK. 3 Food and Veterinary Authority, Tórshavn, Faroe Islands. Page 11 of 11 Page 11 of 11 Aamelfot et al. Vet Res (2015) 46:120 Page 11 of 11 16. Harmache A, LeBerre M, Droineau S, Giovannini M, Bremont M (2006) Bioluminescence imaging of live infected salmonids reveals that the fin bases are the major portal of entry for Novirhabdovirus. J Virol 80:3655–3659 22. McBeath A, Bain N, Fourrier M, Collet B, Snow M (2013) A strand specific real-time RT-PCR method for the targeted detection of the three species (vRNA, cRNA and mRNA) of infectious salmon anaemia virus (ISAV) repli- cative RNA. J Virol Methods 187:65–71 17. Quillet E, Dorson M, Aubard G, Torhy C (2007) In vitro assay to select rain- bow trout with variable resistance/susceptibility to viral haemorrhagic septicaemia virus. Dis Aquat Organ 76:7–16 23. McBeath AJ, Bain N, Snow M (2009) Surveillance for infectious salmon anaemia virus HPR0 in marine Atlantic salmon farms across Scotland. Dis Aquat Organ 87:161–169 24. Steffensen JF, Lomholt JP (1992) The secondary vascular system. In: Randall DJ, Farrell AP, Hoar WS (eds) Fish Physiology. Academic Press, San Diego, pp 185–217ff 18. Hetland DL, Jørgensen SM, Skjødt K, Dale OB, Falk K, Xu C, Mikalsen AB, Grimholt U, Gjøen T, Press CM (2010) In situ localisation of major histo- compatibility complex class I and class II and CD8 positive cells in infec- tious salmon anaemia virus (ISAV)-infected Atlantic salmon. Fish Shellfish Immunol 28:30–39 25. Rasmussen KJ, Steffensen JF, Buchmann K (2013) Differential occurrence of immune cells in the primary and secondary vascular systems in rain- bow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 36:675–679 25. Rasmussen KJ, Steffensen JF, Buchmann K (2013) Differential occurrence of immune cells in the primary and secondary vascular systems in rain- bow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 36:675–679 26. Bergelson JM (2003) Virus interactions with mucosal surfaces: alternative receptors, alternative pathways. Curr Opin Microbiol 6:386–391f 19. Author details 1 Aspehaug V, Falk K, Krossøy B, Thevarajan J, Sanders L, Moore L, Endresen C, Biering E (2004) Infectious salmon anemia virus (ISAV) genomic seg- ment 3 encodes the viral nucleoprotein (NP), an RNA-binding protein with two monopartite nuclear localization signals (NLS). Virus Res 106:51–60 26. Bergelson JM (2003) Virus interactions with mucosal surfaces: alternative receptors, alternative pathways. Curr Opin Microbiol 6:386–391 27. Brudeseth BE, Skall HF, Evensen O (2008) Differences in virulence of marine and freshwater isolates of viral hemorrhagic septicemia virus in vivo correlate with in vitro ability to infect gill epithelial cells and mac- rophages of rainbow trout (Oncorhynchus mykiss). J Virol 82:10359–10365 20. Falk K, Namork E, Dannevig BH (1998) Characterization and applications of a monoclonal antibody against infectious salmon anaemia virus. Dis Aquat Organ 34:77–85 28. Wargo AR, Kell AM, Scott RJ, Thorgaard GH, Kurath G (2012) Analysis of host genetic diversity and viral entry as sources of between-host varia- tion in viral load. Virus Res 165:71–80 21. McBeath AJ, Snow M, Secombes CJ, Ellis AE, Collet B (2007) Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus. Fish Shellfish Immunol 22:230–241 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
W4366984330.txt	http://link.aps.org/pdf/10.1103/PhysRevResearch.5.023056	en		Steering edge currents through a Floquet topological insulator	Physical review research	2,023	cc-by	6,128	PHYSICAL REVIEW RESEARCH 5, 023056 (2023) Steering edge currents through a Floquet topological insulator Helena Drüeke ,* Marcus Meschede ,† and Dieter Bauer ‡ Institute of Physics, University of Rostock, 18051 Rostock, Germany (Received 19 April 2022; revised 5 January 2023; accepted 9 February 2023; published 25 April 2023) Periodic driving may cause topologically protected, chiral transport along edges of a two-dimensional (2D) lattice that, without driving, would be topologically trivial. We study what happens if one adds a different on-site potential along the diagonal of such a 2D grid. In addition to the usual bulk and edge states, the system then also exhibits doublon states, analogous to two interacting particles in one dimension. A particle initially located at an edge propagates along the system’s boundary. Its wave function splits when it hits the diagonal and continues propagating simultaneously along the edge and the diagonal. The strength of the diagonal potential determines the ratio between both parts. We show that for specific values of the diagonal potential, hopping onto the diagonal is prohibited so that the system effectively separates into two triangular lattices. For other values of the diagonal potential, we find a temporal delay between the two contributions traveling around and through the system. This behavior could enable the steering of topologically protected transport of light along the edges and through the bulk of laser-inscribed photonic waveguide arrays. DOI: 10.1103/PhysRevResearch.5.023056 I. INTRODUCTION Topological insulators [1,2] exhibit an insulating bulk and conducting edges. Floquet topological insulators [3–5] are synthetic systems. Periodic driving causes their behavior as topological insulators. Therefore, time becomes an additional system dimension, and modifications of the driving scheme are a practical parameter for tuning Floquet topological insulators. They are realized experimentally with cold atoms in optical lattices [6–8], with photonic platforms [9–14], or even for acoustic waves [15,16]. Theoretically, they are studied in one dimension (1D) [17,18], two dimensions (2D) [19,20], and systematized in a periodic table [21]. For a recent theoretical review, see [22]. Rudner et al. [23] proposed a Floquet topological insulator on a bipartite square lattice. They also derived a topological invariant for that system. In our paper, we investigate a modified version of Rudner’s system. Instead of global, alternating on-site potentials on the two sublattices, we introduce an onsite potential along the diagonal of the lattice. We examine how the diagonal potential can cut the system in half, fully or partially. Without it, a topologically protected edge current can flow along the system’s outer edge. The addition of a diagonal potential introduces an “inner” edge. Depending on the exact choice of parameters, a portion or the whole edge state may flow along this inner edge instead of the outer edge. * helena.drueeke@uni-rostock.de marcus.meschede@uni-rostock.de ‡ dieter.bauer@uni-rostock.de † Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. 2643-1564/2023/5(2)/023056(8) 023056-1 The diagonal potential leads to some eigenstates located (almost) exclusively along the diagonal. We relate these eigenstates to doublon physics. There, two interacting particles on, for example, a one-dimensional [24–27] or two-dimensional [28] lattice may form a bound pair, a so-called doublon state. Doublons are stable due to their energetic separation from other states or because of topology, characterized by a differing topological invariant. They can arise from repulsively as well as from attractively interacting particle pairs. In order to prove that our system is topologically nontrivial, we need a topological invariant. The usual Chern number is insufficient to determine the existence of chiral edge states in Floquet systems. The Chern number of an energy band gives the difference between the numbers of chiral edge modes entering it from above and below. In a static system, the energy spectrum is bound from below, i.e., no edge modes can exist below the lowest-energy band. The number of edge states in any band gap equals the sum of the Chern numbers of all bands below it. However, in Floquet systems, due to their temporal periodicity, the energy becomes a quasienergy, which is also periodic. Edge modes can loop around from below the lowest to above the highest band. This allows chiral edge states to exist in a Floquet system in which all bands have Chern number 0. Rudner et al. [23] derived a winding number as a topological invariant for Floquet systems. In contrast to the Chern number, which only depends on projectors onto Floquet bands, the winding number considers the whole time evolution throughout the driving cycle. This paper is structured as follows: Sec. II contains a description of the system under study, Sec. III its Floquet eigenstates, Sec. IV its band structure, and Sec. V the temporal evolution of states. We give a conclusion in Sec. VI. The Appendices contain derivations of the hopping probability (Appendix A) and the topological invariant (Appendix B), as well as the algorithm for tracking states through (avoided) crossings (Appendix C). Published by the American Physical Society DRÜEKE, MESCHEDE, AND BAUER PHYSICAL REVIEW RESEARCH 5, 023056 (2023) B. Hopping probabilities 1 2 4 3 In each of the four phases of the driving scheme, each lattice site connects to (at most) one neighboring lattice site. Therefore, the Hamiltonian of the whole system breaks down into several two-state Hamiltonians, each describing the interaction between connected sites. In Appendix A, we derive the hopping probability between two connected sites √ V 2 + 4J 2 4J 2 2 sin p(t, V ) = 2 t (5) V + 4J 2 2 FIG. 1. Sketch of the driving scheme for a 4 × 4 system. The dots (squares) indicate the lattice sites on sublattice A (B). The orange dots are the lattice sites on the diagonal (with a modified on-site potential). The four panels show the different hoppings in the four phases of the driving cycle as (blue, orange, green, and red) connecting lines. The gray rectangle indicates the unit cell used for calculating the band structure in Sec. IV. II. SYSTEM We investigate an N × N square-lattice system. The lattice sites si, j are numbered by horizontal and vertical indices i, j ∈ {1, 2, . . . , N}. Index i increases from left to right, and j increases from bottom to top. Sublattice A contains all sites with even sum i + j, and sublattice B those with odd i + j. The periodic driving scheme of this Floquet system is sketched in Fig. 1. We modify the potentials of the sites si, i along the diagonal (as shown in Fig. 1), which we set to Vdia . All other on-site potentials are 0. A. Hamiltonians We will now introduce a mathematical description of the system and its parameters. The hopping amplitudes and potentials are described by the stepwise constant Hamiltonian ⎧ Ĥ1 , 0 < t T /4 ⎪ ⎪ ⎪ ⎪ ⎨Ĥ , T /4 < t T /2 2 Ĥ (t ) = (1) ⎪ Ĥ ⎪ 3 , T /2 < t 3T /4 ⎪ ⎪ ⎩ Ĥ4 , 3T /4 < t T with N Ĥk = Ĥdia + J i, j=1 i+ j even N Ĥdia = Vdia ĥk (i, j) + H.c., (2) as a function of the potential difference V between the sites and the duration t of the respective driving phase. Without a potential (i.e., between the off-diagonal sites), we want the particles to hop completely during each of the four phases, p T4 , 0 = sin2 JT4 := 1. We set J = 1 and choose T = 2π = 2π , such that in the absence of on-site potentials, J the hopping probability between two connected sites during each phase is exactly 1. With on-site potential Vdia along the diagonal, the hopping probability between connected diagonal and off-diagonal sites (and vice versa) becomes p(Vdia ) = p T , Vdia 4 = 4 sin2 +4 2 Vdia π 2 + 4 , (6) Vdia 4 2 + 4 leads to a shown in Fig. 6(a). The prefactor 4/ Vdia decrease in the hopping probability at higher diagonal potentials. As expected, the particle cannot overcome the energetic separation between off-diagonal and diagonal sites at high diagonal potentials. But the probability for hopping onto the diagonal is also vanishing at finite diagonal potentials (n) Vdia,0 = ±2 4n2 − 1 (7) for n ∈ N, determined by the zeros of the sin2 term of (6). At these specific potentials, the duration of each phase cor(n) responds to one complete Rabi period. Therefore, at Vdia,0 ,a particle moves from its starting site to the connected neighboring site and back n times, returning to the starting site at the end of the phase. The √ first zero is at a remarkably low diagonal (1) = 2 3. potential Vdia,0 C. Time evolution operator The time evolution operator (in units where h̄ = 1) t Û (t ) = T exp −i dt Ĥ (t ) , where T is the time-ordering operator, describes the system’s temporal evolution. For a complete driving cycle consisting of the four discrete phases (t = T ), the time evolution operator reads Û (T ) = ecĤ4 ecĤ3 ecĤ2 ecĤ1 , \|i, ii, i\|, (3) with c = i=1 ĥ2 (i, j) = \|i, ji, j + 1\|, ĥ3 (i, j) = \|i, ji − 1, j\|, ĥ4 (i, j) = \|i, ji, j − 1\|, (4) period T , and hopping amplitude J. T . 4i (9) Solving the equation Û (T )ψF = λF ψF ĥ1 (i, j) = \|i, ji + 1, j\|, (8) 0 (10) gives the Floquet eigenstates ψF , and the Floquet energies εF are calculated from the eigenvalues λF = exp(−iεF T ). By following the states (not just their energies) as a function of 023056-2 STEERING EDGE CURRENTS THROUGH A FLOQUET … PHYSICAL REVIEW RESEARCH 5, 023056 (2023) Vdia , we can distinguish state crossings from avoided crossings. Appendix C explains the details of the state-tracking algorithm. Floquet energy εF Without a diagonal potential, the N × N system contains (N − 1)2 bulk states at Floquet energy εF = 0 and 2N − 1 edge states at equally spaced Floquet energies εF = m/(2N − 1), m ∈ Z, \|m\| < N. At nonvanishing diagonal potential, some of the bulk states superpose to form doublon states, resulting in (N − 3)(N − 2) remaining bulk states, N − 1 primary doublon states, and 2(N − 2) secondary doublon states. Particles in a primary doublon state hop on and off the modified diagonal twice during a complete driving cycle. They hop on and off the diagonal once in a secondary doublon state. We call these diagonal states doublons, in analogy to doublons in one-dimensional, interacting systems [24–28]. A system of two interacting particles on a one-dimensional lattice (e.g., a Su-Schrieffer-Heeger chain [29]) can be mapped to a single particle on a two-dimensional lattice. The x and y coordinates of the 2D particle correspond to the position of the first and the second particle on the 1D chain, respectively. Movements of the first (second) particle then amount to horizontal (vertical) hoppings. Their interaction V (\|x − y\|) is captured by an on-site potential in 2D. A local interaction in 1D, through which the particles only affect each other if they are on the same site, corresponds to the diagonal potential along x = y used in this study. Longer-range interactions would require nonvanishing on-site potentials on not only the main but also the secondary, tertiary, etc., diagonal, depending on the maximum interaction distance. However, in our Floquet system, the mapping between the two-particle-1D and the one-particle-2D case is more involved and not that intuitive because the simple driving scheme in 2D corresponds to intricate, time-dependent, nonlocal interactions of two particles in 1D. Figure 2 shows the Floquet energies εF as a function of the diagonal potential Vdia . The four different types of states are indicated in Fig. 2(a): The diagonal potential does not influence the bulk states’ energies because the bulk states remain located on sites whose potentials are zero in each step of the driving cycle. The edge states cross the diagonal in the system’s bottom-left and top-right corners. Therefore, their energies increase with increasing diagonal potential. The doublons cross the diagonal as well, which leads to increasing Floquet energies. The primary doublons’ energies increase faster because they cross the diagonal twice during a driving cycle, unlike the secondary doublons, which only cross it once. At higher diagonal potentials Vdia 2 [shown in Fig. 2(b)], three types of states exist: The bulk states’ energies remain zero, while the edge states’ energies increase slightly with diagonal potential. For the Floquet states located on the diagonal of the system, we find εF ≈ Vdia . All states form pairs with similar energies. These pairs degenerate to identical energies at the zeros of the hopping probability, and we find superpositions of the states confined to either the top-left or bottom-right triangular half of the system. (a) 0.2 0 -0.2 -0.4 0 0.4 Floquet energy εF III. FLOQUET STATES 0.4 0.5 1 1.5 2 (b) 0.2 0 -0.2 -0.4 0 2 4 6 8 diagonal potential Vdia 10 FIG. 2. Floquet energies εF for the 10 × 10 system as a function of the diagonal potential Vdia . (a) The line colors indicate the types of states: bulk states (black), edge states (blue), primary doublon states (orange), and secondary doublon states (green). (b) The dotted gray (1) (2) and Vdia,0 of the hopping probability lines indicate the zeros Vdia,0 p(Vdia ). IV. BAND STRUCTURE In order to calculate a band structure, we must have a system that is infinitely extended in at least one direction. We turn our system by 45◦ and obtain an infinite strip with the former diagonal in the middle. The narrowest viable strip is four sites wide, as shown in Fig. 3. Wider strips would only host more bulk states with identical energies. The unit cell is repeated infinitely in the vertical direction and numbered by an index m. The sites are numbered α = 1, 2, 3, 4. We can write the Hamiltonians for the four phases in real space as Ĥi = V \|m, 3m, 3\| + J (11) ĥi (m) + H.c. m with 023056-3 ĥ1 (m) = \|m, 1m, 2\| + \|m, 3m, 4\|, ĥ2 (m) = \|m, 3m, 2\| + \|m, 1m, 4\|, DRÜEKE, MESCHEDE, AND BAUER 2 PHYSICAL REVIEW RESEARCH 5, 023056 (2023) 4 a 1 3 FIG. 3. Unit cell of the 45◦ -rotated system as outlined in light gray in panel 4 of Fig. 1. The system is infinitely extended in the vertical direction. The height of the unit cell, a, is marked. Periodic boundaries are employed horizontally. The orange circle (3) marks the diagonal. The hoppings of all four phases are shown in the same colors as in Fig. 1. ĥ3 (m) = \|m + 1, 3m, 2\| + \|m + 1, 1m, 4\|, ĥ4 (m) = \|m + 1, 1m, 2\| + \|m + 1, 3m, 4\|. (12) We transform the Hamiltonians to k space by making the Bloch ansatz a dk exp(−ikma) \|k, α, (13) \|m, α = 2π BZ where a is the height of the unit cell (i.e., the distance from site 1 in cell m to site 1 in cell m + 1). We can identify a Ĥi = dk\|kk\|[V \|33\| + J (ĥi (k) + H.c.)] (14) 2π BZ with ĥ1 (k) = \|12\| + \|34\|, ĥ2 (k) = \|32\| + \|14\|, ĥ3 (k) = exp(ika)(\|32\| + \|14\|), ĥ4 (k) = exp(ika)(\|12\| + \|34\|). FIG. 4. Band structure of a four-site-wide diagonal strip at different diagonal potentials Vdia . The bulk states (shown in black) form a flat √ band at εF = 0, irrespective of the diagonal potential. (a) Vdia = 2 3, i.e., p(Vdia ) = 0; the primary diagonal states form a flat band (orange) and the secondary diagonal states form diagonal bands (green and blue). Transferring between bands is impossible. (b) Vdia = 5, i.e., p(Vdia ) = 0.10819: the crossings between (primary and secondary) diagonal states become avoided crossings. (15) We used these Hamiltonians to calculate the band struc(n) tures shown in Fig. 4. At diagonal potentials Vdia = Vdia,0 [Fig. 4(a)], the band structure consists of four separate bands: The flat band at εF = 0 (shown in black) consists of stationary bulk states, and the other flat band (orange) consists of primary diagonal states, which are also stationary. The two diagonal bands (green and blue) consist of secondary diagonal states. Their slope indicates the movement of these states along the diagonal. The bulk band remains unchanged at other diagonal potentials [Fig. 4(b)]. The three other bands, however, connect as their crossings turn into avoided crossings. We employed periodic boundary conditions on the left and right edges of the unit cell when calculating the band structures. The full system is not periodic in this direction. It only contains a single modified diagonal, not a periodically repeated one. For the calculation of the band structure, however, this periodicity (the connection between sites 1 and 4 in Fig. 3) is necessary. Without it, the left and right edges of the unit cell would become edges of the system, hosting edge states (two additional diagonal bands). Such edges, which would lie in diagonal direction in the nonrotated system, do not exist in the full (square) system. It only contains horizontal and vertical edges, whose edge states move with a different velocity than the diagonal ones. These edge states would also result in diagonal bands but with a different slope. The band structures in Fig. 4 do not contain these bands. They are band structures of the bulk, showing stationary states and those moving along the diagonal. A topological invariant for the system is derived in Appendix B. V. TEMPORAL EVOLUTION By definition, the Floquet states ψF remain unchanged after a complete cycle’s evolution, except for a phase factor. However, for an intuitive understanding of the driven quantum dynamics, it is instructive (and closer to experimental realizations on photonic platforms) to study the temporal evolution of (initially) localized states ϕ(r, t ). We initialize these as one at a single site rinitial and zero everywhere else. Specifically, in Fig. 5, we look at a state starting on the left edge of the 10 × 10 system. Without a diagonal potential and therefore perfect hopping p(0) = 1 between diagonal and off-diagonal sites [Fig. 5(b)], the state remains localized and moves counterclockwise along the edges of the system, returning to its origin after 2N − 1 complete driving cycles. The state cannot cross the diagonal at the zeros of the hopping probability p(Vdia ) = 0. Because it started above the diagonal, it will remain in the upper-left triangular half of the system, moving along the edge of that triangle and returning to its origin after 2N − 3 023056-4 STEERING EDGE CURRENTS THROUGH A FLOQUET … PHYSICAL REVIEW RESEARCH 5, 023056 (2023) FIG. 5. Temporal evolution of a localized edge state in the 10 × 10 system. The dots mark the locations of the sites. The orange ones are those on the diagonal. (a) Starting configuration (n = 0). (b)–(d) State after evolution for n = 8 complete cycles. The arrows sketch the path of the state during its evolution. (b) Vdia = 0, i.e., p(Vdia ) = 1; the state moves around the whole system and√will return at its initial site after 2N − 1 = 19 cycles. (c) Vdia = 2 3, i.e., p(Vdia ) = 0; the state moves around the upper left triangle because the diagonal is insurmountable and will return after 2N − 3 = 17 cycles. (d) Vdia = 1.597 37, i.e. p(Vdia ) = 0.5; the state splits. The Supplemental Material [30] contains animations of these three cases for a 20 × 20 system. cycles [Fig. 5(c)]. For all other potentials, which result in 0 < p(Vdia ) < 1, the state splits at the bottom-left corner of the system and delocalizes [Fig. 5(d)]. Most [(N − 3)(N − 2)] of the (N − 1)2 bulk states do not interact with any sites on the diagonal, and therefore their evolution is unaffected by Vdia . The other 3N − 5 bulk states, however, interact with the diagonal at least once and are split up for 0 < p(Vdia ) < 1. For p(Vdia ) = 0, 2N − 3 bulk states turn into edge states on the upper-left and lower-right triangles. The N − 2 bulk and two edge states that start on the diagonal remain stationary for p(Vdia ) = 0. While snapshots of the temporal evolution allow us to understand the general behavior, it is inconvenient to investigate them systematically for varying potentials. Therefore, we record the probability density \|ϕ(rinitial , nT )\|2 (n ∈ N) at the starting location rinitial after each driving cycle. Because we initialize all states perfectly localized, \|ϕ(rinitial , 0)\|2 = 1 for all states. The bulk states return to their starting location after each cycle, and therefore \|ϕbulk (rinitial , nT )\|2 = 1 ∀n ∈ N. FIG. 6. (a) Hopping probability p(Vdia ) as a function of diagonal potential. (b) Probability density \|ϕ(rinitial , nT )\|2 (n ∈ N) at the starting location rinitial for an edge state in a 20 × 20 system, as a function of time and diagonal potential. The dotted gray lines indicate the zeros of p(Vdia ) in (a), which coincide with the perfect earlier returns in (b). As shown in Fig. 6, without a diagonal potential, an edge state returns at its origin after 2N − 1 cycles. At the zeros of p(Vdia ) (7), the system is equivalent to one without sites on the diagonal. The edge states remain confined to the upper-left or bottom-right triangle, which they started in, and return to their origins after 2N − 3 cycles. Other on-site potentials Vdia lead to a splitting of the state. Part of the wave function travels through the diagonal and along the square’s edge, arriving after 2N − 1 cycles, while another part travels around the triangle and arrives after 2N − 3 cycles. Due to the imperfect transfer, there are not two discrete arrival peaks. Instead, the peaks disperse and interfere. VI. CONCLUSION AND OUTLOOK We characterized a Floquet topological insulator on a square lattice with varying diagonal on-site potential. The addition of a diagonal potential causes the appearance of additional bands. We call these states doublons, in analogy to doublons in systems of one-dimensional, interacting particles. There are stationary doublons and those which perform directional motion along the diagonal. We showed how fine-tuning the diagonal potential cuts the Floquet topological insulator into halves. While a small diagonal potential is sufficient to disconnect the system completely, surprisingly, increasing the diagonal potential further will partially reconnect the system. This partial cutting allows switching between the propagation along the edge and the diagonal. In a photonic setting with laser-inscribed waveguides, intense laser light and nonlinearities can modify the 023056-5 DRÜEKE, MESCHEDE, AND BAUER PHYSICAL REVIEW RESEARCH 5, 023056 (2023) diagonal potential (i.e., refractive index) [11,12,31]. In that way, one may switch topologically protected light currents by light, rendering a photonic platform programmable instead of “hardwired.” ACKNOWLEDGMENTS This research and publication were supported by the Studienstiftung des deutschen Volkes (German Academic Scholarship Foundation) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), SFB 1477 “LightMatter Interactions at Interfaces,” Project No. 441234705. APPENDIX A: DERIVATION OF HOPPING PROBABILITY The 2 × 2 Hamiltonian Ĥ = V J J 0 (A1) describes the hopping between two sites with potential difference V and hopping element J. Its eigenstates √ V ± V 2 + 4J 2 ϕ1,2 = (A2) 2J and corresponding eigenenergies ε1,2 = 21 V ± V 2 + 4J 2 (A3) (A4) The solutions of the time-dependent Schrödinger equation Ĥ ψ (t ) = i∂t ψ (t ) (A5) are superpositions of the eigenstates ψ (t ) = a1 exp (−iε1t )ϕ1 + a2 exp (−iε2t )ϕ2 . (A6) For a time-dependent state starting localized on the first lattice site, 1 = ψ (0) = a1 ϕ1 + a2 ϕ2 , 0 (A7) we find 1 a1 = −a2 = √ . (A8) 2 2 V + 4J 2 The hopping probability is the absolute square of the timedependent state at the second lattice site, p(t, V ) = \|ψ2 (t )\|2 √ 4J 2 V 2 + 4J 2 2 = 2 t . sin V + 4J 2 2 Uε (k, r, t ) = U (k, r, t ) exp (itHeff,ε (k, r)) (B1) with i ln−ε (U (k, r, T )). (B2) T The subscript epsilon denotes the branch cut of the complex logarithm so that ln−ε (eiφ ) = iφ for −εT − 2π < φ < −εT . Setting it to a quasienergy value inside the band gap ensures Uε is a continuous function. The topological classification in each symmetry class is then only dependent on the difference between the spatial dimension ds and the defect dimension dd . The dimension of the defect is the dimension of the circle S dd enclosing the defect. For example, a point defect in three dimensions (3D) can be enclosed by a 3D sphere S 3 . A line defect in 2D (such as the modified diagonal in our system) is enclosed by S 0 , which are only two points (±1) in the plane on either side of the defect. Our Hamiltonian and its corresponding time evolution operator Uε do not possess time reversal, charge conjugation, or chiral symmetry, nor do we enforce any other symmetries (e.g., crystal symmetries). Therefore, the system belongs to the complex symmetry class A, for which topologically nontrivial phases exist if ds − dd is even. The 2D winding number with our dd = 0 defect, given by the diagonal potential, is then the difference of the winding numbers of the bulk on the two sides (±1) of the defect: Heff,ε (k, r) = satisfy the time-independent Schrödinger equation Ĥ ϕ1,2 = ε1,2 ϕ1,2 . (n) the diagonal potential However, we argue that at these Vdia,0 decouples the bulk on either side of the diagonal, and the invariant of our Hamiltonian (1) without the diagonal potential can describe the edge state. One might guess that interpreting the diagonal potential as a defect, the state along the diagonal is, in fact, a defect edge state. We explicitly start from the winding number defined on defect Hamiltonians to show that this cannot be the case. The Altland-Zirnbauer classification [32] has been adopted to Floquet systems with defects [33]. The overall structure of the periodic table of static topological insulators/superconductors remains the same. However, the Floquet invariants are now winding numbers associated with the band gaps at quasienergy ε of the time evolution operator U (k, r, t ), where r describes the positional dependence of the time evolution operator. Notably, because the quasienergy spectrum is periodic, there is an additional band gap at εT = ±π . More specifically, because the time evolution operator is generally not periodic in time, one can calculate the Floquet invariant through a periodized time evolution operator Uε [33]: W (Uε ) = W (Uε (k, +1, t )) − W (Uε (k, −1, t )), (A9) (A10) where W (Uε (k, ±1, t )) = dkx dky dt BZ×S 1 × Tr (Uε−1 ∂t Uε ) Uε−1 ∂kx Uε , Uε−1 ∂ky Uε . APPENDIX B: TOPOLOGICAL INVARIANT In the following, we discuss the topological properties of the Hamiltonian (1). Especially the state propagating along the (n) [Eq. (7)] seems peculiar as diagonal potential for specific Vdia,0 the diagonal is not a boundary between topologically different bulks and is therefore not expected to host any edge states. 1 8π 2 (B3) (B4) Here, the square bracket inside the trace denotes the commutator. In our system, there is only a band gap at εT = π , as (in contrast to [23]) we do not have any bipartite on-site potential 023056-6 STEERING EDGE CURRENTS THROUGH A FLOQUET … PHYSICAL REVIEW RESEARCH 5, 023056 (2023) in the bulk. We find the winding number W (Uπ/T (k, ±1, t )) = 1, π < JT < 6.9π 0, else (B5) identical to the one found by Rudner et al. [23] even without the bipartite on-site potential. Because we chose JT = 2π in Sec. II, W (Uπ/T (k, ±1, t )) = 1. As the bulk Hamiltonian on both sides of the diagonal potential is equal, we see from (B3) that the observed edge state cannot be a defect edge state. Still, each bulk on its own is topologically nontrivial with our driving parameters. (n) the probability of As we have shown in Sec. II B, at Vdia,0 the state to hop onto, and hence over the diagonal, is zero after a full period T . Therefore, we interpret a quantum state’s inaccessibility to the other side as an isolation of each bulk to an effective system with open boundary conditions. For this system the edge states at the boundary between the bulk to the vacuum is then clearly protected by the winding number W = 1. APPENDIX C: TRACKING STATES We follow states through changing parameters, e.g., in the closed interval [Vdia,min , Vdia,max ]. As a starting point, we calculate the Floquet energies ε0, j and states ψ0, j ( j = 1, 2, . . . , N 2 ) at V0 = Vdia,min . Then we loop through the following procedure: We increase the diagonal potential to Vi = Vi−1 + δ, with 0 < δ Vdia,max − Vdia,min , and calculate the new Floquet energies εi,k and states ψi,k (k = 1, 2, . . . , N 2 ). We compare each of the states ψi,k to all states ψi−1, j from the previous step by calculating their overlap (scalar product). 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https://openalex.org/W2947999507	https://europepmc.org/articles/pmc6586951?pdf=render	English	null	Dataset on assessment of heavy metals contamination in multi-environmental samples from Patna, India	Data in brief	2,019	cc-by	4,929	Kumari Sonu a, Ishwar Chandra Yadav b, Amrendra Kumar a, Ningombam Linthoingambi Devi a, * Kumari Sonu a, Ishwar Chandra Yadav b, Amrendra Kumar a, Ningombam Linthoingambi Devi a, * a Department of Environmental Science, Central University of South Bihar, SH-7, Gaya-Panchanpur Road District-Gaya 824236, Bihar, India b Department of International Environmental and Agricultural Science (IEAS), Tokyo University of Agriculture and Technology (TUAT) 3-5-8, Saiwaicho, Fuchu, Tokyo 1838509, Japan * Corresponding author. E-mail address: nldevi@cub.ac.in (N.L. Devi). Contents lists available at ScienceDirect Contents lists available at ScienceDirect Contents lists available at ScienceDirect Data in brief 25 (2019) 104079 Data in brief 25 (2019) 104079 https://doi.org/10.1016/j.dib.2019.104079 2352-3409/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). E-mail address: nldevi@cub.ac.in (N.L. Devi). https://doi.org/10.1016/j.dib.2019.104079 2352-3409/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). a r t i c l e i n f o Soil samples were collected in triplicate from the ﬁeld receiving wastewater regularly for irrigation at the depth of 0e20 cm layer which is known to be plough layer. Vegetable samples were collected from the same ﬁeld simultaneously. Care was taken to get samples of the same varieties and age group from different selected sites and delicately analysis of samples were done via AAS, Thermoscientiﬁc, Ice 3000 Series. Experimental features Standard methods were followed for the purpose of collecting samples, taking it to laboratory, preserving water, preparing samples (cutting, drying and grinding) and analysis. Data source location Patna city, Bihar, India. Data accessibility Detailed data are conveyed in this article Subject area Environmental sciences More speciﬁc subject area Geochemistry and heavy metal pollution Type of data Table, ﬁgure and graph How data was acquired Atomic absorption spectroscopy (AAS), Thermoscientiﬁc, Ice 3000 Series Data format Raw and analyzed Experimental factors Water samples were collected using polyethylene bottles immersed in an open drain that was being used for irrigation purposes. 1mL of concentrated HNO3 was added in the bottle ﬁlled with water to avoid microbial utilization of heavy metals immediately after ﬁlling. Soil samples were collected in triplicate from the ﬁeld receiving wastewater regularly for irrigation at the depth of 0e20 cm layer which is known to be plough layer. Vegetable samples were collected from the same ﬁeld simultaneously. Care was taken to get samples of the same varieties and age group from different selected sites and delicately analysis of samples were done via AAS, Thermoscientiﬁc, Ice 3000 Series. Experimental features Standard methods were followed for the purpose of collecting samples, taking it to laboratory, preserving water, preparing samples (cutting, drying and grinding) and analysis. Data source location Patna city, Bihar, India. Data accessibility Detailed data are conveyed in this article Subject area More speciﬁc subject area Type of data How data was acquired Data format Experimental factors Water samples were collected using polyethylene bottles immersed in an open drain that was being used for irrigation purposes. 1mL of concentrated HNO3 was added in the bottle ﬁlled with water to avoid microbial utilization of heavy metals immediately after ﬁlling. Soil samples were collected in triplicate from the ﬁeld receiving wastewater regularly for irrigation at the depth of 0e20 cm layer which is known to be plough layer. Vegetable samples were collected from the same ﬁeld simultaneously. 1. Data Different concentration levels of heavy metals in water, soil and vegetables from sampling sites Gaighat, Paijawa, Danapur, Ranipur and Marchi are given in Table 1 and Table 2. Occurrence of heavy metals in the wastewater was in an order of Mn > Zn > Cu > Pb > Hg. Among all concentration of manganese in soil was found to be higher followed by Zn, Cu, Pb and Hg respectively. Concentrations of heavy metal were below the safe limits prescribed by WHO/FAO (2007) [1] and Indian standards [2]. However, heavy metal concentrations in vegetables are in the order of Zn > Mn > Cu > Pb > Hg respectively (Fig. 1). Value of the data Value of the data This data will be important for the assessment of contamination level of heavy metals by human activities. It will be more informative for researchers for their further research. a r t i c l e i n f o Article history: Received 24 April 2019 Accepted 22 May 2019 Available online 31 May 2019 Keywords: Heavy metals Soil Contamination Risk Article history: Received 24 April 2019 Accepted 22 May 2019 Available online 31 May 2019 Accumulation of heavy metals in vegetables adversely affects the well-being of human health. In this study, we investigated the heavy metals (Hg, Zn, Cu, Pb and Mn) contamination in different environmental samples collected from ﬁve major sites (Gaighat, Paijawa, Danapur, Ranipur and Marchi) of Patna. In all the samples concentration of manganese (Mn) was found to be higher in soil samples. The concentration of heavy metals in soil samples were in the order Mn > Zn > Cu > Pb > Hg in water sample; Mn > Zn > Pb > Cu > Hg, and in vegetables Mn > Zn > Cu > Pb > Hg. In all sites, majority of heavy metal were within the permissible limits except the Zn and Pb. The Zn and Pb contents in vegetables and soil were measured above the permissible limit recommended by WHO/FAO (2007) and Indian standard. The bioconcentration factors (BCFs) for the heavy metal transfer from soils to vegetables are analysed and were ranked in the order of Hg > Pb > Zn > Cu > Mn. The estimated daily intake of metals suggested low health risk despite higher metal content in soil/vegetables. The metal pollution index (MPI) analysis showed Keywords: Heavy metals Soil Contamination Risk K. Sonu et al. / Data in brief 25 (2019) 104079 K. Sonu et al. / Data in brief 25 (2019) 104079 2 high MPI for spinach (15.6) followed by red spinach (14.0) whereas beans (8.6) showed lower metal pollution index. © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). Speciﬁcations table Subject area Environmental sciences More speciﬁc subject area Geochemistry and heavy metal pollution Type of data Table, ﬁgure and graph How data was acquired Atomic absorption spectroscopy (AAS), Thermoscientiﬁc, Ice 3000 Series Data format Raw and analyzed Experimental factors Water samples were collected using polyethylene bottles immersed in an open drain that was being used for irrigation purposes. 1mL of concentrated HNO3 was added in the bottle ﬁlled with water to avoid microbial utilization of heavy metals immediately after ﬁlling. a r t i c l e i n f o Care was taken to get samples of the same varieties and age group from different selected sites and delicately analysis of samples were done via AAS, Thermoscientiﬁc, Ice 3000 Series. p Standard methods were followed for the purpose of collecting samples, taking it to laboratory, preserving water, preparing samples (cutting, drying and grinding) and analysis. Patna city, Bihar, India. Value of the data This data will be important for the assessment of contamination level of heavy metals by human activities. It will be more informative for researchers for their further research. These data's will be important for estimating health risk posed by heavy metal contamination in soil and vegetables. Present data may be useful for policy makers, government ofﬁcial, stakeholder to forumulate health risk managment plan. Subject area More speciﬁc subject area Type of data How data was acquired Data format Experimental factors Table 1 C Table 1 C Table 1 Concentrations of Heavy metals in Water and Soil samples across the sampling sites of Patna city. Heavy metals Water (mg/L) Permissible limit Permissible limit WHO/FAO (2007) Indian Standard (2000) Soil (mg/Kg) WHO/FAO Indian Standard (2000) Hg Range 0.001e0.008 0.18e0.923 Mean 0.006 0.445 Zn Range 0e0.223 2.0 5.0 76.11e347.46 300e600 Mean 0.065 136.952 Pb Range 0.058e0.066 5.0 0.10 14.53e57.42 250e500 Mean 0.061 24.454 Cu Range 0e0.018 0.20 0.05 31.22e119.61 135e270 Mean 0.003 49.604 Mn Range 0.124e0.338 0.2 0.1 298.92e642.48 Mean 0.239 417.73 Table 2 Heavy metal concentration (in milligrams per kilogram dry weight) in vegetable samples grown in wastewater-irrigated agri- cultural ﬁeld. Sampling site Vegetables Heavy Metal concentration in mg/Kg Hg Zn Pb Cu Mn Gaighat Spinach 0.240 137.84 14.61 29.65 41.37 Red Spinach 0.169 118.72 11.66 36.16 64.48 Bean 0.211 85.26 9.42 10.64 26.20 Lady ﬁnger 0.191 80.88 13.17 12.27 27.31 Cabbage 0.154 80.02 24.2 6.30 20.72 Paijawa Spinach 0.189 161.82 30.08 4.56 143.36 Cabbage 0.171 183.72 27.44 28.10 68.48 Cauliﬂower 0.145 68.52 29.34 26.02 13.52 Danapur Spinach 0.269 53.91 13.15 18.87 136.75 Cauliﬂower 0.186 32.88 14.90 5.73 13.09 Ranipur Spinach 0.542 60.32 13.11 13.67 75.28 Cabbage 0.541 42.73 15.84 2.83 26.03 Cauliﬂower 0.705 37.32 13.68 2.17 21.96 Marchi Cabbage 0.455 32.94 12.95 3.11 26.81 Cauliﬂower 0.592 24.66 11.57 36.3 17.96 Beetroot 0.393 50.31 12.61 11.78 40.43 WHO/FAO (2007) 60 5 40 IS (2000) 50 2.5 30 Table 1 Concentrations of Heavy metals in Water and Soil samples across the sampling sites of Patna city. Heavy metals Water (mg/L) Permissible limit Permissible limit WHO/FAO (2007) Indian Standard (2000) Soil (mg/Kg) WHO/FAO Indian Standard (2000) Hg Range 0.001e0.008 0.18e0.923 Mean 0.006 0.445 Zn Range 0e0.223 2.0 5.0 76.11e347.46 300e600 Mean 0.065 136.952 Pb Range 0.058e0.066 5.0 0.10 14.53e57.42 250e500 Mean 0.061 24.454 Cu Range 0e0.018 0.20 0.05 31.22e119.61 135e270 Mean 0.003 49.604 Mn Range 0.124e0.338 0.2 0.1 298.92e642.48 Mean 0.239 417.73 ons of Heavy metals in Water and Soil samples across the sampling sites of Patna city. and Soil samples across the sampling sites of Patna city. Table 2 Heavy metal concentration (in milligrams per kilogram dry weight) in vegetable samples grown in wastewater-irrigated agri- cultural ﬁeld. Table 1 C Sampling site Vegetables Heavy Metal concentration in mg/Kg Hg Zn Pb Cu Mn Gaighat Spinach 0.240 137.84 14.61 29.65 41.37 Red Spinach 0.169 118.72 11.66 36.16 64.48 Bean 0.211 85.26 9.42 10.64 26.20 Lady ﬁnger 0.191 80.88 13.17 12.27 27.31 Cabbage 0.154 80.02 24.2 6.30 20.72 Paijawa Spinach 0.189 161.82 30.08 4.56 143.36 Cabbage 0.171 183.72 27.44 28.10 68.48 Cauliﬂower 0.145 68.52 29.34 26.02 13.52 Danapur Spinach 0.269 53.91 13.15 18.87 136.75 Cauliﬂower 0.186 32.88 14.90 5.73 13.09 Ranipur Spinach 0.542 60.32 13.11 13.67 75.28 Cabbage 0.541 42.73 15.84 2.83 26.03 Cauliﬂower 0.705 37.32 13.68 2.17 21.96 Marchi Cabbage 0.455 32.94 12.95 3.11 26.81 Cauliﬂower 0.592 24.66 11.57 36.3 17.96 Beetroot 0.393 50.31 12.61 11.78 40.43 WHO/FAO (2007) 60 5 40 IS (2000) 50 2.5 30 Fig. 1. Concentration of heavy metals in vegetable samples collected from wastewater irrigated sites of Patna city. 1.1. Bioconcentration factor (BCF) The bioconcentration factors (BCFs) estimated for the heavy metal transfer from soils to vegetables are shown in Table 3. The BCF values of the heavy metals such as Hg, Zn, Pb, Cu, and Mn were found to be in the ranges of 0.2e0.9, 0.2 to 1.5, 0.2 to 1.9, 0.1 to 0.5, and 0.1 to 0.2, respectively. The trend in the BCF for heavy metals in the sampling sites was in the ranking order of Hg > Pb > Zn > Cu > Mn. The food chain (soil-plant-human) is mainly known as one of the major pathways for exposure of human to soil contaminants. K. Sonu et al. / Data in brief 25 (2019) 104079 3 1.2. Daily intake of metals (DIM) Assessing the exposure level and tracing the path of contaminants to the observed organisms are of great value as a result to observe the underlying health risks (see Fig. 2). There may be several pathways but food chain is proved to be the important pathway of human exposure heavy metals. DIM in adults as well as children were calculated using the average amount of vegetable consumed by them on daily routine (Table 4). Fig. 2 shows the DIM in adults and children from different vegetables. DIM values for heavy metals were found to be almost negligible for Hg but were found to be highest in case of Zn, Mn, Pb and Cu from the consumption of spinach, red spinach, cabbage and beans respectively, for both adults and children, grown in wastewater-irrigated soils. DIM suggest that the consumption of vegetables grown inwastewater contaminated soils is high, but it is nearly free of risks, as the dietary intake limits of Cu, Zn and Mn in adults can range from 1.2 to 3.0 mg, 5.0e22.0 mg and 2.0e20.0 mg, respectively [4]. Table 2 ntration (in milligrams per kilogram dry weight) in vegetable samples grown in wastewater-irrigated agri- Table 2 Heavy metal concentration (in milligrams per kilogram dry weight) in vegetable samples grown in wastewater-i cultural ﬁeld. Fig. 1. Concentration of heavy metals in vegetable samples collected from wastewater irrigated sites of Patna city. Fig. 1. Concentration of heavy metals in vegetable samples collected from wastewater irrigated sites of Patna city. K. Sonu et al. / Data in brief 25 (2019) 104079 4 4 Table 3 Bi Table 3 Bio-concentration of different metals in vegetables irrigated with waste water of Patna city. Heavy metals Gaighat Paijawa Danapur Ranipur Marchi Hg 0.209 0.569 1.261 1.928 0.921 Zn 0.289 1.534 0.481 0.577 0.472 Pb 0.254 1.919 0.836 0.769 0.851 Cu 0.158 0.582 0.393 0.195 0.545 Mn 0.12 0.216 0.173 0.111 0.044 Soil-to-plant transfer is one of the key processes of human exposure to toxic heavy metals through the food chain. When BCF <1 or BAF ¼ 1, it denotes that the plant only absorbs the heavy metal but does not accumulate when BCF >1, and this indicates that plant accumulates the heavy metals. BAF values of Hg, Zn and Pb were found to be greater than one which means that they are not only absorbing the heavy metals but also they are accumulating the heavy metals concentration which may cause health problems for human whereas BAF values of Cu and Mn were found to be less than one in the vegetable samples which indicates that plants only absorb the heavy metals not accumulate them [3]. Soil-to-plant transfer is one of the key processes of human exposure to toxic heavy metals through the food chain. When BCF <1 or BAF ¼ 1, it denotes that the plant only absorbs the heavy metal but does not accumulate when BCF >1, and this indicates that plant accumulates the heavy metals. BAF values of Hg, Zn and Pb were found to be greater than one which means that they are not only absorbing the heavy metals but also they are accumulating the heavy metals concentration which may cause health problems for human whereas BAF values of Cu and Mn were found to be less than one in the vegetable samples which indicates that plants only absorb the heavy metals not accumulate them [3]. 1.3. Metal pollution index (MPI) Calculation of Metal pollution index (MPI) is effective method for monitoring of metal pollution in wastewater irrigated areas [5]. Among all collected different vegetable samples, spinach (15.659) showed highest value of MPI followed by red spinach (14.039), and beans (8.607) showed lower metal pollution index (Table 5). Higher MPI value of spinach, red spinach and cabbage recommend that leafy vegetables are potential in causing more human health risk due to the increased accumulation of heavy metals in the vegetables. Table 4 Table 4 Daily intakes of metals (in mg) for individual heavy metal in different vegetables grown in wastewater - irrigated agricultural area. Plants Hg Zn Pb Cu Mn Adult Child Adult Child Adult Child Adult Child Adult Child Spinach 0.000 0.000 0.054 0.062 0.009 0.010 0.008 0.010 0.051 0.059 Red Spinach 0.000 0.000 0.062 0.071 0.006 0.007 0.018 0.021 0.033 0.038 Beans 0.000 0.000 0.044 0.051 0.004 0.005 0.005 0.006 0.013 0.015 L. Finger 0.000 0.000 0.042 0.048 0.006 0.007 0.006 0.007 0.014 0.016 Cabbage 0.000 0.000 0.044 0.054 0.010 0.012 0.005 0.006 0.018 0.021 Cauliﬂower 0.000 0.000 0.021 0.024 0.009 0.010 0.009 0.010 0.008 0.010 Beetroot 0.000 0.000 0.026 0.030 0.006 0.007 0.006 0.007 0.021 0.024 of metals (in mg) for individual heavy metal in different vegetables grown in wastewater - irrigated agricultural Daily intakes of metals (in mg) for individual heavy metal in different vegetables grown in wastewater - irriga area K. Sonu et al. / Data in brief 25 (2019) 104079 5 Fig. 2. Comparison of heavy metals intake by adult and children through vegetables irrigated with waste water of Patna city. Fig. 2. Comparison of heavy metals intake by adult and children through vegetables irrigated with waste water of Patna city. 2.1. Study area The multi-environmental samples were collected from Patna city (25.6N 85.1E), Bihar, located in the southern bank of the river Ganga in Eastern India. The total area of Patna is 136 km2 (53 sq mi). The municipal area constitutes 99 km2 (38 sq mi), while the suburban area constitutes 36 km2 (14 sq mi). It has an average elevation of 53 m (174 ft). It had an estimated population of 1.68 million in 2011, making it the 19th largest city in India. With over 2 million people, its urban agglomeration is the 18th largest in India. Surveillance was done in urban and semi-urban areas of Patna city to ﬁnd out those sites were industrial wastewater, municipal and domestic sewage is used for irrigation purpose. Five sampling sites were identiﬁed were wastewater is direct source of irrigation including Danapur, Gaighat, Ranipur, Paijawa and Marchi were vegetables are grown using wastewater (Fig. 3). 2.2. Sampling of soil, water and vegetables 2.2. Sampling of soil, water and vegetables Samples of water, soil and vegetables were collected from ﬁve different sites namely Danapur, Gaighat, Ranipur, Paijawa and Marchi located in Patna city. Samples were collected in the morning Table 5 Metal pollution index in different foodstuffs from wastewater irrigated site. Samples Metal Pollution Index Spinach 15.659 Red spinach 14.039 Beans 8.607 Lady ﬁnger 9.262 Cabbage 11.505 Cauliﬂower 9.664 Beetroot 10.349 K. Sonu et al. / Data in brief 25 (2019) 104079 6 Fig. 3. Detailed of Sampling sites of Patna city. Fig. 3. Detailed of Sampling sites of Patna city. between 6:00 a.m.e10:00 a.m. from the irrigation sites from December to February in 2016e2017. However, the samples of water were collected immersed in an open drain that was being used for irrigation purposes. 1mL of concentrated HNO3 was added in the bottle ﬁlled with water to avoid microbial utilization of heavy metals immediately after ﬁlling. Soil samples were collected in triplicate from the ﬁeld receiving wastewater regularly for irrigation at the depth of 0e20 cm layer which is known to be plough layer. Vegetable samples were collected from the same ﬁeld simultaneously. Care was taken to get samples of the same varieties and age group from different selected sites. Samples of seven different kinds of vegetables; leafy vegetables included Spinach (Spinacia oleracea), Red spinach (Amaranthus dubius) and Cabbage (Brassica oleracea L. Var. Capatuta). Inﬂorescence vegetable included Cauliﬂower (Brassica oleracea L. Var. botrytis), Fruit vegetables included Lady's Finger (Abelmoschus esculentus L.), Beetroot (Beta vulgaris), and Beans (Phaseolus vulgaris) were taken from the same experimental sites where waters and soils samples were taken. between 6:00 a.m.e10:00 a.m. from the irrigation sites from December to February in 2016e2017. However, the samples of water were collected immersed in an open drain that was being used for irrigation purposes. 1mL of concentrated HNO3 was added in the bottle ﬁlled with water to avoid microbial utilization of heavy metals immediately after ﬁlling. Soil samples were collected in triplicate from the ﬁeld receiving wastewater regularly for irrigation at the depth of 0e20 cm layer which is known to be plough layer. Vegetable samples were collected from the same ﬁeld simultaneously. Care was taken to get samples of the same varieties and age group from different selected sites. Samples of seven different kinds of vegetables; leafy vegetables included Spinach (Spinacia oleracea), Red spinach (Amaranthus dubius) and Cabbage (Brassica oleracea L. Var. Capatuta). 2.2. Sampling of soil, water and vegetables Inﬂorescence vegetable included Cauliﬂower (Brassica oleracea L. Var. botrytis), Fruit vegetables included Lady's Finger (Abelmoschus esculentus L.), Beetroot (Beta vulgaris), and Beans (Phaseolus vulgaris) were taken from the same experimental sites where waters and soils samples were taken. Acknowledgement The present work is funded by the University Grants Commission, Government of India (No.F.30-68/ 2014 (BSR) 2.5.3. Metal pollution index (MPI) 2.5.3. Metal pollution index (MPI) To analyse the overall concentration of heavy metal in all the vegetable samples collected from the different wastewater irrigated site, metal pollution index (MPI) was used. This index was obtained by calculating the geometrical mean of concentrations of all the metals in the vegetables [10]. 1/ Where, Cfn ¼ concentration of metal n in the sample. Where, Cfn ¼ concentration of metal n in the sample. W ¼ Average body weight, W ¼ Average body weight, At ﬁrst, weights of the fresh vegetables were converted into dry weight by using the value of conversion factor 0.085, as described previously [8]. The average body weights of the adult and child were considered to be 55.9 and 32.7 kg, respectively, while the average daily intakes of vegetable for adults and children's were considered to be 0.345 and 0.232 kg/person/day, respectively [9]. 2.3. Preparation of sample Samples of vegetables were then washed using clean water and then air dried till constant weight was achieved. The samples were then crushed separately through a steel grinder and the crushed material was then passed through 2-mm sieve. The sieved samples were kept at ambient temperature before analysis. For the analysis of heavy metals, 100 ml of water samples were digested after adding 10 ml. of concentrated nitric acid (HNO3) at 80 C. The heating was continued, until the solution appeared transparent. After cooling the digested samples were then diluted up to 100 ml with distilled water [6]. 7 K. Sonu et al. / Data in brief 25 (2019) 104079 For Soil and vegetables samples, 0.5 g of dried samples was digested with 10mL of HNO3 at 250 C in tefﬂon bomb on hot plate until a transparent solution was obtained. The solution was then ﬁltered through Whattman No. 42 ﬁlter paper and the solution was ﬁnally diluted to 50mL with distilled water [7]. 2.4. Analysis of sample 2.4. Analysis of sample Conﬂict of interest The authors declare that they have no known competing ﬁnancial interests or personal relation- ships that could have appeared to inﬂuence the work reported in this paper. 2.4.1. Determination of heavy metals 2.4.1. Determination of heavy metals The prepared samples were then analysed by using Atomic Absorption Spectrophotometer (AAS, Thermoscientiﬁc, Ice 3000 Series). The AAS value of blank solution was taken for each heavy metal. food chain transfer, Ecotoxicol. Environ. Saf. 69 (3) (2008) 513e524. [1] WHO/FAO, Joint FAO/WHO Food Standard Programme Codex Alimentarius Commission 13th Session, 2007. [2] N.S. Chary, C.T. Kamala, D.S.S. Raj, Assessing risk of heavy metals from consuming food grown on sewage irrigated soils and food chain transfer, Ecotoxicol. Environ. Saf. 69 (3) (2008) 513e524. 2.5. Data analysis 2.5.1. Bioconcentration factor (BCF) It refers to the ratio of concentration of metal in plant parts to the metal concentration in the soil. In order to assess the concentration of metals from soil to vegetables, the BCF values of metals were calculated as follows: BCF ¼ Cvegetables/Csoil Where, Cvegetable and Csoil represent the concentration of heavy metals in vegetables and soils, respectively [2,10]. f ( ) It refers to the ratio of concentration of metal in plant parts to the metal concentration in the soil. In order to assess the concentration of metals from soil to vegetables, the BCF values of metals were calculated as follows: BCF C /C BCF ¼ Cvegetables/Csoil egetables/ soil vegetable and Csoil represent the concentration of heavy metals in vegetables and soils, [2,10]. 2.5.2. Daily intake of metals (DIM) The daily intake of metals (DIM) was calculated using the following equation: DIM ¼ M K I/W Where, 1 y f ( ) The daily intake of metals (DIM) was calculated using the following equation: DIM ¼ M K I/W Where, 1 , M ¼ Concentration of heavy metals in plants (mg kg1), K ¼ Conversion factor, I ¼ Daily intake of vegetables, [2] N.S. Chary, C.T. Kamala, D.S.S. Raj, Assessing risk of heavy metals from consuming f d h i f i l i S f 69 (3) (2008) 13 24 FAO, Joint FAO/WHO Food Standard Programme Codex Alimentarius Commission 13th Session, 2007. h C T K l D S S R j A i i k f h l f i f d i i d [1] WHO/FAO, Joint FAO/WHO Food Standard Programme Codex Alimentarius Commission 13th [2] N.S. Chary, C.T. Kamala, D.S.S. Raj, Assessing risk of heavy metals from consuming food grown on sewage irrigated soils and food chain transfer, Ecotoxicol. Environ. Saf. 69 (3) (2008) 513e524. 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Rattan, S.P. Datta, P.K. Chhonkar, K. Suribabu, A.K. Singh, Long-term impact of irrigation with sewage efﬂuents on heavy metal content in soils, crops and groundwaterda case study, Agric. Ecosyst. Environ. 109 (3) (2005) 310e322. [8] R.K. Rattan, S.P. Datta, P.K. Chhonkar, K. Suribabu, A.K. Singh, Long-term impact of irrigation with sewage efﬂuents on heavy metal content in soils, crops and groundwaterda case study, Agric. Ecosyst. Environ. 109 (3) (2005) 310e322. [9] Y. Chen, C. Wang, Z. Wang, Residues and source identiﬁcation of persistent organic pollutants in farmland soils irrigated by efﬂuents from biological treatment plants, Environ. Int. 31 (2005) 778e783. [9] Y. Chen, C. Wang, Z. Wang, Residues and source identiﬁcation of persistent organic pollutants in farmland soils irrigated by efﬂuents from biological treatment plants, Environ. Int. 31 (2005) 778e783. [10] A. Singh, R.K. Sharma, M. Agrawal, F.M. Marshall, Health risk assessment of heavy metals via dietary intak from the wastewater irrigated site of a dry tropical area of India, Food Chem. Toxicol. 48 (2) (2010) 611e
https://openalex.org/W3176503772	http://pjms.org.pk/index.php/pjms/article/download/3951/985	English	null	Etiology of Hydronephrosis in adults and children: Ultrasonographic Assessment in 233 patients	Pakistan journal of medical sciences	2,021	cc-by	3,739	doi: https://doi.org/10.12669/pjms.37.5.3951 doi: https://doi.org/10.12669/pjms.37.5.3951 How to cite this: Alshoabi SA, Alhamodi DS, Alhammadi MA, Alshamrani AF. Etiology of Hydronephrosis in adults and children: Ultrasonographic Assessment in 233 patients. Pak J Med Sci. 2021;37(5):1326-1330. doi: https://doi.org/10.12669/pjms.37.5.3951 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Original Article Original Article Etiology of Hydronephrosis in adults and children: Ultrasonographic Assessment in 233 patients Sultan Abdulwadoud Alshoabi1, Dahhan Saleh Alhamodi2, Mohammed Ali Alhammadi3, Abdullah Fahad Alshamrani4 Objectives: Hydronephrosis (HN) is dilatation of the collecting system of the kidney due to obstruction of urine outflow. This study intended firstly, to investigate the efficacy of ultrasound (US) imaging to determine the cause of HN, and secondly, to list the causes of HN. Methods: In this retrospective study, 233 patients with HN were scanned to determine the cause of the HN in the period from 1st January 2016 to 31st October 2017. Categorical results were written as frequencies and percentages. p g Results: Out of 233, 91.41% were adults and 8.58% were children (P<0.001), 66.10% were male and 33.90% were female (P<0.001). In 55.36%, HN was in the right kidney and 44.64% was in the left (P=0.116). Exactly 58% of patients were suffering from grade-2, 21.5% grade-3, 11.6% grade-1, and 8.2% grade-4 HN. US imaging can determine the cause of HN in 70.4% of patients. Kidney or ureteric calculi were the cause of HN in 54.1% of cases, reflux was in 7.3%, and pelviureteric junction (PUJ) stenosis was in 3.9%. In cases of calculi induced HN, 25.3% of the calculi were in the vesicoureteric junction (VUJ), 21.5% were in the renal pelvis, 6.4% were in the PUJ or upper ureter, and only 0.9% were in the middle ureter. Conclusion: Ultrasound imaging can determine the cause of HN in more than two thirds of patients. Calculi are the most common cause of HN even in children and are most common in the VUJ. KEYWORDS: Etiology; Hydronephrosis; Calculi; Pregnancy-induced hydronephrosis; Ultrasound imaging. i i KEYWORDS: Etiology; Hydronephrosis; Calculi; Pregnancy-induced hydronephrosis; Ultrasound imaging. Correspondence: Abbreviations: N: Hydronephrosis, US: Ultrasound, PUJ: Pelviureteric Junction, HN: Hydronephrosis, US: Ultrasound, PUJ: Pelviureteric Junction, y p UJ: Vesicoureteric Junction, SFU: Society of Fetal Urology, y p VUJ: Vesicoureteric Junction, SFU: Society of Fetal Urology, , y gy, MHz: Megahertz, SPSS: Statistical Package for the Social sciences, y gy MHz: Megahertz, SPSS: Statistical Package for the Social sciences, IBM: International Business Machines, NY: New York, CI: confidence interval. doi: https://doi.org/10.12669/pjms.37.5.3951 RESULTS This study involved 233 patients who underwent abdominal US imaging and diagnosed as HN. 91.41% were adults and 8.58% were children, 66.10% were male and 33.90% were female. HN was unilateral in 88.85% and bilateral in 11.5%. HN was in the right kidney in 55.36% and 44.64% was in the left (Table-I). Table-II shows that 58% of patients were suffering from mild (Grade-2) HN, 21.5% moderate (Grade-3), 11.6% minimal (Grade-1), and 8.2% severe (Grade-4) HN (Fig.1). This study also elucidate the distribution of HN in the right and left kidneys and in male and female gender and explained the cause of predilection of HN to right side in female. Vol 37 No 5 www pjms org pk 1327 Fig.1: Ultrasound images of right kidney of different patients shows a) grade-1, b) grade-2, c) grade-3, and d) grade-4 hydronephrosis according to SFU grading system. INTRODUCTION Patients were scanned by the same highly experienced radiologist following the protocol of ultrasound imaging of the kidneys. g g y Statistical Analysis: The collected data were analyzed using the statistical package for social sciences (SPSS), IMB, version 23 for windows (Armonk, NY: IBM Corp. 2015). Chisquare test was used to compare urinary calculi in gender and side. P value was assumed to be significant when <0.05. Cross tabulation was used to analyze relationship between gender and side distribution of urinary calculi. Results were reported as frequencies and percentages in categorical data. Ethical Approval: Institutional ethical approval was obtained for this study. Confidentiality of the patients was assured during and after this study. The most common reported causes of HN are kidney or ureteric calculi, pregnancy, pelviureteric junction (PUJ) stenosis or bladder outlet obstruction.4 Ultrasound (US) imaging is commonly used, universally available, non- invasive and radiation free imaging modality. It is highly valuable in diagnosing and grading of HN.5 This study was designed to investigate the efficacy of US in determining the cause of HN which is a common medical problem in all age groups and the detection of the cause of HN is a critical point for planning of the management. Moreover, the study was carried out to examine the causes of HN in adults and children due to lack of enough similar previous studies about causes of HN in adults and children which is a common and important topic and many previous studies were concentrated only on prenatal and neonatal HN. This work will be beneficial for ultrasonographists, radiologists, urologists and emergency practitioners who are usually interested in detecting the cause of HN as a frequent and important problem corresponds to their daily work. The most common reported causes of HN are kidney or ureteric calculi, pregnancy, pelviureteric junction (PUJ) stenosis or bladder outlet obstruction.4 Ultrasound (US) imaging is commonly used, universally available, non- invasive and radiation free imaging modality. It is highly valuable in diagnosing and grading of HN.5 i g y g g g g This study was designed to investigate the efficacy of US in determining the cause of HN which is a common medical problem in all age groups and the detection of the cause of HN is a critical point for planning of the management. INTRODUCTION Moreover, the study was carried out to examine the causes of HN in adults and children due to lack of enough similar previous studies about causes of HN in adults and children which is a common and important topic and many previous studies were concentrated only on prenatal and neonatal HN. This work will be beneficial for ultrasonographists, radiologists, urologists and emergency practitioners who are usually interested in detecting the cause of HN as a frequent and important problem corresponds to their daily work. INTRODUCTION Hydronephrosis (HN) is dilatation of the renal collecting system of the kidney due to obstruction of urine outflow in any part of the urinary tract. It can present solely or together with dilatation of the ureter in an entity called hydroureternephrosis. HN can present as unilateral or bilateral, acute or chronic at any age.1 Correspondence: Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1326 g y y of urine outflow in any part of the urinary can present solely or together with dilatatio ureter in an entity called hydroureterne HN can present as unilateral or bilateral, chronic at any age.1 According to the Society of Fetal Urolog classification system, HN is classified in grades; Grade-1; dilatation of the rena only. Grade-2; dilatation of the renal pel p Sultan Abdulwadoud Alshoabi, Associate Professor of Radiology, Department of Diagnostic Radiology Technology, College of Applied Medical Sciences, Taibah University, Almadinah Almunawarah, Postal Code: 42312, Kingdom of Saudi Arabia. E-mail: alshoabisultan@yahoo.com * Received for Publication: November 26, 2020 * Accepted for Publication: April 28, 2021 Pak J Med Sci September - October 20 Sultan Abdulwadoud Alshoabi, Associate Professor of Radiology, Department of Diagnostic Radiology Technology, College of Applied Medical Sciences, Taibah University, Almadinah Almunawarah, Postal Code: 42312, Kingdom of Saudi Arabia. E-mail: alshoabisultan@yahoo.com * Received for Publication: November 26, 2020 * Accepted for Publication: April 28, 2021 Pak J Med Sci September - Oct Sultan Abdulwadoud Alshoabi, Associate Professor of Radiology, Department of Diagnostic Radiology Technology, College of Applied Medical Sciences, Taibah University, Almadinah Almunawarah, Postal Code: 42312, Kingdom of Saudi Arabia. E-mail: alshoabisultan@yahoo.com * Received for Publication: November 26, 2020 * Accepted for Publication: April 28, 2021 According to the Society of Fetal Urology (SFU) classification system, HN is classified into four grades; Grade-1; dilatation of the renal pelvis only. Grade-2; dilatation of the renal pelvis and Sultan Abdulwadoud Alshoabi et al. major calyces. Grade-3; dilatation of the renal pelvis and major and minor calyces. Grade-4; dilatation of the renal pelvis and all calyces with thinning of the renal parenchyma. The SFU classification system is used also in adults.2 This classification system has good intra observer and interobserver reliability and is recommended for assessment of neonatal HN.3 transverse sections with an examination of the whole ureter and urinary bladder to demonstrate the cause of HN. METHODS This cross-sectional retrospective study involved 233 patients who underwent abdominal US imaging and diagnosed with HN at the US unit of Amran hospital in Yemen during the period from 1st January 2016 to 31st October 2017. This study involved both adults and children. Exclusion criteria include prenatal HN because a lot of previous studies satisfy this topic. Fig.1: Ultrasound images of right kidney of different patients shows a) grade-1, b) grade-2, c) grade-3, and d) grade-4 hydronephrosis according to SFU grading system. Procedure: All patients were scanned by the same radiologist who has eight years of experience in abdominal ultrasonography. A 3.5 MHz curved transducer of Medison, Sono ex-model six color Doppler machine was used in scanning of all patients. Each patient was scanned in supine and oblique positions to demonstrate and grading HN. Each kidney was examined in longitudinal and Fig.1: Ultrasound images of right kidney of different patients shows a) grade-1, b) grade-2, c) grade-3, and d) grade-4 hydronephrosis according to SFU grading system. Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1327 1327 Etiology of Hydronephrosis in adults and children Table-I: Age, sex and side groups of hydronephrosis. Variables Categories Numbers Percentage P-value Age Adult 213 91.41 <0.001 Child 20 8.58 Total 233 100% Sex Male 154 66.10 <0.001 Female 79 33.90 Total 233 100% Kidney Unilateral 207 88.85 <0.001 Bilateral 26 11.15 Total 233 100% Side Right 129 55.36 0.116 Left 104 44.64 Total 233 100% Table shows significance association between hydronephrosis with adults and male gender (P<0.001), but no significant association between hydronephrosis and any side (P=0.116). Table-I: Age, sex and side groups of hydronephrosis. Table shows significance association between hydronephrosis with adults and male gender (P<0.001), but no significant association between hydronephrosis and any side (P=0.116). In cases of calculi induced HN, 25.3% of calculi were in the vesicoureteric junction (VUJ) or distal part of ureter, 21.5% were in the renal pelvis, 6.4% were in the PUJ or upper ureter, and only 0.9% were in the middle ureteric part (Table-V). Ultrasound imaging can determine the cause of HN in 70.4% of patients with HN. Kidney or ureteric calculi was the cause of HN in 54.1% of cases, 7.3% of cases were reflux HN, PUJ stenosis was the cause in 3.9%, and residual HN was determined in 1.7% of patients (Table-III). DISCUSSION HN is a widespread health problem worldwide that can be present at any age because of multiple causes. Determining the cause of HN is an essential when planning the treatment. In a previous study by Riddell et al., the sensitivity of bedside US imaging to detect unilateral HN was 72-83%.6 In this study, we found that US imaging can determine the cause in 70.4% of all patients with HN. Another previous study by Moş et al. reported that transabdominal US imaging can identify HN in 88.94% and can identify ureteric calculi in nearly 73% of patients.7 Sternberg et al. reported that HN on US imaging has a 77% positive predictive value for diagnosis of ureteric calcui with 71% negative predictive value.8 Another similar study by Hansen et al. reported that calculi were the most common cause of HN in adults. He reported that renal pelvic, PUJ and VUJ calculi can be detected by US. However, calculi in the ureter are difficult to be detected due to obscuration by bowel gases.10 This explains the low detection rate of ureteric calcui by US which reported in the results of this study. Calculi are the cause of HN in 54.1% of patients in the current study. This is consistent with another previous study by Alshoabi, who reported that calculi were the cause of HN in 60% of cases.11 P k J M d S i S t b O t b 2021 Table-V: Common sites of detected calculi in cases of hydronephrosis. Categories Numbers Percentage Renal pelvis 50 21.5 Upper ureter 15 6.4 Mid ureter 2 .9 Lower ureter 59 25.3 Other causes 38 16.3 Not determined 69 29.6 Total 233 100 Table reveals that calculi were common in distal part of ureter, then renal pelvis, followed by upper ureter. Table-V: Common sites of detected calculi in cases of hydronephrosis. This study showed predilection of HN to male gender. This predilection was explained by the results of a previous study by Ahmed et al. who reported that urinary tract calculi, which are the most common cause of HN, are formed more common in males due to hormonal effects.12 The high prevalence of nephrolithiasis in males is attributed to the effect of sex hormones such as androgens on some lithogenic risk factors which increases excretion and deposition of calcium oxalate in the pelvicalyceal system and kidney stone formation. METHODS Regarding causes in children, calculi were the cause in 30% of cases, PUJ stenosis in 20%, reflux HN in 15%, residual in 5%, and 25% with no determined cause (Table-IV). The cross-tabulation test between sex and side of HN shows predilection of HN to the right side only in female but not statistically significant (p=0.072), (Odds ratio= 0.832), (95% confidence interval (CI) 0.666-1.054), (Table-VI). Table-II: Grades of hydronephrosis. Pak J Med Sci September - October 202 Table-II: Grades of hydronephrosis. Categories Numbers Percentage Grade-1 27 11.6 Grade-2 137 58.8 Grade-3 50 21.5 Grade-4 19 8.2 Total 233 100 Table reveals that grade-2 hydronephrosis was the most common then grade-3. Grade-1 (Mild): Dilatation of the renal pelvis only. Grade-2 (Mild): Grade-1+ dilatation of major calyces. Grade-3 (Moderate): Grade-2+ dilatation of all calyces. Grade-4 (Severe): Grade-3+ thinning of the renal parenchyma. Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1328 (p 0.072), (Odds ratio 0.832), (95% confidence interval (CI) 0.666-1.054), (Table-VI). Table-II: Grades of hydronephrosis. Categories Numbers Percentage Grade-1 27 11.6 Grade-2 137 58.8 Grade-3 50 21.5 Grade-4 19 8.2 Total 233 100 Table reveals that grade-2 hydronephrosis was the most common then grade-3. Grade-1 (Mild): Dilatation of the renal pelvis only. Grade-2 (Mild): Grade-1+ dilatation of major calyces. Grade-3 (Moderate): Grade-2+ dilatation of all calyces. Grade-4 (Severe): Grade-3+ thinning of the renal parenchyma. Table-III: Causes of hydronephrosis in all patients. Categories Numbers Percentage Calculi 126 54.1 Reflux 17 7.3 PUJ stenosis 9 3.9 Pregnancy 8 3.4 Residual 4 1.7 Not determined 69 29.6 Total 233 100 Table reveals that ultrasound can detect the cause of hydronephrosis in 164 (70.38%) of cases of hydronephrosis and calculi were the cause in 126 (54.10%) of patients. Vol. 37 No. 5 www.pjms.org.pk 1328 Table-III: Causes of hydronephrosis in all patients. Categories Numbers Percentage Calculi 126 54.1 Reflux 17 7.3 PUJ stenosis 9 3.9 Pregnancy 8 3.4 Residual 4 1.7 Not determined 69 29.6 Total 233 100 Table reveals that ultrasound can detect the cause of hydronephrosis in 164 (70.38%) of cases of hydronephrosis and calculi were the cause in 126 (54.10%) of patients. Table-III: Causes of hydronephrosis in all patients. Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1328 Sultan Abdulwadoud Alshoabi et al. Table-VI: Cross-tabulation test between sex and side of hydronephrosis. METHODS Categories Right Left Male 80 (51.9%) 74 (48.1%) 154 Female 49 (62%) 30 (38%) 79 Total 129 (55.4%) 104 (44.6%) 233 Table reveals slight predilection of hydronephrosis to the right side only in female but not statistically significant (p=0.072). Table-VI: Cross-tabulation test between sex and side of hydronephrosis. Table-IV: Causes of hydronephrosis in children. Categories Numbers Percentage Calculi 6 30 PUJ stenosis 4 20 Reflux 3 15 Residual 1 5 Not determined 5 25 Total 20 100 Table reveals that ultrasound can detect the cause of hydronephrosis in 164 (70.38%) of cases of hydronephrosis and calculi were the cause in 126 (54.10%) of patients. Table-IV: Causes of hydronephrosis in children. Table reveals slight predilection of hydronephrosis to the right side only in female but not statistically significant (p=0.072). In this study, we found that HN was more common in male than in female, mild (grade-2) HN was the most common and ureteric or kidney calculi were the most common cause even in children. These results were consistent with that of Nuraji and Hyseni., who reported that HN was more common in grade-2 and in males with kidney and ureteric stones were the most common cause.4 These results are also consistent with a previous study by Abu-Ghazzeh and Abdu-Alro’f, who reported that calculi were the most common cause of obstructive HN and were most common in the VUJ.9 Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1329 DISCUSSION In addition, estrogen decreases excretion of urinary oxalate and contribute in nephrolithiasis.12 Table reveals that calculi were common in distal part of ureter, then renal pelvis, followed by upper ureter. d Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1329 Etiology of Hydronephrosis in adults and children 5. Rykkje A, Carlsen JF, Nielsen MB. Hand-Held Ultrasound Devices Compared with High-End Ultrasound Systems: A Systematic Review. Diagnostics (Basel). 2019;9(2):61. doi: 10.3390/diagnostics9020061 The current study revealed predilection of HN to the right side in female gender. This can be explained by that pregnancy-induced HN was the cause in 3.4% of cases and was reported more common on the right side.13 g 6. Riddell J, Case A, Wopat R, et al. Sensitivity of emergency bedside ultrasound to detect hydronephrosis in patients with computed tomography-proven stones. West J Emerg Med. 2014;15(1):96-100. doi: 10.5811/westjem.2013.9.15874 CONCLUSION ( ) / j 7. Moş C, Holt G, Iuhasz S, Moş D, Teodor I, Halbac M. The sensitivity of transabdominal ultrasound in the diagnosis of ureterolithiasis. Med Ultrason. 2010;12(3):188-197. Ultrasound imaging is an effective method to diagnose hydronephrosis which is a very common medical problem predominantly in adults, more in male, and slightly predominate on the right side. Ultrasound imaging can determine the cause of hydronephrosis in more than two thirds of patients. Ureteric or kidney calculi were the most common cause of hydronephrosis followed by reflux. Pelviureteric junction stenosis and residual hydronephrosis were uncommon causes. Calculi were determined in vesicoureteric junction, then in the renal pelvis and upper ureteric part and rarely in the middle ureteric part. ( ) 8. Sternberg KM, Pais VM Jr, Larson T, Han J, Hernandez N, Eisner B. Is Hydronephrosis on Ultrasound Predictive of Ureterolithiasis in Patients with Renal Colic? J Urol. 2016;196(4):1149-1152. doi: 10.1016/j.juro.2016.04.076 9. Abu-Ghazzeh Y, Abdu-Alro’f S. The role of ultrasound in initial evaluation of renal colic. Saudi J Kidney Dis Transpl. 2000;11(2):186-190. ( ) 10. Hansen KL, Nielsen MB, Ewertsen C. Ultrasonography of the Kidney: A Pictorial Review. Diagnostics (Basel). 2015;6(1):2. doi: 10.3390/diagnostics6010002 11. Alshoabi SA. Association between grades of Hydronephrosis and detection of urinary stones by ultrasound imaging. Pak J Med Sci. 2018;34(4):955-958. doi: 10.12669/pjms.344.14602 12. Ahmad S, Ansari TM, Shad MS. Prevalence of renal calculi; type, age and gender specific in Southern Punjab, Pakistan. Professional Med J. 2016;23:389-395. Significant statement: This study focused on study- ing the ability of US imaging to determine the cause of HN which is a common medical problem world- wide. US is a widely available and safe medical im- aging modality that can frequently determine the cause of HN and calculi are the most common de- termined cause. This data is highly valuable to ul- trasonographists, radiologists, urologists and phy- sicians who commonly using ultrasound imaging. 13. Rajaei Isfahani M, Haghighat M. Measurable changes in hydronephrosis during pregnancy induced by positional changes: ultrasonic assessment and its diagnostic implication. Urol J. 2005;2(2):97-101. Authors’ Contribution: SAA Prepared the manuscript and critically reviewed and approved the final draft and is responsible for the accuracy of the work. Conflicts of interest: The authors declare no conflicts of interest. Conflicts of interest: The authors declare no conflicts of interest. DSA collected and organized data. DSA collected and organized data. MAA interpreted data.i MAA interpreted data.i Financial support: None AFA revised and final approval of the manuscript. AFA revised and final approval of the manuscript. Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1330 REFERENCES 1. Ilgi M Sr, Bayar G, Abdullayev E, et al. Rare Causes of Hydronephrosis in Adults and Diagnosis Algorithm: Analysis of 100 Cases During 15 Years. Cureus. 2020;12(5):e8226. doi: 10.7759/cureus.8226 1. Ilgi M Sr, Bayar G, Abdullayev E, et al. Rare Causes of Hydronephrosis in Adults and Diagnosis Algorithm: Analysis of 100 Cases During 15 Years. Cureus. 2020;12(5):e8226. doi: 10.7759/cureus.8226 Authors: Authors: 1. Sultan Abdulwadoud Alshoabi, Department of Diagnostic Radiology Technology, 2. Dahhan Saleh Alhamodi, Unit of Ultrasound Imaging, Department of Radiology, Amran Hospital, Amran, Republic of Yemen 3. Mohammed Ali Alhammadi, Department of Radiology and Medical Imaging, Prince Mohamad bin Abdulaziz Hospital, National Guard, Almadinah Almunawarah, Kingdom of Saudi Arabia. 4. Abdullah Fahad Alshamrani, Department of Diagnostic Radiology Technology, 1,4: College of Applied Medical Sciences, Taibah University, Almadinah Almunawarah, Kingdom of Saudi Arabia. ( ) / 2. Onen A. Grading of Hydronephrosis: An Ongoing Challenge. Front Pediatr. 2020;8:458. doi: 10.3389/ fped.2020.00458 ( ) / 2. Onen A. Grading of Hydronephrosis: An Ongoing Challenge. Front Pediatr. 2020;8:458. doi: 10.3389/ fped.2020.00458 1. Sultan Abdulwadoud Alshoabi, Department of Diagnostic Radiology Technology, 1. Sultan Abdulwadoud Alshoabi, Department of Diagnostic Radiology Technology, p 3. Choi YH, Cheon JE, Kim WS, Kim IO. Ultrasonography of hydronephrosis in the newborn: A practical review. Ultra- sonography. 2016;35(3):198-211. doi: 10.14366/usg.15073 2. Dahhan Saleh Alhamodi, g p y ( ) g 4. Nuraj P, Hyseni N. The Diagnosis of Obstructive Hydronephrosis with Color Doppler Ultrasound. Acta Inform Med. 2017;25(3):178-181. doi: 10.5455/ aim.2017.25.178-181 Amran Hospital, Amran, Republic of Yemen Department of Radiology and Medical Imaging, Prince Mohamad bin Abdulaziz Hospital, National Guard, Almadinah Almunawarah, Kingdom of Saudi Arabia. 4. Abdullah Fahad Alshamrani, Department of Diagnostic Radiology Technology, 1,4: College of Applied Medical Sciences, Almadinah Almunawarah, Kingdom of Saudi Arabia. Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1330 Pak J Med Sci September - October 2021 Vol. 37 No. 5 www.pjms.org.pk 1330
https://openalex.org/W1983272723	https://europepmc.org/articles/pmc3861486?pdf=render	English	null	Whole-Exome Sequencing to Identify a Novel LMNA Gene Mutation Associated with Inherited Cardiac Conduction Disease	PloS one	2,013	cc-by	8,209	Abstract * E-mail: hsula@adm.cgmh.org.tw Funding: This study was supported by research grants from the Chang Gung Memorial Hospital (CMRPG 300121 and CMRPG 3B0991), the National Science Council (NSC101-2325-B-007-001), and the "Aim for the Top University Project" of the National Tsing Hua University and the Ministry of Education, Taiwan, R.O.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This study was supported by research grants from the Chang Gung Memorial Hospital (CMRPG 300121 and CMRPG 3B0991), the National Science Council (NSC101-2325-B-007-001), and the "Aim for the Top University Project" of the National Tsing Hua University and the Ministry of Education, Taiwan, R.O.C. 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. ☯ These authors contributed equally to this work. mutations identified in genes encoding ion channels (Voltage- Gated Sodium Channel Subunit Alpha (SCN5A), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 4 (HCN4), Potassium Inwardly-Rectifying Channel Subfamily J Member 2 (KCNJ2)), cardiac transcription factors (Homeobox protein Nkx-2.5 (NKX2-5), T-box transcription factor (TBX5)), gap junctions (Connexin 40 (Cx40)), energy metabolism regulators (5'-AMP-activated protein kinase subunit gamma-2 (PRKAG2)) and structural proteins (lamin A/C (LMNA)) that cause progressive conduction system disease in the absence of structural heart disease [1,2]. Inherited forms of CCD are rare, but each new mutation provides helpful insights into the molecular mechanisms Whole-Exome Sequencing to Identify a Novel LMNA Gene Mutation Associated with Inherited Cardiac Conduction Disease Chun-Chi Lai1☯, Yung-Hsin Yeh2☯, Wen-Ping Hsieh3, Chi-Tai Kuo2, Wen-Ching Wang4,5, Chia-Han Chu5, Chiu-Lien Hung4,5, Chia-Yang Cheng1,5, Hsin-Yi Tsai2, Jia-Lin Lee4, Chuan-Yi Tang1, Lung-An Hsu2* 1 Department of Computer Science, National Tsing Hua University, Hsinchu, Taiwan, 2 First Cardiovascular Division, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Tao-Yuan, Taiwan, 3 Institute of Statistics, National Tsing Hua University, Hsinchu, Taiwan, 4 Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing-Hua University, Hsinchu, Taiwan, 5 Biomedical Science and Engineering Center, National Tsing Hua University, Hsinchu, Taiwan Abstract Background: Inherited cardiac conduction diseases (CCD) are rare but are caused by mutations in a myriad of genes. Recently, whole-exome sequencing has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases. Objective: To investigate the genetic background of a family affected by inherited CCD. Methods and Results: We used whole-exome sequencing to study a Chinese family with multiple family members affected by CCD. Using the pedigree information, we proposed a heterozygous missense mutation (c.G695T, Gly232Val) in the lamin A/C (LMNA) gene as a candidate mutation for susceptibility to CCD in this family. The mutation is novel and is expected to affect the conformation of the coiled-coil rod domain of LMNA according to a structural model prediction. Its pathogenicity in lamina instability was further verified by expressing the mutation in a cellular model. Conclusions: Our results suggest that whole-exome sequencing is a feasible approach to identifying the candidate genes underlying inherited conduction diseases. Citation: Lai C-C, Yeh Y-H, Hsieh W-P, Kuo C-T, Wang W-C, et al. (2013) Whole-Exome Sequencing to Identify a Novel LMNA Gene Mutation Associated with Inherited Cardiac Conduction Disease. PLoS ONE 8(12): e83322. doi:10.1371/journal.pone.0083322 Editor: Andrew Dewan, Yale School of Public Health, United States of America Copyright: © 2013 Lai 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: © 2013 Lai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by research grants from the Chang Gung Memorial Hospital (CMRPG 300121 and CMRPG 3B0991), the National Science Council (NSC101-2325-B-007-001), and the "Aim for the Top University Project" of the National Tsing Hua University and the Ministry of Education, Taiwan, R.O.C. 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. Introduction The functional components of the cardiac conduction system can be broadly divided into the impulse-generating nodes and the impulse-propagating His-Purkinje system. In cardiac conduction disease (CCD), the integrity of the conduction system is impaired such that impulse generation, impulse propagation, or both will be slowed or even blocked and life- threatening rhythm disturbances may ensue. CCD may result from acquired injury such as ischemia or drug toxicity, be associated with heart diseases such as congenital defects or cardiomyopathy, be associated with neuromuscular disease, or rarely be an isolated finding [1,2]. The familial clustering of idiopathic CCD has led to the discovery of a large number of PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 1 1 Exome Sequencing in Inherited Conduction Disease Figure 1. Novel LMNA mutation (G232V) segregates with inherited cardiac conduction disease. (A) Pedigree of the studied family. The pedigree shows the affection statuses, individual identifiers, and genotypes at LMNA c.G695T. The samples (marked by arrows) I-3, II-2, II-3, II-4 and III-1 were exome sequenced. The I-1, I-3 and II-1 samples were from healthy individuals, and the phenotype of sample III-1 was uncertain. (B) Sequencing result showing the heterozygous LMNA c.G695T (G232V) mutation. WT, wild type allele; MT, mutant allele. (C) Schematic of Lamin A protein. G232V mutation in the 1B−2 linker region. doi: 10.1371/journal.pone.0083322.g001 Figure 1. Novel LMNA mutation (G232V) segregates with inherited cardiac conduction disease. (A) Pedigree of the studied family. The pedigree shows the affection statuses, individual identifiers, and genotypes at LMNA c.G695T. The samples (marked by arrows) I-3, II-2, II-3, II-4 and III-1 were exome sequenced. The I-1, I-3 and II-1 samples were from healthy individuals, and the phenotype of sample III-1 was uncertain. (B) Sequencing result showing the heterozygous LMNA c.G695T (G232V) mutation. WT, wild type allele; MT, mutant allele. (C) Schematic of Lamin A protein. G232V mutation in the 1B−2 linker region. doi: 10.1371/journal.pone.0083322.g001 were conducted in concordance with the Declaration of Helsinki Principles. Written informed consent was obtained from each subject. controlling cardiac conduction system development and function [3]. Clinical genetic testing can help identify patients at risk for CCD before its manifestation. However, genetic testing techniques, knowledge about the candidate genes and the size of the pedigrees may compromise the ability to uncover relevant genetic defects. Introduction The analysis is often restricted to previously identified candidate genes and may fail to uncover previously unidentified causal genes for familial CCD. Linkage analysis may be compromised by small pedigree sizes. Therefore, next-generation whole-genome or exome sequencing may help overcome these limitations. Clinical Assessment Clinical data, including medical history, physical examination, 12-lead electrocardiogram (ECG), 24-hour Holter recordings and echocardiographic data on the study family, were collected and evaluated (Table 1). All affected family members (the father and 3 siblings; I-2, II-2, II-3 and II-4) presented with a combination of at least two of the following phenotypes: sinus nodal dysfunction, paroxysmal atrial fibrillation, or atrioventricular conduction block. Three affected family members (the father and 2 brothers; I-2, II-2, and II-3) had permanently implanted pacemakers. The diagnosis of sinus node dysfunction was also confirmed by an electrophysiology study in two affected members (II-3 and II-4). Sinus node Study Population Genomic DNA was prepared from the venous blood of a variety of affected and unaffected individuals following standard procedures. The first set of analyses was performed on a Chinese family with inherited CCD (Family TS; pedigree in Figure 1A). CCD was characterized by early onset, symptomatic arrhythmia, including a long pause after atrial fibrillation and a combination of sino-atrial and atrioventricular nodal conduction blocks, which led to pacemaker implantation between the ages of 44 and 64. The study family consisted of one affected father, three affected siblings, one unaffected mother, one unaffected uncle and a grandson with an uncertain phenotype. The validation study included a cohort of 252 additional unrelated controls recruited from a population receiving routine health examinations and a population receiving regular hypertension treatment in an outpatient clinic. Among them, 137 subjects displayed sinus rhythm and 115 subjects displayed atrial fibrillation. Many of the recent studies analyzed variant data from exome sequencing to identify causal genes for Mendelian diseases [4-8]. Exome sequencing is a powerful tool for characterizing DNA sequences surrounding target regions at a much lower cost compared to the whole-genome sequencing technique [9]. In some cases, researchers have analyzed exome sequencing data from only one patient [4,5], whereas most studies have used exome sequencing data from several members of an afflicted family [6-8]. Some studies have summarized the computational workflow from those reports [10,11]. In this study, we investigated a Chinese family with inherited CCD that was characterized by sinus arrest, atrial fibrillation and atrioventricular conduction disturbance. Using emerging whole- exome sequencing techniques, we identified a novel point mutation located in LMNA, which encodes the inner nuclear membrane protein lamin A/C. Structural modeling predicts that this mutation may affect LMNA function. Cellular modeling further verifies its pathogenicity in lamina stability. Ethics Statement The protocols were approved by the Human Research Ethics Committee at Chang Gung Memorial Hospital (Chang Gung Medical Foundation Institutional Review Board 99-2468B) and PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 2 Exome Sequencing in Inherited Conduction Disease Table 1. Demographic and phenotypic information on the study family. Table 1. Demographic and phenotypic information on the study family. Table 1. Demographic and phenotypic information on the study family. Sex Conduction defect Paf Age at echo LV EF, % LA, mm LV, mm Age at death Age at PPM I-1 M No No 85 66 36 56 I-2 M SND, 2nd-degree AVB yes 64 76 35 41 78 64 I-3 F No No 73 58 34 34 II-2 M SND, 3rd-degree AVB yes 48 70 34 49 44 II-3 M SND, 1st- & 2nd-degree AVB yes 45 69 36 48 48 II-4 F SND yes 44 74 29 40 III-1 M No No 31 77 36 48 Paf, paroxysmal atrial fibrillation; SND, sinus node dysfunction; AVB, atrioventricular block; PPM, permanent pacemaker implantation doi: 10.1371/journal.pone.0083322.t001 to Binary Alignment and Mapping files (BAM) using SAMtools [13]. Picard (http://picard.sourceforge.net/) was used to mark and remove the polymerase chain reaction (PCR) duplicates detected from the BAM files. The Genome Analysis Toolkit (GATK) [14] was then used for base quality recalibration and local realignment around the potential insertion/deletion (Indel) sites. The UnifiedGenotyper in the GATK was used in the final step for variant calling using the Bayesian model. The loci with more than 3 alleles were filtered, and the bi-allelic variants matching any of the three inheritance models in the subsequent step were reserved to pass through the workflow. dysfunction was defined as sinus bradycardia, sino-atrial exit block and/or sinus pause or arrest. The echocardiographic findings from the 4 affected family members showed normal left and right ventricular sizes and contractile function (LV ejection fraction >50%). None of the findings indicated significant valvular disease. The affected father died from a stroke at the age of 78 and was screened for a mutation in the SCN5A gene before this study. He did not carry any mutations in the entire protein-coding region or the intron/exon boundaries of the SCN5A gene, and his DNA was not available for further exome sequencing. Exome Sequencing Three micrograms of DNA from each of the 5 family members (the 3 affected siblings, the healthy mother and the grandson) were subjected to exome enrichment with the TruSeq Exome Enrichment Kit, according to the protocol provided by the manufacturer. The Illumina TruSeq Enrichment kit was designed to target 201,121 exons within the 20,794 human gene regions. The kit allowed us to target 62.1 million bases of the human genome and enabled the capture of 97.2% of the exons defined by the consensus coding sequence (CCDS). Enriched DNA libraries were then sequenced on an Illumina Genome Analyzer IIx at the Biomedical Science and Engineering Center, National Tsing Hua University, Hsinchu, Taiwan. The original data files have been deposited in the NIH Short Read Archive (http://www.ncbi.nlm.nih.gov/sra) under the accession number SRP030761. Matching Genotypes with Pedigree Analysis Figure 2A illustrates the workflow for the identification of candidate variants. The first filtering step was performed to infer the genotypes of each family member from the pedigree according to the possible inheritance models (Figure S1). We filtered out the variants that did not match any reasonable inheritance pattern. One of the affected members (sample II-4) was female, so the Y chromosome was excluded. The mitochondrial chromosome was excluded because mitochondria are transmitted almost exclusively through the mother. X-linked dominant inheritance was also excluded by the male-to-male transmission. Although autosomal dominant inheritance is the most likely transmission model, we also analyzed variants matching the criteria for the autosomal recessive and X-linked recessive inheritance models. Ethics Statement The grandson, whose phenotype was not established, was enrolled at the age of 31 and did not show abnormal findings in a 12-lead ECG, 24-hour Holter recordings, or echocardiography examination. The unaffected uncle was enrolled at the age of 85 and did not show abnormal findings in a 12-lead ECG and echocardiography examination. Annotation Analysis Variants that passed the pedigree analysis were annotated using ANNOVAR [15] for function (exonic or splicing); gene; exonic function (synonymous, nonsynonymous, stop gain, nonframeshift or frameshift indel); amino acid change; conservation; allele frequency in ESP 5400 Exome Project; dbSNP (version 132) reference number; allele frequency in 1000 Genomes Project (2012 February version); dbNSFP [16] functional prediction scores that integrated and normalized scores for PhyloP, SIFT, Polyphen2, LRT and GERP++; and chromosome position. ANNOVAR processed the combined variants with the three transmission models. The first step in the pipeline was to extract and annotate single nucleotide variants (SNVs) in the exonic regions and then to exclude the synonymous variants (Figure 2B). ANNOVAR then selected the Sequence Alignment and Variant Calling To maximize the information retrieved and improve confidence in the results, we used the Burrows-Wheeler Alignment tool (BWA) [12] as a second alignment tool in addition to the Consensus Assessment of Sequence and Variation (CASAVA) default tool for the sequencing system. The poor quality short reads were filtered out and aligned to the human reference genome (NCBI Build 37/hg19). The Sequence Alignment and Mapping (SAM) files were converted PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 3 Exome Sequencing in Inherited Conduction Disease Figure 2. Workflow and annotation pipeline for the identification of candidate variants. (A) Workflow for finding candidate variants. (B) The number of SNV and Indel variants from GATK-called data sorted following the autosomal dominant model through the ANNOVAR pipeline. There were 52 SNV and 2 Indel variants remaining, and each variant was located in different genes. doi: 10.1371/journal.pone.0083322.g002 Figure 2. Workflow and annotation pipeline for the identification of candidate variants. (A) Workflow for finding candidate variants. (B) The number of SNV and Indel variants from GATK-called data sorted following the autosomal dominant model through the ANNOVAR pipeline. There were 52 SNV and 2 Indel variants remaining, and each variant was located in different genes. doi: 10.1371/journal.pone.0083322.g002 (residues 81−383) were built from the generated MSA profile using Modeller [24]. variants located in the genomic regions that are highly conserved among 46 vertebrate model organisms. The SNVs and Indels that appeared in the segmental duplication regions, dbSNP and the 1000 Genomes Project were subsequently filtered out. We used a maximal minor allele frequency (MAF) <0.05 filter for the 1000 Genome project because our target was a rare variant. The effects of the remaining nonsynonymous SNVs on protein function were then predicted using dbNSFP [16] and SIFT 2013 (updated version 5.0) [17]. LMNA wild-type/mutation Structural Modeling Homology detection and structural prediction by HMM-HMM comparison (HHpred) [18] was employed to build the structural models of wild-type (WT) and Gly232Val lamin A/C (LMNA) based on the crystal structures of intermediate filaments (tropomyosin from Sus scrofa, PDB ID: 1C1G [19]; human lamin coil 2B, PDB ID: 1X8Y [20]; human vimentin coil 2B fragment, PDB ID: 1GK4 [21] and human vimentin coil 2 fragment, PDB ID: 3TRT [22]). HHblits [23], a HMM-HMM- based lightning-fast iterative sequence search tool in the HHpred suite, was used to generate a multiple sequence alignment (MSA) profile between the templates and WT/mutant with a maximum of three MSA generation iterations. The structural models of WT LMNA and the G232V mutant Expression Analysis To characterize the consequences of the LMNA mutation at the cellular level, transient cell transfections were performed in HL-1 atrial cardiomyocytes with WT or mutated lamin A (NM_170707.3) and lamin C (NM_005572.3) mRNA expressed as fusions to the C-terminus of green fluorescent protein ( pCMV6-AC-GFP, RG204970 and RG201809 for Lamin A and Lamin C repectively, OriGene Technologies). Mutations were introduced via site-directed mutagenesis (Q5®Site-directed mutagenesis kit, New England Biolabs) with the forward primer 5′-GTGGAGATTGACAATGTGAAGCAGCGTGAGTTTG-3′ and the reverse primer 5′- CAAACTCACGCTGCTTCACATTGTCAATCTCCAC-3′. All of the inserts were systematically verified by sequencing. HL-1 myocytes, a cell line derived from atria of adult mice, were cultured in Claycomb medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mmol/L L-glutamine and 0.1 mmol/L norepinephrine and incubated at 37°C with 5% CO2. The transfection was performed by incubating 2 μg of fusion protein construct using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were grown for 48 hours. HL-1 images were captured using a Leica confocal laser-scanning microscope (Leica TCS SP2, Wetzlar, Germany). To characterize the consequences of the LMNA mutation at the cellular level, transient cell transfections were performed in HL-1 atrial cardiomyocytes with WT or mutated lamin A (NM_170707.3) and lamin C (NM_005572.3) mRNA expressed as fusions to the C-terminus of green fluorescent protein ( pCMV6-AC-GFP, RG204970 and RG201809 for Lamin A and Lamin C repectively, OriGene Technologies). Mutations were introduced via site-directed mutagenesis (Q5®Site-directed mutagenesis kit, New England Biolabs) with the forward primer 5′-GTGGAGATTGACAATGTGAAGCAGCGTGAGTTTG-3′ and the reverse primer 5′- Mutation Validation All of the candidate variants were reconfirmed by Sanger sequencing with the new PCR product for all 3 affected siblings, the healthy uncle, mother and grandson. Oligonucleotide primers were generated to amplify the fragments of genomic DNA containing identified variants (Table S1). The novel variants that were validated in the affected members were then examined in at least 100 unrelated healthy persons with normal phenotypes to exclude the possibility of polymorphisms. The genotyping was performed by PCR and restriction enzyme digestion, as described in Table S1. CAAACTCACGCTGCTTCACATTGTCAATCTCCAC-3′. All of the inserts were systematically verified by sequencing. HL-1 myocytes, a cell line derived from atria of adult mice, were cultured in Claycomb medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mmol/L L-glutamine and 0.1 mmol/L norepinephrine and incubated at 37°C with 5% CO2. The transfection was performed by incubating 2 μg of fusion protein construct using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were grown for 48 hours. HL-1 images were captured using a Leica confocal laser-scanning microscope (Leica TCS SP2, Wetzlar, Germany). Candidate Genes The 2 remaining Indels were located in the chondroitin polymerizing factor 2 (CHPF2) and hephaestin-like 1 (HEPHL1) genes (Table S2). According to the Online Mendelian Inheritance in Man (OMIM) and PubMed databases, neither gene has been linked to heart disease. Of the 52 remaining SNVs (Table S3), we filtered out another 33 variants that had been reported in the 1000 Genomes Project (MAF <0.05) and the ESP5400 Exome Project and were considered known variants. Nineteen candidate variants remained and were entered into function prediction programs. Among them, only the LMNA mutation (c.G695T, p.Gly232Val, Figures 1B and 1C) was consistently predicted to be a deleterious mutation across both the dbNSFP [16] and SIFT 2013 programs (Table 4). Exome Sequencing, Sequence Alignment and Variant Calling We listed the number of short reads mapped to the reference genome (NCBI Build 37, hg19) for each sample in Table 2. Approximately 9.36 Gb of average sequence was generated per sample as 76-bp paired-end reads. Although only PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 4 Exome Sequencing in Inherited Conduction Disease Table 2. Number of reads, variants and variant coverage by sample. Total reads number Average read depth outside the target regions Average read depth in the target regions Mapping ratio outside the target regions Mapping ratio in the target regions Unmapped reads ratio I-3 137213408 11.92 72.40 44.23% 48.96% 6.81% II-2 141576846 12.37 78.61 41.65% 52.07% 6.28% II-3 98710290 10.64 48.02 47.44% 45.22% 7.34% II-4 130819190 12.26 68.47 45.41% 48.49% 6.10% III-1 107741382 10.83 55.96 44.67% 48.29% 7.04% doi: 10.1371/journal.pone.0083322.t002 Table 3. Number of combined SNVs and Indels remaining after each filtering step. Step 0 Step 1 Step 2 GATK calling Inheritance model filtering ANNOVAR filtering SNVs 381264 Autosomal Dominant SNVs 15875 SNVs 52 Indels 34937 Indels 1228 Indels 2 Autosomal Recessive SNVs 2070 SNVs 0 Indels 195 Indels 0 X-linked Recessive SNVs 2 SNVs 0 Indels 2 Indels 0 doi: 10.1371/journal.pone.0083322.t003 Table 3. Number of combined SNVs and Indels remaining after each filtering step. doi: 10.1371/journal.pone.0083322.t003 each step of the ANNOVAR automatic pipeline are shown in Figure 2B. Only 52 SNVs and 2 Indels that matched the autosomal dominant model passed through the ANNOVAR pipeline (Table 3). In contrast, all of the variants sorted by the autosomal recessive or X-linked recessive models were excluded after ANNOVAR filtering (Table 3). approximately 50% of the mapped reads were aligned to the target regions encompassing the exons, the average read depths in the target regions were much higher than those outside of the target regions. After removing the PCR- duplicated reads, 2.58 Gb of average sequence per sample mapped to the targeted exome sequences with a mean depth of 42.78-fold. On average, 87.02% of the exome was covered at least 10-fold. Overall, we identified approximately 381,264 SNVs and 34,937 Indels from 5 study samples (150,000 to 180,000 variants per subject) (Table 2). Variant filtration and prioritization according to inheritance models Our first selection strategy was to find variants that matched possible inheritance models. When a genotyped variant from the 5 studied family members did not fit the assumed inheritance models, this gene was excluded from further investigation. As shown in Table 3, 15,875 SNVs and 1,228 Indels fit the autosomal dominant inheritance pattern. Only 2,070 SNVs and 195 Indels and 2 SNVs and 2 Indels fit the autosomal recessive and X-linked recessive inheritance patterns, respectively (Table 3). Annotation Analysis doi: 10.1371/journal.pone.0083322.t004 N, not conserved; C, conserved; D, deleterious; T, tolerated; P, possible damage; B, benign; &N, neutral; #N, polymorphism; N/A, not applicable; Table S3 shows the detailed dbNSFP [16] functional prediction scores for each SNV. doi: 10.1371/journal.pone.0083322.t004 Table 5. Candidate SNVs for a causal mutation in the family. Gene Name LMNA MMP2 ZFHX3 ANGPTL5 SNV position Chromosome 1, 156104651 Chromosome 16, 55519584 Chromosome 16, 72830334 Chromosome 11, 101773401 Variants c.G695T, p.G232V c.G577A, p.E193K c.G3505A, p.V1169M c.C491T, p.P164L III-1 genotype GG GA GA CC doi: 10.1371/journal.pone.0083322.t005 Table 5. Candidate SNVs for a causal mutation in the family. 5 (ANGPTL5), Myosin Heavy Chain 7B (MYH7B) and Potassium voltage-gated channel subfamily H member 6 (KCNH6) have been previously linked to heart disease. Although the intronic splicing site mutation in the SACM1L gene has not been linked to heart disease, its effect on protein function could not be predicted by either dbNSFP or SIFT 2013. Therefore, we conducted direct sequencing validation for these 7 potential candidates in all 3 affected siblings, the mother, the uncle, the grandson and the 100 unrelated healthy controls. The MYH7B variant (Chr20:33588956 G/A) was present in 2 of the 100 controls and was thus considered a polymorphism. The LMNA, ZFHX3, MMP2 and ANGPTL5 SNVs were all novel and family-specific mutations (Table 5). The LMNA mutation was not present in the healthy mother, uncle and grandson. However, both of the ZFHX3 and MMP2 mutations were also present in the grandson, whose phenotype was not established and is most likely unaffected. Furthermore, the other three novel mutations, excluding LMNA, were predicted to be benign mutations. The LMNA gene remained the most likely candidate gene for CCD requiring further investigation. Additionally, both the KCNH6 and SACM1L gene mutations were not validated by direct sequencing and were thus categorized as calling errors. Annotation Analysis All of the fitted variants were then subjected to ANNOVAR [15] for annotation. The ANNOVAR automatic pipeline filtered out most of the synonymous SNVs that were considered neutral variants and the nonsynonymous SNVs that were reported in the dbSNP135 and 1000 Genomes Project (MAF >0.05) databases. The variant numbers following the autosomal dominant inheritance model and remaining after All of the 19 candidate variants, except the SAC1 Suppressor of Actin Mutations 1-Like (SACM1L) gene mutation, were missense mutations. We searched for potential links between the 19 candidate genes and cardiovascular diseases using the OMIM and PubMed databases. Six candidate genes including LMNA, Zinc Finger Homeobox 3 (ZFHX3), Matrix Metallopeptidase 2 (MMP2), Angiopoietin-Like December 2013 \| Volume 8 \| Issue 12 \| e83322 PLOS ONE \| www.plosone.org 5 Exome Sequencing in Inherited Conduction Disease Table 4. Family-specific variants sorted by autosomal dominant inheritance and annotated by ANNOVAR. SNV PhyloP SIFT PolyPhen LRT& MutationTaster# GERP++ SIFT2013 SLC39A1 C D P D D C T LMNA C D D D D C D SACM1L † N/A N/A N/A N/A N/A C N/A ADH4 N D D N D C D MFSD8 N T P N D N T DLK2 C D D D N C T MDN1 C T B D N C T ODZ4 N/A N/A N/A N/A N/A C B ANGPTL5 C T D D D C T L2HGDH C T P D D C T ZNF646 C T D N/A N/A C T LONP2 C T D D D C T MMP2 C T P D D C T ZFHX3 C N/A P D D C T MVD C D B D D C T KCNH6 C T D D D C T USF2 C T B N N C T MYH7B C N/A N/A N N C T ARHGAP40 N/A N/A N/A N/A N/A C T †. SACM1L intronic splicing site mutation; N, not conserved; C, conserved; D, deleterious; T, tolerated; P, possible damage; B, benign; &N, neutral; #N, polymorphism; N/A, not applicable; Table S3 shows the detailed dbNSFP [16] functional prediction scores for each SNV. doi: 10.1371/journal.pone.0083322.t004 † p g ; N, not conserved; C, conserved; D, deleterious; T, tolerated; P, possible damage; B, benign; &N, neutral; #N, polymorphism; N/A, not applicable; Table S3 shows the detailed dbNSFP [16] functional prediction scores for each SNV. LMNA wild-type/mutation Structural Modeling Intermediate filament structures are characterized as a central alpha-helical coiled-coil rod domain consisting of three alpha-helical segments (1A, 1B and 2) bridged by two linker regions [25]. Notably, the G232V mutation in LMNA is located in the 1B−2 linker region, also referred to as linker 2, and is hypothesized to play a crucial rule in stabilizing the assembly of intermediate filaments [4,25]. Therefore, we sought to build a structural model containing this site. To accomplish this, we utilized HHpred to search for the structures with the highest sequence identity across this region: tropomyosin from Sus scrofa (PDB ID: 1C1G) [19], human lamin coil 2B (PDB ID: 1X8Y) [20], human vimentin coil 2B fragment (PDB ID: 1GK4) [21] and human vimentin coil 2 fragment (PDB ID: 3TRT) [22]. Structural models of LMNA were built using Modeller [24] with the HHblits-generated parameters. Glycine 232 is located at PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 6 Exome Sequencing in Inherited Conduction Disease Figure 3. Structural models of LMNA. (A) The model of wild type LMNA (residues 81−383) was built using the HHblits-generated parameters from Modeller. (B) The G232V LMNA mutant model. (C) Higher magnification of (A). (D) Higher magnification of (B). G232 and V232 are shown as sticks. Modeling visualization was performed using PyMol (http://www.pymol.org). doi: 10.1371/journal.pone.0083322.g003 Figure 3. Structural models of LMNA. (A) The model of wild type LMNA (residues 81−383) was built using the HHblits-generated parameters from Modeller. (B) The G232V LMNA mutant model. (C) Higher magnification of (A). (D) Higher magnification of (B). G232 and V232 are shown as sticks. Modeling visualization was performed using PyMol (http://www.pymol.org). doi: 10.1371/journal.pone.0083322.g003 Figure 4. Expression of lamin A/C in cultured atrial cardiomyocytes. HL-1 atrial cardiomyocytes transfected with wild-type lamin A (A) and lamin C (B) in fluorescent expression vectors, showing the cell nucleus with homogenous nuclear veil. HL-1 atrial cardiomyocytes transfected with p.G232V mutant lamin A (C) and lamin C (D), showing the cell nucleus with aggregates. doi: 10.1371/journal.pone.0083322.g004 Figure 4. Expression of lamin A/C in cultured atrial cardiomyocytes. HL-1 atrial cardiomyocytes transfected with wild-type lamin A (A) and lamin C (B) in fluorescent expression vectors, showing the cell nucleus with homogenous nuclear veil. HL-1 atrial cardiomyocytes transfected with p.G232V mutant lamin A (C) and lamin C (D), showing the cell nucleus with aggregates. doi: 10.1371/journal.pone.0083322.g004 the tip of a loop in WT LMNA (Figure 3). LMNA wild-type/mutation Structural Modeling Interestingly, a glycine-to-valine substitution at this site led to a dramatic conformational change, possibly because of the large valine side chain. Subsequently, this mutant form may assemble more loosely as a dimer or assemble into a dimer of a different configuration, which hinders the subsequent formation of the coiled-coil rod domain. LMNA gene as the most likely candidate mutation. The ANNOVAR program predicted that this mutation would be damaging, and the HHpred software predicted that it would affect the conformation of the LMNA coiled-coil rod domain. We further verified its pathogenicity by expressing the mutation in a cellular model. Subsequently, we confirmed that the mutation showed a complete cosegregation with the disease phenotype in the family; it was carried by all three affected individuals and was absent in the three healthy individuals. We further confirmed the absence of the mutation in 137 additional healthy controls and 115 patients with atrial fibrillation. Expression of the p.Gly232Val Mutation in the HL-1 Cell Line Lamin A and lamin C constructs were transfected separately into HL-1 cells. As shown on Figure 4, cells transfected with WT constructs (Figure 4A for lamin A; Figure 4B for lamin C) displayed fluorescence homogeneously distributed at the inner nuclear lamina. After transfection of the p.Gly232Val mutated lamin A and lamin C constructs (Figure 4C for lamin A; Figure 4D for lamin C), we observed characteristic aggregates indicating an uneven distribution of lamin protein within the nucleus. More than 450 different mutations have been identified in the LMNA gene that can cause a variety of diseases [26], called laminopathies, and have been categorized into 4 major types of disorders: striated and cardiac muscle diseases, lipodystrophy syndromes, peripheral neuropathy and premature aging. Since Bonne et al. identified the first LMNA gene mutation in patients affected with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM) and conduction defects in 1999 [27], mutations in LMNA have subsequently been identified in individuals with isolated DCM, usually with a conduction defect [28]. Atrial or ventricular arrhythmias are often present and even precede the onset of DCM [28-33]. The heterogeneity of LMNA mutations was further established by the lack of clear genotype-phenotype Exome Sequencing in Inherited Conduction Disease Exome Sequencing in Inherited Conduction Disease correlations [26,34]. Interestingly, the major phenotype of our study family was isolated CCD characterized by extensive atrial disease, tachy-brady arrhythmias and an atrioventricular conduction block. Similar to a previous observation that isolated cardiac involvement was predominantly found in patients with LMNA mutations located in the a-helical rod domain [28], none of the affected family members presented with muscular dystrophy or other organ system involvement. However, none of them had evidence of cardiomyopathy and LV dysfunction. There was also no history of sudden cardiac death in the family members. In contrast, there was a report of a patient with EDMD [35] whose mutation was identical to the one found in the study family except that the mutation in the patient was G to A instead of T and an amino acid change from glycine to glutamate instead of valine. However, the ECG and echocardiography of this patient at 11 years of age was normal in that report. More recently, another study reported a patient with EDMD whose mutation (c.694GC) also affected the codon 232 and changed glycine to arginine. Interestingly, this patient had DCM, right bundle branch block and ventricular arrhythmia at 18 years of age in that report [36]. Thus, the clinical manifestations of LMNA mutations are complex and most likely reflect the interaction of background genetic modifiers and environmental factors for the development of the phenotype. mutation in the SCN5A gene. With the development of whole- exome sequencing, we could obviate the extensive laboratory work and expenses incurred by subsequent targeted sequencing of many previously identified candidate genes. However, the main challenge of using exome sequencing to find novel disease genes for our study was distinguishing between disease-related alleles and non-pathogenic polymorphisms and sequencing errors. For Mendelian disorders, the use of pedigree information can substantially narrow the search. Interestingly, subsequent discrete filtering indicated the autosomal dominant inheritance of the trait. Although the discrete filtering method we adopted has been well-established and used successfully to identify disease- causing mutations, these filtering strategies do not remove false positive variants that cannot be logically eliminated by filtering alone [10]. This limitation could be overcome if linkage mapping data were available. Furthermore, the Holter recording and echocardiography studies on the young grandson, 31 years of age, whose phenotype was still not established, were normal. Exome Sequencing in Inherited Conduction Disease Although 92% of carriers with 30+ years of age had conduction disease, including atrioventricular conduction disturbances, atrial arrhythmias and ventricular arrhythmias in a meta-analysis of 299 patients with LMNA mutations [41], the possibility of a false-positive call for LMNA G232V still cannot be completely ruled out. Moreover, genetic heterogeneity has been observed in inherited CCD. False negative calls might occur in the regions of genes such as SCN5A, HCN4, KCNJ2, NKX2-5, TBX5, Cx40, PRKAG2 and others that are known to be involved in CCD if the depth and quality of reads were insufficient to confidently discover a variant at that site. Finally, technical failure could occur when causative mutations occur in coding sequences outside the target region, such as in the intronic or regulatory regions or outside the gene. The pathogenic mechanism underlying the association between the LMNA mutations and laminopathies remains unclear. Our structural model prediction suggested a dominant negative effect resulting from the G232V mutation. The structural modeling of the LMNA mutation showed a conformational change in the linker 2 region, which may destabilize the rod of the natural homodimer. The linker 2 region of LMNA is homologous to the linker L12 regions in other intermediate filaments proteins [25]. One hypothesis [37] is that the conformational change alters the quaternary structure of intermediate filament proteins. The heterozygous LMNA mutation genotypes also suggest that the conformational change is caused by the formation of a heterodimer between the normal LMNA protein and the mutated protein. The pathogenicity of the G232V mutation predicted by the structure model was further supported by the fact that the transient transfection of the mutated cDNA in HL-1 atrial mocyteses leads to abnormal lamin aggregates in the nucleus and to the loss of internal nuclear lamin organization, which are in agreement with the findings of previous reports [38]. Previous autopsy studies revealed marked fibrosis and fatty metamorphosis of the sinoatrial and atrioventricular nodes as well as the atrioventricular bundle in patients with mutations in the LMNA gene and conduction defect [28,39]. Thus, more studies are needed to determine whether the G232V mutation could further cause lamina instability in vivo as well as subsequent cell apoptosis in the cardiac conduction system or the loss of lamin-dependent gene regulatory function that was previously reported in some LMNA missense mutations [40]. Discussion We used whole-exome sequencing to evaluate a family with autosomal dominant CCD and identified a heterozygous missense mutation (NM_005572, c.G695T, G232V) in the 7 7 December 2013 \| Volume 8 \| Issue 12 \| e83322 PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 References 1. Wolf CM, Berul CI (2006) Inherited conduction system abnormalities-- one group of diseases, many genes. J Cardiovasc Electrophysiol 17: 446–455. doi:10.1111/j.1540-8167.2006.00427.x. PubMed: 16643374. 1. Wolf CM, Berul CI (2006) Inherited conduction system abnormalities-- one group of diseases, many genes. J Cardiovasc Electrophysiol 17: 446–455. doi:10.1111/j.1540-8167.2006.00427.x. PubMed: 16643374. 11. Stitziel NO, Kiezun A, Sunyaev S (2011) Computational and statistical approaches to analyzing variants identified by exome sequencing. Genome Biol 12: 227. doi:10.1186/gb-2011-12-9-227. PubMed: 21920052. j 2. Smits JP, Veldkamp MW, Wilde AA (2005) Mechanisms of inherited cardiac conduction disease. Europace 7: 122–137. doi:10.1093/ europace/7.s1.122-b. PubMed: 15763526. j 2. Smits JP, Veldkamp MW, Wilde AA (2005) Mechanisms of inherited cardiac conduction disease. Europace 7: 122–137. doi:10.1093/ europace/7.s1.122-b. PubMed: 15763526. 12. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26: 589–595. doi:10.1093/ bioinformatics/btp698. PubMed: 20080505. p 3. Park DS, Fishman GI (2011) The cardiac conduction system. Circulation 123: 904–915. doi:10.1161/CIRCULATIONAHA. 110.942284. PubMed: 21357845. p 3. Park DS, Fishman GI (2011) The cardiac conduction system. Circulation 123: 904–915. doi:10.1161/CIRCULATIONAHA. 110.942284. PubMed: 21357845. 13. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079. doi:10.1093/bioinformatics/btp352. PubMed: 19505943. 4. Gilissen C, Arts HH, Hoischen A, Spruijt L, Mans DA et al. (2010) Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87: 418–423. doi:10.1016/ j.ajhg.2010.08.004. PubMed: 20817137. 4. Gilissen C, Arts HH, Hoischen A, Spruijt L, Mans DA et al. (2010) Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87: 418–423. doi:10.1016/ j.ajhg.2010.08.004. PubMed: 20817137. 14. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20: 1297–1303. doi:10.1101/gr.107524.110. PubMed: 20644199. 5. Hoischen A, van Bon BW, Gilissen C, Arts P, van Lier B et al. (2010) De novo mutations of SETBP1 cause Schinzel-Giedion syndrome. Nat Genet 42: 483–485. doi:10.1038/ng.581. PubMed: 20436468. 15. Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38: e164. doi:10.1093/nar/gkq603. PubMed: 20601685. 6. Roach JC, Glusman G, Smit AF, Huff CD, Hubley R et al. (2010) Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science 328: 636–639. doi:10.1126/science.1186802. PubMed: 20220176. 16. Supporting Information Conceived and designed the experiments: LAH YHY JLL. Performed the experiments: CHC CLH CYC HYT. Analyzed the data: CCL WPH LAH. Contributed reagents/materials/analysis tools: CTK WCW CYT. Wrote the manuscript: CCL YHY LAH. Figure S1. Deduced genotypes in the pedigrees according to the possible inheritance models. “A” represents the dominant trait, and “a” represents the recessive trait. “XA” Exome Sequencing in Inherited Conduction Disease Exome Sequencing in Inherited Conduction Disease arguable relationship with the disease. However, there has been a lack of suitable research methods to study the molecular mechanisms linking cardiac laminopathy to electrophysiological abnormalities. We anticipate that future studies utilizing patient-specific induced pluripotent stem cells cardiomyocyts models will elucidate electrophysiological features of cardiac laminopathies. Second, the uncertain phenotype for the young grandson and the lack of genotypic information on the affected father throw into doubt the causal relationship between the LMNA mutation and the disease. Third, only mutations in genes previously linked to cardiovascular disease were considered. There is a possibility that the 13 genes with mutations (including 2 indels) not known as cardiovascular genes could potentially contribute to CCD in this family. Finally, replicating LMNA mutations in an independent family or a cohort of unrelated patients with CCD would improve the strength of the analysis. represents the dominant trait on chromosome X, and “Xa” represents the recessive trait on chromosome X. (A) Type I, autosomal dominant model. We considered the disease trait to be the dominant trait in the pedigree. (B) Type II, autosomal recessive model. We considered the disease trait to be the recessive trait in the pedigree. (C) Type IV: X-linked recessive model. We assumed that the disease trait was the recessive trait on the X chromosome in the pedigree. Exome Sequencing in Inherited Conduction Disease We did not attempt to perform linkage analysis on our study Rare monogenic diseases are of substantial interest because the identification of their genetic bases provides important knowledge about disease mechanisms, biological pathways and potential therapeutic targets. Our studies support the previous observations that cardiac manifestation of laminopathies is heterogeneous, and the diagnosis of laminopathy should be suspected in a patient with an early onset of atrial fibrillation, progressive conduction disease with or without DCM and a family history of sudden cardiac death. The identification of LMNA as the candidate gene underlying CCD in the study family enables presymptomatic genetic diagnosis and early disease management. The affected siblings should be monitored closely for the development of DCM and lethal arrhythmias. Additionally, identifying a familial mutation can be valuable in reproductive planning and in the identification of family members who are not carriers of the mutation and will thus not require lifelong surveillance. Even if the young grandson turns out to be affected, we still can use the ZFHX3 and MMP2 family-specific mutations as markers associated with the trait for future monitoring. We did not attempt to perform linkage analysis on our study family because of the small pedigree size. We conducted direct sequencing analysis on the SCN5A gene in the affected father prior to this study because the sick sinus syndrome 1 gene is the sodium channel SCN5A gene, and we did not find a causal Apart from the intrinsic limitations associated with a whole- exome sequencing study, our study has several other limitations. The main limitation is that our study was only analyzed in an in vitro functional manner and showed only an PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83322 8 Table S1. Primer sequences and restriction enzyme (RE) used in the analysis of novel variants. (DOCX) Table S1. Primer sequences and restriction enzyme (RE) used in the analysis of novel variants. (DOCX) Table S2. Indel variants that pass the filtering criteria and match to the autosomal dominant pedigree. (DOCX) In conclusion, we used whole-exome sequencing to evaluate a family with autosomal dominant CCD and proposed a missense mutation in the LMNA gene as the most likely candidate mutation. Our results implicated whole-exome sequencing as a feasible approach for the identification of candidate genes underlying inherited conduction diseases. Table S3. SNVs that pass the filtering criterions and match to the autosomal dominant pedigree. (DOCX) Exome Sequencing in Inherited Conduction Disease 21. 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Chernyatina AA, Nicolet S, Aebi U, Herrmann H, Strelkov SV (2012) Atomic structure of the vimentin central alpha-helical domain and its implications for intermediate filament assembly. Proc Natl Acad Sci U S A 109: 13620–13625. doi:10.1073/pnas.1206836109. PubMed: 22869704. 35. Bonne G, Mercuri E, Muchir A, Urtizberea A, Bécane H et al. (2000) Clinical and molecular genetic spectrum of autosomal dominant Emery‐ Dreifuss muscular dystrophy due to mutations of the lamin A/C gene. Ann Neurol 48: 170–180. doi:10.1002/1531-8249(200008)48:2. PubMed: 10939567. 26. Cattin M-E, Muchir A, Bonne G (2013) 'State-of-the-heart' of cardiac laminopathies. Curr Opin Cardiol 28: 297–304. doi:10.1097/HCO. 0b013e32835f0c79. PubMed: 23455585. 36. Rudenskaya GE, Polyakov AV, Tverskaya SM, Zaklyazminskaya EV, Chukhrova AL et al. (2008) Laminopathies in Russian families. Clin Genet 74: 127–133. doi:10.1111/j.1399-0004.2008.01045.x. PubMed: 18564364. 27. 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https://openalex.org/W1690650007	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-3-18	English	null	Conventional and molecular epidemiology of Tuberculosis in Manitoba	BMC infectious diseases	2,003	cc-by	6,716	BioMed Central BioMed Central BioMed Central Open Acce Research article Conventional and molecular epidemiology of Tuberculosis in Manitoba Kym S Blackwood1, Assaad Al-Azem2, Lawrence J Elliott2, Earl S Hershfield3 and Amin M Kabani1,3,4 Open Access Address: 1National Reference Center for Mycobacteriology, National Microbiology Laboratory, Health, Canada, Winnipeg, MB, Canada, 2Department of Community Health Sciences, University of Manitoba, Winnipeg, MB, Canada, 3Department of Medicine, University of Manitoba, Winnipeg, MB, Canada and 4Department of Clinical Microbiology, Health Sciences Center, Winnipeg, MB, Canada : Kym S Blackwood - kym_blackwood@hc-sc.gc.ca; Assaad Al-Azem - umalmoua@cc.umanitoba.ca; ll ll l b l hf ld h h b b Email: Kym S Blackwood* - kym_blackwood@hc-sc.gc.ca; Assaad Al-Azem - umalmoua@cc.umanitoba.ca; Lawrence J Elliott - elliottl@cc.UManitoba.CA; Earl S Hershfield - ehersh@ccu.umanitoba.ca; Amin M Kabani - amin_kabani@hc-sc.gc.ca * Corresponding author Lawrence J Elliott - elliottl@cc.UManitoba.CA; Earl S Hershfield - ehersh@ccu.umanitoba.ca; Amin M Kabani - amin_kabani@hc-sc.gc.ca * Corresponding author * Corresponding author Received: 11 June 2003 Accepted: 13 August 2003 This article is available from: http://www.biomedcentral.com/1471-2334/3/18 This article is available from: http://www.biomedcentral.com/1471-2334/3/18 © 2003 Blackwood et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. © 2003 Blackwood et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. © 2003 Blackwood et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistributio all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract Background: To describe the demographic and geographic distribution of tuberculosis (TB) in Manitoba, thus determining risk factors associated with clustering and higher incidence rates in distinct subpopulations. Methods: Data from the Manitoba TB Registry was compiled to generate a database on 855 patients with tuberculosis and their contacts from 1992–1999. Recovered isolates of M. tuberculosis were typed by IS6110 restriction fragment length polymorphisms. Bivariate and multivariate logistic regression models were used to identify risk factors involved in clustering. Results: A trend to clustering was observed among the Canadian-born treaty Aboriginal subgroup in contrast to the foreign-born. The dominant type, designated fingerprint type 1, accounts for 25.8% of total cases and 75.3% of treaty Aboriginal cases. Among type 1 patients residing in urban areas, 98.9% lived in Winnipeg. In rural areas, 92.8% lived on Aboriginal reserves. Statistical models revealed that significant risk factors for acquiring clustered tuberculosis are gender, age, ethnic origin and residence. Those at increased risk are: males (p < 0.05); those under age 65 (p < 0.01 for each age subgroup); treaty Aboriginals (p < 0.001), and those living on reserve land (p < 0.001). Conclusion: Molecular typing of isolates in conjunction with contact tracing data supports the notion of the largest ongoing transmission of a single strain of TB within the treaty-status population of Canada recorded to date. This data demonstrates the necessity of continued surveillance of countries with low prevalence of the disease in order to determine and target high- risk populations for concentrated prevention and control measures. bally, TB is considered the most common cause of death from a single infectious organism, Mycobacterium tubercu- losis [1–3]. Canadians have a long history of suffering Page 1 of 11 (page number not for citation purposes) Methods Setting tremendously from TB. In the 19th century, one fifth of Canadians were infected with this disease and large num- bers died. In the beginning of the 20th century, the mortal- ity rate among Aboriginal people of Saskatchewan and Alberta reached as high as 9,000 per 100,000 [4,5]. g Manitoba has a population of 1.14 million (Statistics Canada, 1996). 78.1% are Canadian-born (CB) non- treaty, 8.9% are treaty Aboriginals, and the remainder is foreign-born (FB) [12,13]. It is unknown how many Abo- riginal individuals have not claimed treaty status. All diag- nosed cases of tuberculosis in Manitoba are registered in the Manitoba Central Tuberculosis Registry (MCTR) at the Health Sciences Center (HSC) in Winnipeg. Case finding in Manitoba is done by clinical diagnosis of symptomatic patients and by contact tracing. Administered treatment is drug therapy of a combination of first line antituberculo- sis drugs: rifampin, isoniazid, pyrazinamide, ethambutol, and/or streptomycin. Direct observed therapy (DOT) ver- ifies a completed course of treatment. Ninety-five percent of patients under DOT complete treatment in Manitoba [8]. Improvements in socioeconomic conditions, public health services, drug therapy and medical surveillance in Canada markedly reduced the mortality rate [0.4 per 100,000 by 1987] and overall incidence rates (5.9 per 100,000 by 1998) [6]. However, certain groups in Canada are at higher risk for acquiring TB, such as the Aboriginal population (defined as status and non-status Indian, Metis, Inuit, and Innu), foreign-born individuals from countries with high prevalence rates of TB, the homeless and the HIV-infected population [6]. In 1998, the inci- dence rate among non-Aboriginal Canadians was 1.5 per 100,000; in contrast, the status Indian, total Aboriginal and foreign-born population had incidence rates of 35.4, 22.5 and 21.4 per 100,000 respectively [7]. Furthermore, the proportion of TB cases reported in Canada from the foreign-born population has increased, from 35% in 1980 to 64% in 1998 [7]. In an attempt to eliminate tuberculo- sis in Canada, the Tuberculosis Prevention and Control Unit of Health Canada implemented guidelines recom- mended by the World Health Organization in 1997 [6]. Despite adoption of these guidelines, incidence rates of TB are still high in certain geographic and demographic zones, making elimination of the disease by recom- mended time periods put forth by Health Canada an increasingly difficult goal [6,8]. Currently, incidence rates in Canada have leveled off and remain fairly constant due to these sustaining population subgroups. Background Background Tuberculosis (TB) has infected one-third of the world population and kills three million people each year. Glo- Page 1 of 11 (page number not for citation purposes) Page 1 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 http://www.biomedcentral.com/1471-2334/3/18 http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 Sample Data ll Data on all TB patients and identified contacts from Janu- ary 1, 1992 to December 31, 1999 was obtained from the MCTR. Data fields such as the patient's unique identifier number, date of birth, birthplace, sex, country of birth, postal code, origin (CB treaty / non-treaty or FB), date of diagnosis, case finding method, TB status (new, active or relapse), BCG status, year of landing (foreign patients), smear results, culture results, TB DNA fingerprint type, drug resistance, chest X-rays and cavity status were extracted from the patient records and the information was entered into a database. This data set did not include patient name, allowing data analysis while maintaining the patient confidentiality. Specimens were collected by designated medical staff at the Respiratory Clinic, HSC, and other health care centers in Manitoba. The collected samples were sent to the Clinical Microbiology Labora- tory at HSC for isolation and identification of M. tubercu- losis. The Accuprobe M. tuberculosis complex kit (Gen- Probe Incorporated, San Diego, CA, USA) was used to identify M. tuberculosis in the clinical samples. Samples were subsequently processed for DNA sub-typing accord- ing to the internationally standardized methodology of IS6110 restriction fragment length polymorphisms (RFLP) [14]. Procedures outlined by Small et. al. to mini- mize the possibility of cross-contamination [15] was strictly adhered to by staff at the HSC and no evidence of cross-contamination was observed. The majority (75%) of TB patients in Canada live in three provinces: Ontario, Quebec and British Columbia. Fur- thermore, 90% of all TB cases in the country attributed to the foreign-born population reside in these provinces [9]. In Manitoba however, the composition of TB patients is distinctly different: the majority of TB cases are among Canadian-born, with the highest incidence among treaty Aboriginals (48.4 per 100,000 overall, with rates as high as 496.3 per 100,000 in select communities [10,11]. These distinct epidemiological patterns merited a further examination. Consequently, in this study, both conven- tional in conjunction with molecular methods were used to describe the epidemiology of tuberculosis in Manitoba from 1992 to 1999. The objectives of the study were to describe subgroups within Manitoba at high risk for developing TB, and to define risk factors and possible rea- sons for the ongoing transmission in order to better target prevention and control programs. RFLP Analysis All gel pictures containing the IS6110 RFLP patterns were analyzed to determine clustering using Molecular Analyst® Software Fingerprinting version 1.12, BioRad, Hercules, CA, USA. Clustering establishes groups of patients from which isolates with identical or near identical fingerprints are found and are likely to be linked epidemiologically Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 Table 1: Bivariate logistic regression analysis of risk factors associated with clustering. Characteristic Incidence Clustered Non-Clustered Odds Ratio p value (person-year/100 000) n = 426 (%) n = 203 (%) (95%) CI Sex Female 8.4 167 (39.2) 102 (50.2) 1 (reference) Male 10.1 259 (60.8) 101 (49.8) 1.57 (1.12–2.19) 0.011 Age * 0 to 14 3.1 18 (4.2) 4 (2.0) 4.61 (1.39–16.84) 0.004 15 to 24 5.8 51 (12.0) 17 (8.4) 3.07 (1.58–6.03) 0.000 25 to 44 10.4 170 (39.9) 47 (23.1) 3.70 (2.33–5.89) 0.000 45 to 64 10.7 102 (23.9) 48 (23.6) 2.17 (1.35–3.52) 0.000 65 + 18.4 85 (20.0) 87 (42.9) 1 Origin Canadian-Born (non-treaty) 3.3 97 (22.8) 60 (29.6) 1 Canadian-Born (treaty) 48.4 254 (59.6) 27 (13.3) 5.82 (3.46–9.54) 0.000 Foreign-Born 20 75 (17.6) 116 (57.1) 0.340 (0.261–0.620) 0.000 Residence Winnipeg 9.7 235 (55.1) 153 (75.4) 1 other urban 4.1 14 (3.3) 11 (5.4) 0.829 (0.369–1.829) 0.809 reserves 33.7 148 (34.7) 15 (7.4) 6.424 (3.563– 10.947) 0.000 other rural 9.2 29 (6.9) 24 (11.8) 0.787 (0.443–1.392) 0.051 Site of Infection Extrapulmonary 111 (26.1) 66 (32.5) 1 Pulmonary 315 (73.9) 137 (67.5) 1.367 (0.951–1.969) 0.112 * χ2 test for overall linear trend in age = 32.6, p < 0.001 Table 1: Bivariate logistic regression analysis of risk factors associated with clustering. * χ2 test for overall linear trend in age = 32.6, p < 0.001 ate model (table 1) were gender, age (five subgroups), eth- nic origin (three subgroups), residence (four subgroups) and site of infection. A chi-squared test for linear trend was performed on the subgroups. Multivariate models using gender, age, ethnic origin, residence, site of infec- tion, status (new or relapse), drug resistance, BCG status, chest x-ray and cavity were analyzed and potential interac- tions were assessed for significance. Variables significant at the p < 0.05 level were kept in the final multivariate model, summarized in Table 2. [16]. RFLP Analysis Clustered cases were identified by searching each fin- gerprint against the created database containing all other patterns over the eight-year period using the Dice coeffi- cient with 1.5% position tolerance and verified visually. Isolates with identical patterns or differing by a single band were considered the same [17]. All created databases were stored in Microsoft Access® and linked together uti- lizing the MCTR unique identifier numbers. Statistical Analysis y Population figures from the 1996 Statistics Canada Cen- sus were used to calculate tuberculosis incidence rates. Since the study was conducted over an eight-year period, the person-year population of Manitoba throughout this time was determined by multiplying the 1996 population by eight. To determine the incidence rate for a specific population subgroup, we added all TB cases in that sub- group from 1992 to 1999 then divided by the total eight- year subgroup person-years. The resulting number was multiplied by 100,000 to obtain the incidence rate per 100,000 person-years. To compare risk factors for recent TB acquisition of the subpopulation of interest, we used clustering versus non-clustering (unique) as the outcome variable. Bivariate and multivariate logistic regression analyses were carried out using NCSS 2000 software (NCSS, Kaysville, UT, USA). Variables used in the bivari- Tuberculosis Trends For the treaty status subgroup, the incidence in the oldest (65+) age group was 17.3 times that in the youngest (0–14) age group, whereas this ratio Page 3 of 11 (page number not for citation purposes) Page 3 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 Table 2: Multivariate logistic regression analysis of risk factors associated with clustering Characteristic Incidence Clustered Non-Clustered (unique) Odds Ratio p value (person-year/100 000) n = 426 (%) n = 203 (%) (95%) CI Sex Female 8.4 167 (39.2) 102 (50.2) 1 (reference) Male 10.1 259 (60.8) 101 (49.8) 1.959 (1.306–2.938) 0.0011 Age * 0 to 14 3.1 18 (4.2) 4 (2.0) 2.55 (0.74–8.88) 0.1401 15 to 24 5.8 51 (12.0) 17 (8.4) 2.53 (1.20–5.34) 0.0151 25 to 44 10.4 170 (39.9) 47 (23.1) 3.26 (1.92–5.54) 0.0000 45 to 64 10.7 102 (23.9) 48 (23.6) 1.47 (0.87–2.50) 0.1523 65 + 18.4 85 (20.0) 87 (42.9) 1 Origin† Canadian-Born (non-treaty) 3.3 97 (22.8) 60 (29.6) 1 Canadian-Born (treaty) 48.4 254 (59.6) 27 (13.3) 4.108 (2.163–7.803) 0.0000 Foreign-Born 20.0 75 (17.6) 116 (57.1) 0.248 (0.149–0.412) 0.0000 Residence‡ Winnipeg 9.7 235 (55.1) 153 (75.4) 1 other urban 4.1 14 (3.3) 11 (5.4) 0.490 (0.195–1.230) 0.1289 reserves 33.7 148 (34.7) 15 (7.4) 1.206 (0.546–2.665) 0.6433 other rural 9.2 29 (6.9) 24 (11.8) 0.410 (0.203–0.830) 0.0132 * age overall effect, χ2 test = 21.83 with 4 different factors, p < 0.001 † origin overall effect, χ2 test = 81.54 with 2 different factors, p < 0.001 ‡ residence overall effect, χ2 test = 9.26 with 3 different factors, p < 0.05 Table 2: Multivariate logistic regression analysis of risk factors associated with clustering * age overall effect, χ2 test = 21.83 with 4 different factors, p < 0.001 † origin overall effect, χ2 test = 81.54 with 2 different factors, p < 0.001 ‡ residence overall effect, χ2 test = 9.26 with 3 different factors, p < 0.05 was 10.9 for the non-treaty population, and only 1.3 for the foreign-born population (Figure 3). was 10.9 for the non-treaty population, and only 1.3 for the foreign-born population (Figure 3). by means other than microbiological and thus no culture was available for sub-typing. The patient characteristics of these unavailable isolates were compared to those availa- ble to observe any significant differences that may affect estimation of clustering. Tuberculosis Trends The distribution of patients was roughly the same for both and was deemed not signifi- cant. Fingerprint patterns of less than six RFLP bands were observed in 15 isolates. These fingerprints did not cluster, and therefore secondary sub-typing of these isolates, i.e. spoligotyping, was not performed [16]. Dividing Manitoba into rural and urban categories indi- cates that the majority of FB individuals and non-treaty Canadians with TB reside within the city of Winnipeg, while the majority of treaty-status individuals with TB reside in rural areas (figure 1). Winnipeg, as the major urban center of Manitoba, hosts 98.4% of all FB TB patients, and 62.8% of total patients. Aboriginal reserve land includes 22.1% (189) of total patients, with nine reserves in northern Manitoba harboring 159 (84%) of these cases. The TB incidence rate per 100,000 person- years for these nine isolated communities ranged from 19.9 to 496.3 per community. Almost all of these cases were in registered treaty individuals. The remaining 15.1% of total patients live in urban areas other than Win- nipeg and rural areas other than reserves. Individuals liv- ing in urban places other than Winnipeg or rural areas other than reserves experienced a rate of only 4.1 per 100,000 person-year. The overall Winnipeg incidence rate (9.7 per 100,000 person-years) was comparable to the overall Manitoba's rate (9.2 per 100,000 person-years). In total, 249 different fingerprints were obtained, and 203 of them were unique. The 46 that were not unique formed clusters that linked patients into groups containing as lit- tle as two and as many as 162 individuals per cluster. The dominant fingerprint type over the study period was fin- gerprint type 1 (FP1), a strain containing a 12-band IS6110 pattern that was tested sensitive to primary drugs. This strain afflicted 162 (25.8%) individuals of the total TB population, with a very high proportion of treaty indi- viduals (75.3%) residing in both Winnipeg and reserves being infected. By contrast, the second most frequent fingerprint is type 2, infecting only 32 (5.1%) of the total population with TB. The top five fingerprint patterns in terms of prevalence are distributed as seen in figure 5. Tuberculosis Trends Between January 1st 1992 and December 31st 1999, there were 855 reported cases of tuberculosis in Manitoba. The majority of individuals with tuberculosis were born in Canada (70.6%) and almost half of the total patients were CB with treaty status (44.4%). Non-treaty CB individuals contributed 26.2% of total cases and FB population con- tributed 29.4% (figure 3). The age distribution of TB cases varies between the three population subgroups. Tuberculo Figure 3 Tuberculosis incidence rates by age group and origin, 1992–1999 Figure 3 Tuberculosis incidence rates by age group and origin, 1992–1999. females (table 2). Treaty status patients are at four times higher risk of developing clustered TB than those without treaty status (reference subgroup), while in comparison, foreign-born patients are at one-quarter the risk for devel- oping clustered TB compared to the reference population. TB patients in the four age groups under age 65 had risks of developing clustered TB that ranged from 1.5 to 3.3 times that of those over the age of 65 (table 2). Residence on a reserve was significant in the bivariate analysis: these patients have a five times higher risk of having clustered TB than the patient group that lives in Winnipeg. In the multivariate model, however, controlling for the other variables revealed that residence is not a significant inde- pendent risk factor, although living in a rural non-reserve area is protective (table 2). Finally, there was no evidence that patients with pulmonary TB had a higher risk of developing clustered TB than those with extra pulmonary TB. subpopulation contributed 254 (59.6%) of clustered cases; in contrast, the FB population comprised the major- ity of unique cases (57.2%). Comparison of clustering among geographical subgroups also showed the highest proportion of clustering among the reserves and Winni- peg. Of the 162 FP1 cases, 93 (57.4%) lived in urban areas, and of these, 98.9% were in Winnipeg. Of the 69 (42.6%) living in rural areas, 92.8% resided on reserve land. Furthermore, Winnipeg presented the majority of unique fingerprint types, due to the large proportion of foreign-born persons residing in the city. Molecular typing and clustering of cases Isolates of M. tuberculosis were obtained from a total of 629 patients (73.6%) for molecular typing to establish clustering. The remaining balance of cases were diagnosed The distribution of clustering by the distinct sub-popula- tions is also graphically represented in figure 4. The treaty Page 4 of 11 (page number not for citation purposes) Page 4 of 11 (page number not for citation purposes) BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/18 Tuberculosis incidence rates by age group and origin, 1992–1999 Figure 3 Tuberculosis incidence rates by age group and origin, 1992–1999. 3.3 9.2 48.4 22.0 0 50 100 150 200 Manitoba (855 total cases) Canadian non treaty (26.2%) Canadian treaty (44.4%) Foreign (29.4%) origin subgroup(% of total) rates per 100 000 0 - 14 15 - 24 25 - 44 45 - 64 >65 overall rates 3.3 9.2 48.4 22.0 0 50 100 150 200 Manitoba (855 total cases) Canadian non treaty (26.2%) Canadian treaty (44.4%) Foreign (29.4%) origin subgroup(% of total) rates per 100 000 0 - 14 15 - 24 25 - 44 45 - 64 >65 overall rates rates per 100 000 origin subgroup(% of total) Tuberculosis incidence rates by age group and origin, 1992–1999 Figure 3 Tuberculosis incidence rates by age group and origin, 1992–1999. Contact Tracing The largest cluster contained 162 cases that displayed FP1. Contact investigations were indicated on 121 of these cases, determining a total of 2195 contacts. 145 of those contacts presented with disease. Of the 145 contacts, 92 (63.5%) harbored the FP1 strain of TB. 4 (2.8%) had a fin- gerprint type other than FP1, and the remainder of cases did not have isolates available for typing purposes. Most exposures were from family members (57.5%)or friends (24.5%) in the home (71.7%). Preliminary social network analysis using software packages Ucinet and Pajek revealed the spread of TB from Winnipeg to reserve land and vice versa. Risk Factor Analysis Analysis of risk factors for clustering using both bivariate and multivariate analysis is summarized in tables 1 and 2. Significant independent risk factors for clustering included gender, age, geographic residence and ethnic ori- gin. Males are twice as likely to develop clustered TB than Page 5 of 11 (page number not for citation purposes) BMC Infectious Diseases 2003, 3 http://www.biomedcentral.com/1471-2334/3/18 Geographic distribution of tuberculosis patients in Manitoba, 1992–1999 Figure 1 Geographic distribution of tuberculosis patients in Manitoba, 1992–1999. Top chart represents rural Manitoba, in contrast, the bottom depicts urban Manitoba. 30.4% 24.8% 44.7% Canadian born treaty Canadian born non-treaty Foreign born 1.3% 18.5% 80.2% Canadian born treaty Canadian born non-treaty Foreign born y Canadian born non-treaty Foreign born Geographic distribution of tuberculosis patients in Manitoba, 1992–1999 Figure 1 Geographic distribution of tuberculosis patients in Manitoba, 1992–1999. Top chart represents rural Manitoba, in contrast, the bottom depicts urban Manitoba. Geographi Figure 1 Geographic distribution of tuberculosis patients in Manitoba, 1992 1999 Figure 1 Geographic distribution of tuberculosis patients in Manitoba, 1992–1999. Top chart represents rural Manitoba, in contrast, the bottom depicts urban Manitoba. p , of tuberculosis patients in Manitoba, 1992–1999. Top chart represents rural Manitoba, in contrast, the anitoba. the last 10 years have shown a leveling off of total inci- dence rates at approximately 7 per 100,000, with no appreciable decline (figure 2) [6,7,9]. Manitoba shares the same national trends [7,10]. Although rates have declined in every subpopulation, comparative rates among CB treaty individuals in Manitoba (48.4 per 100,000) are still more than ten times than those of the non-treaty sub- group (3.3 per 100,000), and more than two times those of the FB subgroup (22.0 per 100,000) (figure 3) [10,11,18]. Furthermore, whereas the overall majority of TB cases in Canada are attributed to immigration, the population distribution of tuberculosis in Manitoba is distinctly different than in the other provinces. While for- eign-born cases represent 81% of Ontario TB patients, 48% of Quebec TB patients and 60% of TB patients in British Columbia, in contrast, they represent only 29% of the total TB cases in Manitoba between 1992 and 1999 Page 6 of 11 (page number not for citation purposes) Discussion The end of World War II marked the beginning of a sharp decline in TB incidence rates in Canada. Unfortunately, Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999 Figure 5 Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999. 0 20 40 60 80 100 120 140 160 180 1 2 5 72 71 fingerprint type n cases TOTAL Canadian treaty Canadian non treaty Foreign born 0 20 40 60 80 100 120 140 160 180 1 2 5 72 71 fingerprint type n cases TOTAL reserve other rural Winnipeg other urban 0 20 40 60 80 100 120 140 160 180 1 2 5 72 71 fingerprint type n cases TOTAL Canadian treaty Canadian non treaty Foreign born 0 20 40 60 80 100 120 140 160 180 1 2 5 72 71 fingerprint type n cases TOTAL reserve other rural Winnipeg other urban fingerprint type Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999 Figure 5 Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999. p five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999 p five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999 Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999 Figure 5 Distribution of the top five prevalent fingerprint types by (a) population subgroup and (b) geographic subgroup, 1992–1999. [9,18]. It is well established that the foreign-born and Aboriginal groups seem more susceptible to tuberculosis than others within Canada [6,9]. In Manitoba, however, these sub-populations are sustaining the steady level of incidence. Our data demonstrates that TB in Manitoba is a multifaceted problem that can be examined in two distinct, parallel categories. These categories are Canadian TB, where the source of the strains and the patients is Canadian, and foreign TB, where both the patients and strains are from outside Canada. als who have not claimed treaty status. Discussion Moreover, the incidence rate of TB among the treaty status population (48.4 per 100,000) was higher than both the provincial rate (9.2 per 100,000) and the national rate of (5.9 per 100,000) by five and eight times respectively. Certain northern First Nation communities have TB incidence rates that are markedly elevated (up to 496.3 per 100 000 person-years). Statistical analysis showed the treaty sub- group to be at relatively high risk for the development of TB, with significant independent risk factors for clustering to include male gender, treaty status, and under age 65. The major single contributor to the current burden of inci- dent cases is the treaty status subpopulation. The treaty subgroup, with 44.4% of all tuberculosis cases in Mani- toba, comprises the largest group of cases, despite the fact that they represent only 8.9% of Manitoba's population. This figure is probably an underestimate of all Aboriginal cases due to an unknown number of Aboriginal individu- More than 75% of the treaty status population group was clustered together by the FP1 strain. Contact tracing revealed only 92 (57%) confirmed epidemiological links within this cluster, living in both Winnipeg and reserve land. Although using both molecular and conventional techniques verifies that linked cases are most likely linked, Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 254 426 97 75 203 116 60 27 0 100 200 300 400 500 TOTAL Canadian born treaty Canadian born non-treaty Foreign born subpopulation n cases 239 15 11 29 426 148 14 153 24 203 0 100 200 300 400 500 TOTAL reserve other rural Winnipeg other urban residence n cases clustered unique 239 15 11 29 426 148 14 153 24 203 0 100 200 300 400 500 TOTAL reserve other rural Winnipeg other urban residence n cases clustered unique 254 426 97 75 203 116 60 27 0 100 200 300 400 500 TOTAL Canadian born treaty Canadian born non-treaty Foreign born subpopulation n cases clustered unique Clustering of fingerprint patterns for (a) population subgroups, (b) geographic subgroups, 1992–1999 Figure 4 Clustering of fingerprint patterns for (a) population subgroups, (b) geographic subgroups, 1992–1999. Clustering of fingerprint patterns for (a) population subgroups, (b) geographic subgroups, 1992–1999 Figure 4 Clustering of fingerprint patterns for (a) population subgroups, (b) geographic subgroups, 1992–1999. Discussion transmitted more efficiently than in other subgroups. Potential factors contributing to increased risk in this sub- population include: relative poverty, crowded housing, dense social networks, delays in diagnosis and treatment, increased susceptibility to infection due to poorer health status, and increased prevalence of substance abuse. Data on these variables was not available for analysis in the present study. In addition, while the HIV positive group was emerging as an at-risk group for TB in the US, it has not yet been recognized as a risk group in Canada [20]. Thus, the HIV status of tuberculosis patients was not rou- tinely determined in Manitoba until 1997; therefore, complete data on this variable was not available for the current study [21]. there are limitations to using these techniques [19]. Firstly, transmission can occur through brief, casual con- tacts, leading to patients having the same fingerprint pattern, yet no definable connection. Secondly, identical RFLP patterns can be obtained from patients that are not linked through recent transmission/reinfection, but due to reactivation. Thirdly, the strain can be inherent in a population, with an unidentifiable source. However, as clustering is assumed to reflect recent trans- mission within in a population [16,19], supported by the considerable transmission between family and friends as illustrated by contact investigations, it is highly probable that there is an ongoing, propagating TB outbreak in this subpopulation. The presence of a high percentage of res- piratory tuberculosis cases and the large number of posi- tive smear results (data not shown) among FP1 patients suggests that TB in this population subgroup may be The reason for the dominance of FP1 remains unknown, although it has been speculated that perhaps this strain was the first strain introduced to Manitoba. Host genetics, Page 8 of 11 (page number not for citation purposes) Page 8 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998 Figure 2 Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998. . . Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998 Figure 2 Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998. Reported Figure 2 Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998 Figure 2 Reported new active and relapsed tuberculosis cases and incidence rate per 100,000 – Canada: 1928–1998. more than six times as likely to have clustered TB compared to patients in Winnipeg using bivariate analy- sis. Sixty percent of Manitoba's entire population lives in Winnipeg and there is considerable mobility between Winnipeg and the reserve communities. Since neither geography nor physical distance can provide a barrier to stop the spread of this disease, the types of contact rela- tionships are important factors in elucidating the spread of TB and differentiating active transmission versus reactivation. strain virulence or a combination thereof may play a role [22,23]. Routine BCG administration to treaty status infants could have further fostered the natural selection of FP1 by increasing body protection against other suscepti- ble strains through selective immunity. While it is difficult to disentangle social, economic and host risk factors from our analysis, we speculate that this particular strain may be more virulent than other strains introduced to the pop- ulation. It is possible that this strain shares some features in common with putatively 'hyper virulent' strains CDC 1551 and/ or HN878 [24,25]. Bacterial virulence factors, their expression, regulation and mutation, and host immune system evasion mechanisms may play a role, and this remains to be studied [23]. The epidemiological separation between CB and FB tuber- culosis cases is the result of many different characteristics. The dominance of unique (non-clustered) fingerprints among foreign-born individuals suggests that these patients may have contracted the disease outside the prov- ince and have subsequently imported tuberculosis into Manitoba through immigration. This was found true of Winnipeg and nine Aboriginal reserves of northern Mani- toba appear to be the two reservoirs that maintain the bulk of clustered TB cases. Patients on reserves were found Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/3/18 BMC Infectious Diseases 2003, 3 the use of the data for the manuscript. All authors read and approved of the final manuscript. imported TB in province of Alberta [26]. The age distribu- tion of cases in the foreign-born subgroup reflects the age distribution of immigrants, some of whom for unknown reasons progress to active TB after arrival into Canada. References References 1. Fanning EA: Globalization of tuberculosis. CMAJ 1998, 158:611-612. 2. Piheu JA: Tuberculosis 2000: problems and solutions. Int J Tuberc Lung Dis 1998, 2:696-703. g 3. Raviglione MC, Snider DE and Kochi A: Global epidemiology of t b l i bidit d t lit f ld id 3. Raviglione MC, Snider DE and Kochi A: Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic. JAMA 1995, 273:220-226. p J 4. Grzybowski S and Allen EA: Tuberculosis: 2. History of the dis- ease in Canada. CMAJ 1999, 160:1025-1028. 5. Wherret GJ: Tuberculosis in Canada. Ottawa: Royal Commission on Health Services 1964. 6. Canada Communicable Disease Report. Supplement: Proceedings of the national consensus conference on tuberculosis. Health Canada 1997, 24S2:. of the national consensus conference on tuberculosis. Health Canada 1997, 24S2:. 7. Long R and ed: Canadian Tuberculosis Standards. Canadian Lung Association and Health Canada 52000. While the foreign-born population may not seem to be actively spreading TB in Manitoba, incidence rates are still relatively high, and the number of FB cases reported annu- ally is increasing. Screening procedures for immigrating individuals from countries with high or low prevalence of TB need further examination for effectiveness and cost- effectiveness [27]. 8. Health Canada: Tuberculosis in the First Nations, 1999. Health Canada, Canada 1999. (Cat.H35-4/7-1999E) (ISBN 0-662-30044-0) ( ) ( ) 9. Long R, Njoo H and Hershfield E: Tuberculosis: 3. Epidemiology of the disease in Canada. CMAJ 1993, 160:1185-1190. 10. Al-Azem A, Abomoelak B, Wolfe J, MacMorran J, Hershfield E and Kabani A: Tuberculosis in Manitoba: Conventional and molec- ular epidemiology (1992–1999) [abstract]. Public Health Poster Session (ATS) 2000. CDC-NCHSTP-DTBE ( ) 11. Al-Azem A, Abomoelak B, Wolfe J, MacMorran J, Hershfield E and Kabani A: Rural and localized tuberculosis outbreaks in Mani- toba (1992–1999) [abstract]. Public Health Poster Session (ATS) 2000. CDC-NCHSTP-DTBE In the present study, information derived from both con- ventional and molecular epidemiology methods allowed assessment of current trends and risk factors for subpopulation groups in Manitoba for contracting TB. Retrospective studies such as this one using stored RFLP data have shown invaluable to identify both clinical and epidemiological risk factors for clustering, and have been used extensively to study other populations within San Francisco, New York and the Netherlands [17,19,28,29]. This information helps assess the effectiveness and possi- ble limitations of the Manitoba TB control programs. p g None declared. 20. Gaudette LA and Ellis E: Tuberculosis in Canada: a focal disease requiring distinct control strategies for different risk groups. Tuber Lung Dis 1993, 74:244-53. References Elimination of TB has been defined as less than 1 case per 100,000 population per year [6,8]. By this standard, recent transmission to Canadian-born non-treaty status individuals has been nearly eliminated, reaching inci- dence rates of only 3.3 per 100,000. Complete elimina- tion of TB in Manitoba has been targeted for 2010, with 5% reduction in incidence per year goal [6,8], but with the high incidence rates in the remaining two sub-popula- tions, this will be a difficult task. Targeting and intensify- ing control measures in the immigrant and treaty status Aboriginal subpopulations should be a priority in striving for the goal of tuberculosis elimination in Manitoba. 12. Dimensions Series: Portrait of Aboriginal Population in Canada. Ottawa, ON: Statistics Canada 1999. [94F0011XCB] [ ] 13. Live Database 1992–1997, Indian Registry System, Indian and Northern Affairs Canada. Manitoba Region . 14. van-Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gic- quel B, Hermans P, Martin C, McAdam R and Shinnick TM: Strain identification of Mycobacterium tuberculosis by DNA finger- printing: recommendations for a standardized methodology. J Clin Microbiol 1993, 31:406-409. gy J 15. Small PM, McClenny NB, Singh SP, Schoolnik GK, Tompkins LS and Mickelsen PA: Molecular strain typing of Mycobacterium tuber- culosis to confirm cross- contamination in the mycobacteri- ology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J Clin Microbiol 1993, 31:1677-82. 16. Rhee JT, Tanaka MM, Behr MA, Agasino CB, Paz EA, Hopewell PC and Small PM: Use of multiple markers in population-based molec- ular epidemiologic studies of tuberculosis. Int J Tuberc Lung Dis 2000, 4:1111-1119. 17. Shafer RW, Small PM, Larkin C, Singh SP, Kelly P, Sierra MF, Schoolnik G and Chirgwin KD: Temporal trends and transmission pat- terns during the emergence of multidrug-resistant tubercu- losis in New York City: a molecular epidemiologic assessment. J Infect Dis 1995, 171:170-76. J f 18. Al-Azem A, Abomoelak B, Wolfe J, MacMorran J, Hershfield E and Kabani A: The impact of foreign-born tuberculosis in Mani- toba (1992–1999) [abstract]. Public Health Poster Session (ATS) 2000. CDC-NCHSTP-DTBE 19. 19. Small PM, Hopewell PC, Singh SP, Paz A, Parsonnet J, Ruston DC, Schechter GF, Daley CL and Schoolnik GK: The epidemiology of tuberculosis in San Francisco. A population based study using conventional and molecular methods. N Eng J Med 1994, 330:1703-1709. Reported Figure 2 New arrivals into the country may for some time have lim- ited close contact with Canadian-born individuals, which reduces the chance of spread from immigrants to the CB population. 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Borgdorff MW, Nagelkerke N, van Soolingen D, de Haas PE, Veen J and van Embden JD: Analysis of tuberculosis transmission between nationalities in the Netherlands in the period 1993– 1995 using DNA fingerprinting. Amer J Epidem 1998, 147:187-195. Pre-publication history The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/3/18/prepub Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W1830002900	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0135490&type=printable	English	null	Primary Productivity and Precipitation-Use Efficiency in Temperate Grassland in the Loess Plateau of China	PloS one	2,015	cc-by	8,881	RESEARCH ARTICLE Primary Productivity and Precipitation-Use Efficiency in Temperate Grassland in the Loess Plateau of China Xiaoxu Jia1, Baoni Xie2, Ming’an Shao1,3, Chunlei Zhao2,3 1 Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China, 2 College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China, 3 State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Northwest A&F University, Yangling 712100, China shaoma@igsnrr.ac.cn * shaoma@igsnrr.ac.cn * shaoma@igsnrr.ac.cn Abstract Clarifying spatial variations in aboveground net primary productivity (ANPP) and precipita- tion-use efficiency (PUE) of grasslands is critical for effective prediction of the response of terrestrial ecosystem carbon and water cycle to future climate change. Though the combi- nation use of remote sensing products and in situ ANPP measurements, we quantified the effects of climatic [mean annual precipitation (MAP) and precipitation seasonal distribution (PSD)], biotic [leaf area index (LAI)] and abiotic [slope gradient, aspect, soil water storage (SWS) and other soil physical properties] factors on the spatial variations in ANPP and PUE across different grassland types (i.e., meadow steppe, typical steppe and desert steppe) in the Loess Plateau. Based on the study, ANPP increased exponentially with MAP for the entire temperate grassland; suggesting that PUE increased with increasing MAP. Also PSD had a significant effect on ANPP and PUE; where more even PSD favored higher ANPP and PUE. Then MAP, more than PSD, explained spatial variations in typical steppe and desert steppe. However, PSD was the dominant driving factor of spatial variations in ANPP of meadow steppe. This suggested that in terms of spatial variations in ANPP of meadow steppe, change in PSD due to climate change was more important than that in total annual precipitation. LAI explained 78% of spatial PUE in the entire Loess Plateau temperate grassland. As such, LAI was the primary driving factor of spatial variations in PUE. Although the effect of SWS on ANPP and PUE was significant, it was nonetheless less than that of precipitation and vegetation. We therefore concluded that changes in vegetation structure and consequently in LAI and/or altered pattern of seasonal distribution of rainfall due to global climate change could significantly influence ecosystem carbon and water cycle in temperate grasslands. Introduction Precipitation is the key factor in controlling primary production of most of the world’s grass- land ecosystems, especially in arid and semiarid regions [1,2,3]. It is predicted that global climate change will lead to more frequent extreme precipitation and drought events, with sig- nificant effects on ecosystem processes [4,5]. Aboveground net primary productivity (ANPP), a key integrative measure of ecosystem function, is reported to be greatly affected by changes in mean annual precipitation (MAP) and/or precipitation seasonal distribution (PSD) [6,7,8,9,10,11]. There is a growing interest to explore relations between both ANPP-MAP and ANPP-PSD under global climate change. While the patterns of MAP-ANPP relations are highly variable, the underlying processes of these relations are not yet fully understood. For example, some studies have reported simple linear relation between MAP and ANPP [12,13,14,15] and others have noted exponential relation [10,16,17] for the same temperate grasslands in China. The response of grassland ANPP to change in PSD is still controversial. For example, Tho- mey et al. [18] and Guo et al. [10] noted that more concentrated PSD (i.e., large, infrequent rainfall events) attenuated water stress which, in turn improved Chihuahuan desert and Eur- asian temperate grassland ANPP. However, the reverse was observed for Kansas tallgrass prai- rie where more even PSD favored higher ANPP [3,19]. Although extreme rainfall regimes induce greater dry intervals between rainfall events, semiarid steppes are not particularly sensi- tive to increase in rainfall event intervals because semiarid steppe plants are well adapted to extended periods of intense water stress [2]. Besides, several studies have shown that concen- trated rainfall regimes lead to deep infiltration of precipitation water into the soil layer and less proportional loss to evaporation, thus increasing the amount and duration of water in the soil for subsequent plant uptake [10,18,19,20]. However, all of these studies assumed no dramatic increase in runoff with larger rain events [9]. The conditions in the Loess Plateau are possibly different from those in other regions. The Loess Plateau is highly complex in topography and landform and the loess soil is easily prone to erosion. Also the few larger rainfall events could induce excessive water loss as overland flow. Thus the effects of altered PSD pattern on grass- land ANPP in the Loess Plateau could be different from those in other areas. However, the relative effects of MAP and PSD on ANPP variations in the Loess Plateau are still largely unknown. OPEN ACCESS OPEN ACCESS Citation: Jia X, Xie B, Shao M, Zhao C (2015) Primary Productivity and Precipitation-Use Efficiency in Temperate Grassland in the Loess Plateau of China. PLoS ONE 10(8): e0135490. doi:10.1371/ journal.pone.0135490 Editor: Shiping Wang, Institute of Tibetan Plateau Research, CHINA Received: November 27, 2014 Accepted: July 22, 2015 Published: August 21, 2015 Copyright: © 2015 Jia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Jia X, Xie B, Shao M, Zhao C (2015) Primary Productivity and Precipitation-Use Efficiency in Temperate Grassland in the Loess Plateau of China. PLoS ONE 10(8): e0135490. doi:10.1371/ journal.pone.0135490 Citation: Jia X, Xie B, Shao M, Zhao C (2015) Primary Productivity and Precipitation-Use Efficiency in Temperate Grassland in the Loess Plateau of China. PLoS ONE 10(8): e0135490. doi:10.1371/ journal.pone.0135490 Data Availability Statement: Data can be found on Figshare at figshare.com/s/ 469bfb503a6f11e58d9806ec4bbcf141 and at http:// dx.doi.org/10.6084/m9.figshare.1501438. Funding: This study was supported by the National Natural Science Foundation of China (No. 41390461 and No. 51179180). 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. 1 / 16 1 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Introduction Precipitation-use efficiency (PUE), the ratio of ANPP to precipitation, is a useful index that explains the relation between ecosystem carbon and water cycle [15,17]. PUE is closely linked to both plant physiological characteristics and physical water loss processes [14,21,22]. In gen- eral, PUE tends to decrease spatially with increasing aridity and potential evapotranspiration [23]. However, recent reports have strongly challenged this point [7,13]. For example, Huxman et al. [7] showed decreasing mean PUE from deserts, grasslands to forests with increasing MAP in North and South America. Hu et al. [17] observed that when the range of MAP and ecosystem type narrows toward dry end, the spatial variation in PUE shifts from decreasing to increasing one with increasing MAP. Spatiotemporal variations in PUE are therefore variable due to the controlling physiological and physical processes of PUE which vary with the scale of analysis. While previous studies have mainly focused on the relation between PUE and MAP, little remains known about the response of PUE to altered PSD patterns. Also the lack of knowledge on PUE-PSD relation inhibits the understanding of how climate change affects eco- system carbon and water cycle. PUE is linked to many factors, including vegetation composition, edaphic conditions and biogeochemical constraints [7,13,23,24]. For example, communities with higher diversity of plant species and functional groups could have greater PUE due to increased ANPP [25]. Hu 2 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland et al. [17] showed that vegetation cover significantly affects spatial variations in PUE along 4500 km of grassland transect. Leaf area index (LAI) is a primary factor controlling spatiotem- poral variations in ecosystem water use efficiency. Studies in four grassland ecosystems in China have shown that LAI has a regulatory effect on the ratio of transpiration to evapotranspi- ration [22,26]. Soil properties, such as texture, water-holding capacity and depth are major determinants of soil water availability and could therefore have significant effect on PUE [1,6,23]. Although the effects of above biotic and/or abiotic factors on ANPP and PUE have been widely studied, the relative importance of the factors in the spatiotemporal patterns of ANPP and PUE is not fully understood [15]. PUE patterns and the associated driving factors generally vary from site to site. The Loess Plateau is ca. 35% comprised of grassland. Materials and Methods Ethics statement The sampling fields belong to “Grain-for-Green” program and permission for plant and soil sampling was obtained by the Institute of Soil and Water Conservation, Chinese Academy of Sciences & Ministry of Water Resources of PRC. The sampling fields in this study did not include endangered or protected species. Introduction It is a vital component of China’s grasslands and it is critical for controlling soil and water erosion. Despite its significance, little has been done to address the spatial variations in ANPP and PUE in the grasslands in the Loess Plateau. Precipitation is the only source of water for plant productivity in the arid Loess Plateau region. Plants in the region use only a small fraction of the rainfall because of high evaporation and runoff. Thus this study hypothesized that ANPP and PUE increase with increasing MAP. This is because plants have high growth rates with increasing MAP and subsequently more dense vegetation canopy. Dense canopies induce more precipitation water use in transpiration which in turn results in higher productivity. Besides, there is generally severe soil and water erosion in the Loess Plateau due to complex landforms, loose soil and sparse vegetation conditions. In the region, there is excessive waste of extreme and concentrated precipitation as overland flow [27,28]. Thus this study also hypothe- sized that more even PSD will favor higher ANPP and PUE. With few but large rainfall events, more precipitation water is lost to runoff and less used for plants growth. This condition is likely to decrease ANPP and PUE in the Loess Plateau. The main objective of this study was to analyze spatial variations in ANPP and PUE using environmental and ANPP data from differ- ent grassland types in the Loess Plateau. ANPP measurement and regional estimation A simple conventional method based on empirical relationship between in situ measurement of ANPP and the corresponding remotely sensed vegetation index (i.e., Normalized Difference Vegetation Index, NDVI) was used to estimate regional multi-year ANPP in the study area [30]. The in situ measurement of ANPP was taken as the peak aboveground biomass (including live biomass and standing dead biomass) during the growing season. This method is widely used to estimate grassland ANPP [31,32]. Vegetation surveys were conducted during peak growing seasons in 2011–2013 when aboveground biomass was maximum (i.e., in August). The sampling sites were randomly selected at intervals of c. 10–50 km across the entire Loess Plateau region to ensure representativeness. The sampling plots with natural re-vegetation were protected from human disturbances in order to prevent soil erosion and further land deg- radation. In particular, all sampling sites were protected from grazing herbivores. At each sam- pling site, aboveground biomass was clipped in three to five randomized 1 m × 1 m quadrats. The sampled biomass was dried at 65°C in an oven for 72 h and the dry weight then estimated. For the sites with shrub species, the twigs biomass for the current year was not measured. Thus shrub steppe ANPP was not estimated for the respective regions. ANPP data were collected from 188 sites, covering the three grassland types in the study area (Fig 1). The range of varia- tion in ANPP was 25.4–569.8 g m-2 yr-1. Based on statistical analysis, the standard deviations of ANPP for the replicates at 82% of the sites were less than 20% of the mean value. This sug- gested that heterogeneity at each site was negligible in analysis of spatial patterns of ANPP at regional scale [10,17]. The peak monthly NDVI in 2011–2013 (in August) was used to determine the relation between in situ measurement of ANPP and the corresponding NDVI. Then the peak monthly NDVI in 2000–2013 (also in August) relative to the established ANPP-NDVI relation was used to estimate ANPP for each pixel of the study area. The NDVI data were derived from the Mod- erate Resolution Imaging Spectroradiometer (MODIS; https://lpdaac.usgs.gov). The peak monthly NDVI data for 2011–2013 (with 250 m × 250 m resolution) were obtained from MOD09GQ products, which provided daily surface-reflectance and NDVI datasets. The composite 1-day NDVI for August (corresponding to field sampling time) was averaged to get the monthly NDVI for August. Primary Production and Rain-Use Efficiency in Loess Plateau Grassland “Grain-for-Green” program), in 1999 in the region. The program has been operating for 15 years now and the natural ecology in most parts of the Plateau is gradually improving. From southeast to northwest, the vegetation changes from forest through forest steppe to typical steppe and then to desert steppe type. Meadow steppe is mainly in the west of the Pla- teau (Fig 1). The meadow steppe has the highest plant biodiversity, with dominant species of Festuca ovina, Filifoliumsibiricum Kitam, Stipa Baicalensis, Bothriochloa ischaemum, Artemisia giraldii, Spodiopogon sibiricus, and Leymus secalinus. The typical steppe has moderate plant biodiversity that is dominated by Stipa bungeana, Artemisia giraldii, Thymus mongolicus, Les- pedeza davurica, Artemisia scoparia, and Heteropappus altaicus. Then the desert steppe has the lowest plant biodiversity that is dominated by Stipa breviflora, Stipa bungeana, Peganum har- mala, Salsola collina, and Lespedeza davurica. Study area The Loess Plateau is in Northwestern China and covers latitudes 33°43’–41°16’N and longi- tudes 100°54’–114°33’E with a total area of 620,000 km2, accounting for some 6.5% of the area of China. The region lies in the temperate, arid and semi-arid continental monsoon zones. The mean annual temperature range is 3.6 to 14.3°C and MAP from northwest to southeast is 150– 800 mm. About 55–78% of MAP falls in June through September as high intensify rainstorm. The soil is mainly loess, with clay-loam as the most common texture and then sandier soils in the northwest and clayier soils in the southeast. The region has a complex topography, includ- ing sub-plateaus, basins, hills and gullies of altitude range of 200–3000 m above mean sea level [29]. Much of the study area experiences severe soil and water erosion, resulting in land degra- dation and loss of soil fertility. For soil and water erosion control and also for ecosystem resto- ration, Chinese government initiated an extensive ecological rehabilitation program (the 3 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 ANPP measurement and regional estimation The peak monthly NDVI data for 2000–2013 were from MOD13Q1 products (with the same spatial resolution of 250 m × 250 m) provided the 16-day composite NDVI. In this study, the 1 km × 1 km NDVI were extracted from MOD13Q1 products with 4 raster cells for the whole study area. This was used to explore the spatial patterns of ANPP to match with corresponding climatic and LAI data for further analysis. The two 16-day NDVI compos- ites for August were averaged to represent monthly NDVI for August. A variety of processing 4 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Fig 1. Location of the Loess Plateau in China (left plate) and the distribution of the 188 sampling sites where in situ measurements of aboveground net primary productivity were taken in the study area (right plate). doi:10 1371/journal pone 0135490 g001 Fig 1. Location of the Loess Plateau in China (left plate) and the distribution of the 188 sampling sites where in situ measurements of aboveground net primary productivity were taken in the study area (right plate). Fig 1. Location of the Loess Plateau in China (left plate) and the distribution of the 188 sampling sites where in situ measurements of aboveground net primary productivity were taken in the study area (right plate). doi:10.1371/journal.pone.0135490.g001 doi:10.1371/journal.pone.0135490.g001 doi:10.1371/journal.pone.0135490.g001 techniques (e.g., orbit calibration, sensor calibration, cloud screening, geo-referencing correction and atmospheric correction) were used to reduce the effects of cloud contamination, atmo- spheric perturbations and variable illumination and viewing geometry on the data products. A significant exponential correlation was noted between in situ measurement of ANPP and the corresponding NDVI for August—ANPP = 27.48e3.70NDVI (R2 = 0.71, n = 188, P < 0.001). The spatial pattern of ANPP for the entire region for 2000–2013 was thus estimated using this rela- tionship and NDVI data for August. The regional ANPP data were thus used to explain the spa- tial variations in PUE. PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Precipitation interpolation MAP data for 2000–2013 were from the China Meteorological Data Sharing Service System at http://cdc.cma.gov.cn/. This dataset contains climate data from 73 stations, 64 of which were selected based on the European Climate Assessment standards. The kriging method was used to interpolate the station-specific data at 1 km × 1 km resolution to create continuous data sur- faces for meteorological factors. Then the data surface was re-sampled in ArcInfo GIS (v. 9.2) to derive specific meteorological factor for each sampling site and pixel. Precipitation seasonal distribution (PSD) in the growing season (May to September) was quantified as coefficient of variation of the monthly precipitation (CVmp) as follows [10]: CVmp ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 5 X 9 i¼5 ðMi MÞ 2 s M ð1Þ M ¼ 1 5 X 9 i¼5 ðMiÞ ð2Þ ð1Þ M ¼ 1 5 X 9 i¼5 ðMiÞ ð2Þ ð2Þ where Mi is the averaged precipitation for month i (i from May to September); and M is the mean precipitation for the 5 months (May to September). where Mi is the averaged precipitation for month i (i from May to September); and M is the mean precipitation for the 5 months (May to September). where Mi is the averaged precipitation for month i (i from May to September); and M is the mean precipitation for the 5 months (May to September). PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 5 / 16 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Spatial variations in ANPP and PUE Spatial variations in ANPP and PUE ANPP and PUE generally decreased with increasing aridity (P < 0.01) across the Loess Plateau. ANPP for the three grassland types varied significantly, in the range of 33.8–656.2 g m-2 yr-1 (Table 1). Meadow steppe had the highest average ANPP (380.2 g m-2 yr-1) and the lowest coef- ficient of variation (CV; 26.3%). Then typical steppe had medium ANPP (153.0 g m-2 yr-1) and the highest CV (60.0%). The lowest ANPP was for desert steppe (80.0 g m-2 yr-1) and with CV of 39.7%. Consistent with ANPP, there were significant differences in PUE among the three grassland types (Table 1). Meadow steppe had the highest PUE (0.92 g m-2 mm-1), followed by typical steppe (0.41 g m-2 mm-1) and then desert steppe (0.28 g m-2 mm-1). Soil, topography and vegetation measurement properties From the 188 sampling sites, 101 were long-term observation sites for monitoring changes in deep soil water content and ANPP. Soil samples at the 101 sites were collected after clipping aboveground biomass. At each sampling site, a 40 cm deep pit was excavated and undisturbed soil samples taken at three layer depths (0–10 cm, 10–20 cm and 20–40 cm) to measure satu- rated soil hydraulic conductivity (Ks, cm min-1) and soil bulk density (BD, g cm-3). Disturbed soil samples were also collected for laboratory analysis. The disturbed soil samples were air- dried and passed through 1 mm sieve and then analyzed for soil particle sizes using laser dif- fraction Mastersizer2000 (Malvern Instruments, Malvern, England). Volumetric soil water contents were simultaneously measured at the 101 sites using neutron probes placed a 20 cm interval to the depth of 500 cm. This gave the measure of the effect of soil water storage on ANPP and PUE. The slope gradient (°) and aspect (°) at each of the 101 sites was measured using geological compass. LAI data were from MODIS LAI product (1 km × 1 km; http:// lpdaac.usgs.gov). Mean LAI was calculated for the peak growing season (August) in 2000–2013 as the maximum mean LAI for each pixel, corresponding to NDVI data. Data analysis To determine the effects of MAP, PSD, vegetation and soil properties on spatial variations in ANPP and PUE in the entire Loess Plateau region, the 400, 1200 and 3000 sites were randomly selected respectively for meadow steppe, desert steppe and typical steppe based on the area of each grassland type. Prior to the site sampling, land use and land cover data (1 km × 1 km) for the Loess Plateau were used to eliminate non-grassland pixels. One-way ANOVA was used to determine the differences in ANPP and PUE for the three grassland types. For sites with signif- icant differences, the least significant difference (LSD) test was conducted at P < 0.05. Regres- sion analysis was used to test the associations among ANPP, PUE, vegetation, and soil properties. All statistical analyses were performed in SAS software (v. 8.0) (SAS Institute Inc., Cary, NC, USA). Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Table 1. Statistical values of aboveground net primary productivity (ANPP) and precipitation-use efficiency (PUE) for the three grassland types: MS, meadow steppe (n = 400); TS, typical steppe (n = 3000); DS, desert steppe (n = 1200). CV% is the percent coefficient of variation: (Standard devia- tion/Mean). MAPavg is the average mean annual precipitation (MAP) for the grassland types. Values followed by different lower-case letters are significantly different for different grassland types at P < 0.05. ANPP (g m-2 yr-1) PUE (g m-2 mm-1) Variable MS TS DS MS TS DS Minimum 54.8 43.0 33.8 0.17 0.10 0.11 Maximum 656.2 617.7 394.4 1.69 1.77 0.88 Mean 380.2a 153.0b 80.0c 0.92a 0.41b 0.28c CV% 26.3 60.0 39.7 24.3 47.2 31.5 MAPavg 415.0 372.0 280.0 415.0 372.0 280.0 doi:10 1371/journal pone 0135490 t001 Table 1. Statistical values of aboveground net primary productivity (ANPP) and precipitation-use efficiency (PUE) for the three grassland types: MS, meadow steppe (n = 400); TS, typical steppe (n = 3000); DS, desert steppe (n = 1200). CV% is the percent coefficient of variation: (Standard devia- tion/Mean). MAPavg is the average mean annual precipitation (MAP) for the grassland types. Values followed by different lower-case letters are significantly different for different grassland types at P < 0.05. Table 1. Statistical values of aboveground net primary productivity (ANPP) and precipitation-use efficiency (PUE) for the three grassland types: MS, meadow steppe (n = 400); TS, typical steppe (n = 3000); DS, desert steppe (n = 1200). CV% is the percent coefficient of variation: (Standard devia- tion/Mean). MAPavg is the average mean annual precipitation (MAP) for the grassland types. Values followed by different lower-case letters are significantly different for different grassland types at P < 0.05. ANPP (g m-2 yr-1) PUE (g m-2 mm-1) Variable MS TS DS MS TS DS Minimum 54.8 43.0 33.8 0.17 0.10 0.11 Maximum 656.2 617.7 394.4 1.69 1.77 0.88 Mean 380.2a 153.0b 80.0c 0.92a 0.41b 0.28c CV% 26.3 60.0 39.7 24.3 47.2 31.5 MAPavg 415.0 372.0 280.0 415.0 372.0 280.0 doi:10.1371/journal.pone.0135490.t001 analysis) of the spatial variations in ANPP in typical steppe, 31% in desert steppe and 21% in meadow steppe. Inconsistent with MAP, there was significant negative correlation between ANPP and CVmp (R2 = 0.29, P < 0.001), suggesting that PSD had a strong impact on spatial variations in ANPP at regional scale (Fig 2b). Although negative correlations were also noted for all the grassland types, R2 values were different (P < 0.001). Regression analysis showed that CVmp accounted for 29% and 30% of the spatial variations in ANPP respectively in meadow steppe and typical steppe. However, CVmp only explained 7% of the spatial variations in ANPP in des- ert steppe. Using in situ measurement of ANPP and soil moisture for the 101 sites, the effects of SWS at various soil layer depths on ANPP were determined (Fig 4a). Consistent with MAP, ANPP was positively correlated with SWS for the various soil layer depths and it increased exponen- tially with increasing SWS (P < 0.001). However, the ability of SWS to explain the variations in ANPP decreased with increasing soil depth. For example, the 0–1 m layer SWS accounted for 29% of the spatial variations in ANPP and the 4–5 m layer SWS for only 9%. Note that MAP explained more spatial variations in ANPP (49%) than SWS (Fig 4a). This implied that regional patterns of ANPP were controlled more by precipitation than by soil moisture in the Loess Plateau. Consistent with ANPP, PUE was positively correlated with MAP (R2 = 0.13, P < 0.001) and negatively correlated with PSD (R2 = 0.29, P < 0.001) for the Loess Plateau temperate grassland (Fig 5a and Fig 3). Besides, a positive correlation existed between PUE and LAI. LAI explained 78% of the variations in PUE in temperate Loess Plateau grasslands (Fig 5b). SWS for various soil depths also had a positive effect on PUE, but the ability to explain PUE decreased with increasing soil depth (Fig 4b). The above results suggested that the ability of climatic and soil factors to explain spatial variations in ANPP was much weaker than that of biotic factors. In contrast to the above results, soil texture and other soil physical properties (e.g., Ks, BD) were not significantly correlated with ANPP and PUE (data not shown). Furthermore, the effects of slope gradient and aspect on ANPP and PUE were determined using in situ measurement of ANPP, slope gradient and aspect at the 101 sites. The analysis showed that ANPP was negatively correlated with slope gradient (P = 0.03, n = 101). PUE was also negatively correlated with slope gradient, although not statistically significant (P = 0.063, n = 101). Driving factors of ANPP and PUE Regression analysis was used to determine the effects of climatic, biotic and soil factors on the spatial variations in ANPP and PUE. ANPP for the entire temperate grassland in the Loess Pla- teau increased exponentially with increasing MAP (ANPP = 22.63e0.048MAP, n = 4600; R2 = 0.50, P < 0.001), implying that an increase in PUE increased MAP (Fig 2a). The correlation was also exponential for typical steppe and desert steppe. However, a linear MAP-ANPP corre- lation (R2 = 0.21, P < 0.001) was observed for meadow steppe. MAP accounted for 56% (by R2 6 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Discussions Characteristics of ANPP and PUE No significant correlation was noted between aspect and ANPP or PUE (data not shown). 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Fig 2. Correlation between (a) aboveground net primary productivity (ANPP) and mean annual precipitation (MAP) and that between (b) ANPP and precipitation seasonal distribution [PSD, quantified herein with coefficient of variation (CVmp)] and for each grassland type in the Loess Plateau temperate grassland. Each data point in the figure represents 14-year average value for 2000 2013. R2 is the coefficient of determinant of the regression functions between MAP, CVmp and ANPP fo grassland type. Fig 2. Correlation between (a) aboveground net primary productivity (ANPP) and mean annual precipitation (MAP) and that between (b) ANPP and precipitation seasonal distribution [PSD, quantified herein with coefficient of variation (CVmp)] and for each grassland type in the Loess Plateau temperate grassland. Each data point in the figure represents 14-year average value for 2000– 2013. R2 is the coefficient of determinant of the regression functions between MAP, CVmp and ANPP for each grassland type. doi:10.1371/journal.pone.0135490.g002 Characteristics of ANPP and PUE Compared with both typical steppe and desert steppe, meadow steppe had the highest ANPP and PUE in the Loess Plateau. This trend could be attributed to favorable environmental condi- tions in the region. Similarly, previous studies noted that drier sites tend to have lower PUE because of low plant density, low production potential, high evaporation potential and high 8 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Fig 3. Correlations between (a) precipitation use efficiency (PUE) and mean annual precipitation (MAP) and that between (b) PUE and leaf area index (LAI) for the entire Loess Plateau temperate grasslands. d i 10 1371/j l 0135490 003 Fig 3. Correlations between (a) precipitation use efficiency (PUE) and mean annual precipitation (MAP) and that between (b) PUE and leaf area index (LAI) for the entire Loess Plateau temperate Fig 3. Correlations between (a) precipitation use efficiency (PUE) and mean annual precipitation (MAP) and that between (b) PUE and leaf area index (LAI) for the entire Loess Plateau temperate grasslands. doi:10.1371/journal.pone.0135490.g003 tolerance to water stress [1,13,15,17]. Meadow steppe is mainly in the west of the Loess Plateau where water and heat conditions (driven by southeast monsoon winds) favor plant growth [33]. Other studies also noted that higher precipitation, lower temperature and thus lower potential evapotranspiration rates favor higher ANPP and PUE in meadow steppes [10,17]. PUE for temperate grasslands in the Loess Plateau (0.28–0.94 g m-2 mm-1) is similar to that (0.05–1.81 g m-2 mm-1) reported by Le Houérou et al. [34] for grassland ecosystems in arid and semiarid regions in the world. Furthermore, mean PUE for temperate grasslands in the Loess Plateau is also close to those reported by Bai et al. [15] and Hu et al. [17]. PUE for meadow steppe was significantly different from that for typical steppe under similar precipitation condi- tion (Table 1). This could be because typical steppe is in areas with the highest water and soil erosion in the Loess Plateau [35], resulting in severe precipitation loss by runoff and soil water tolerance to water stress [1,13,15,17]. Meadow steppe is mainly in the west of the Loess Plateau where water and heat conditions (driven by southeast monsoon winds) favor plant growth [33]. Other studies also noted that higher precipitation, lower temperature and thus lower potential evapotranspiration rates favor higher ANPP and PUE in meadow steppes [10,17]. Characteristics of ANPP and PUE PUE for temperate grasslands in the Loess Plateau (0.28–0.94 g m-2 mm-1) is similar to that (0.05–1.81 g m-2 mm-1) reported by Le Houérou et al. [34] for grassland ecosystems in arid and semiarid regions in the world. Furthermore, mean PUE for temperate grasslands in the Loess Plateau is also close to those reported by Bai et al. [15] and Hu et al. [17]. PUE for meadow steppe was significantly different from that for typical steppe under similar precipitation condi- tion (Table 1). This could be because typical steppe is in areas with the highest water and soil erosion in the Loess Plateau [35], resulting in severe precipitation loss by runoff and soil water tolerance to water stress [1,13,15,17]. Meadow steppe is mainly in the west of the Loess Plateau where water and heat conditions (driven by southeast monsoon winds) favor plant growth [33]. Other studies also noted that higher precipitation, lower temperature and thus lower potential evapotranspiration rates favor higher ANPP and PUE in meadow steppes [10,17]. PUE for temperate grasslands in the Loess Plateau (0.28–0.94 g m-2 mm-1) is similar to that (0.05–1.81 g m-2 mm-1) reported by Le Houérou et al. [34] for grassland ecosystems in arid and semiarid regions in the world. Furthermore, mean PUE for temperate grasslands in the Loess Plateau is also close to those reported by Bai et al. [15] and Hu et al. [17]. PUE for meadow steppe was significantly different from that for typical steppe under similar precipitation condi- tion (Table 1). This could be because typical steppe is in areas with the highest water and soil erosion in the Loess Plateau [35], resulting in severe precipitation loss by runoff and soil water PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 9 / 16 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Fig 4. Correlation between precipitation use efficiency (PUE) and precipitation seasonal distribution (PSD, quantified herein with CVmp) for each grassland type and for the entire Loess Plateau temperate grassland. doi:10 1371/journal pone 0135490 g004 Fig 4. Correlation between precipitation use efficiency (PUE) and precipitation seasonal distribution (PSD, quantified herein with CVmp) for each grassland type and for the entire Loess Plateau temperate grassland. doi:10.1371/journal.pone.0135490.g004 evaporation. This reduces the ratio of transpiration to annual precipitation, which in turn reduces PUE [17]. Furthermore, severe soil and water erosion could cause loss of nutrients, renders soils infertile and thus lower ANPP. Characteristics of ANPP and PUE It was therefore conclude that PUE of grassland ecosystems depended on land surface patterns such as topography and landforms. Factors affecting variations in ANPP Precipitation, a proxy for water availability, is a major determinant of vegetation production at regional and global scales, especially in arid and semiarid regions [1,10,13,36]. This study showed an exponential positive relation between ANPP and MAP for the Loess Plateau tem- perate grasslands. This suggested that increase in MAP causes increase in PUE, which sup- ported the first hypothesis in this study. This result is also consistent with that reported in most previous studies [10,12,17,37]. Studies on grassland ecosystems in other regions of the world reported linearity as the most common relation between ANPP and MAP [6,12,13]. Hu et al. [17] concluded that inconsistencies could be due to insufficient sampling sites in arid regions, which conclusion was also supported by Guo et al. [10]. The findings of this study also confirmed this point. Using data from a large randomly-selected sample sites, a linear MAP-ANPP relation was observed for meadow steppe. However, the function was exponential for the sites in the three grassland types. This was due to possible differences in plant functional characteristics such as sensitivity to changes in rainfall [7,10,13,14]. Obviously, the explanatory exponential nature of MAP for variations in ANPP of desert steppe (31%) and meadow steppe (20%) was substantially weaker than that for the whole grassland types (50%) and/or typical steppe (56%). This trend could be related to the range of variation in MAP. MAP range was 265–520 mm for meadow steppe and 150–500 mm for desert steppe. However, typical steppe had the widest range of variation in MAP (140–640 mm). Note that range included that of MAP for the entire temperate grassland in the Loess Plateau. The results suggested that the PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 10 / 16 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Fig 5. Correlation between (a) aboveground net primary productivity (ANPP) and mean annual precipitation (MAP) and that separately between (b) precipitation-use efficiency (PUE) and MAP and also soil water storage (SWS) at various soil depths at the 101 soil water observation sites in the Fig 5. Correlation between (a) aboveground net primary productivity (ANPP) and mean annual precipitation (MAP) and that separately between (b) precipitation-use efficiency (PUE) and MAP and also soil water storage (SWS) at various soil depths at the 101 soil water observation sites in the Loess Plateau grassland. doi:10.1371/journal.pone.0135490.g005 effects of MAP on ANPP somehow depended on the spatial scale used. Factors affecting variations in ANPP The results of this study are in agreement with those reported in other studies. Several other studies have observed significant correlation between ANPP and MAP at regional scale, but relatively weak correla- tion at site scale [10,17,24]. Like MAP, PSD had a significant effect on spatial variations in ANPP in the Loess Plateau temperate grassland ecosystems. The results showed that more even PSD favored higher ANPP in the Loess Plateau, supporting the second hypothesis of this study. However, this finding was PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 11 / 16 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland inconsistent with others where more concentrated PSD improved ANPP [10,18,20]. The differ- ences in the ANPP patterns could be due to the differences in the characteristics of the climate, topography, landforms, soil and parent materials. As loess is subject to high erosion, a unique morphology has developed in the Loess Plateau. The typical landforms in the region are Loess “Yuan, Liang and Mao” and valleys of different magnitudes of erosion. Hills, deep gullies and undulating loess slopes are very common and are the characteristic landscapes of the Plateau region [35]. Subsequently, infrequent, but more extreme rainfall events, without concurrent changes in total precipitation could intensify soil and water erosion in the region [27,28,35]. This increases water and nutrient loss via runoff, lowers precipitation water infiltration into deep soil layers and reduces soil water content during growing season, thereby limiting plants photosynthesis. The negative correlations between slope gradient and measured ANPP for the 101 observa- tion sites could explain the issue of water and nutrient loss via runoff. The spatial variations in ANPP could be related with the complexity of the terrain. Other studies noted that concen- trated PSD could enhance precipitation water infiltration into deep soil layers and thereby lower water loss via evaporation [10,18,19,20]. However, weak evaporation water loss under concentrated PSD could not compensate for enhanced precipitation water loss via runoff because of complex terrain, sparse vegetation and loose soil in the Loess Plateau. Longer dry spells between rainfall events due to enhanced CVmp plus excessively waste of concentrated precipitation water via overland flow could extend the period of below-average soil water con- tent. Decrease in soil water availability could increase plant water stress, and thereby force plants to maintain low rates of photosynthesis. PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Factors affecting variations in PUE MAP, PSD, LAI and SWS had significant effects on the spatial variations in PUE in Loess Pla- teau temperate grassland. The results of this study were in good agreement with the results of several other studies [15,17,23,41] where PUE increased with increasing MAP. McNaughton [42] showed that PUE increased within 0.17–0.94 g m-2 mm-1 along a precipitation gradient of 480–1150 mm yr-1 in the Serengeti grassland of East Africa. Paruelo et al. [13] investigated spa- tial variations in PUE across a precipitation gradient of 200–1200 mm yr-1 and noted that PUE increased initially before decreasing with increasing MAP, peaking at MAP = 475 mm. The results here agreed with this pattern of annual precipitation range of 280–415 mm across the three grassland types (Table 1). However, other reports observed constant spatial PUE [2,6,24,43], while Huxman et al. [7] noted negative correlation between PUE and MAP across North America. Hu et al. [17] observed that PUE increased initially and then decrease with increasing MAP at continental and global scales. These discrepancies could be ascribed to the use of different MAP ranges and ecosystem types. Irrespectively, PUE for temperate Loess Pla- teau grasslands increased with increasing MAP. This is because in water-limited regions, vege- tation canopies grow close together and plants have much higher growth rates with increasing MAP. Thus plants use more precipitation for productivity and for transpiration. Contrary to MAP, PUE decreased with increasing CVmp. This implied that concentrated PSD weakened PUE of the temperate Loess Plateau grasslands. This is because there was more precipitation lost (via runoff) with increasing rainfall variability. Also severe soil and water ero- sion [35] meant less available precipitation water for plant use. These conditions reduced ANPP and thus PUE. The contribution of PSD to the variations in PUE was higher under meadow steppe and typical steppe than under desert steppe (Fig 5). This also suggested that ecosystem carbon and water cycle was less sensitive to changes in PSD in drier environments. This could be ascribed to the relatively low PUE and high water stress tolerance at direr sites. Compared with MAP, PSD explained more variations in PUE for the entire temperate Loess Plateau grassland. Thus changes in PSD due to climate change could have more effect on PUE than changes in total annual precipitation. This aspect hydrology is largely understudied. Factors affecting variations in ANPP This is especially holds for low-stress tolerance plants, a condition that could limit ANPP [19]. Also intensified soil and water erosion due to concentrated PSD could cause loss of nutrients and plant seeds in slope lands, thereby reducing plant species diversity and thus ANPP [15,32]. This study showed that MAP contribution to spatial variations in ANPP was greater than that of PSD for the Loess Plateau temperate grassland. This suggested that changes in PSD due to climate change are not commensurate to changes in total annual precipitation. For the entire temperate grassland, spatial variations in ANPP for typical and desert steppes were more affected by MAP than by PSD. However, the reverse trend was noted for meadow steppe. Thus as noted by Guo et al. [10], the relative significance of MAP and PSD depended on grassland type. Guo et al. [10] attributed this difference to the different compositions of functional plant types in different grassland types. In addition to MAP and PSD, the effects of SWS at various soil depths on spatial variations in ANPP were determined using in situ measurement of ANPP. Consistent with MAP, ANPP increased exponentially with increasing SWS. However, the effect of SWS on the spatial varia- tions in ANPP was less than that of MAP. Furthermore, the ability of SWS to explain the varia- tions in ANPP decreased with increasing soil depth. This could be related with the distribution of plant roots in the soil. With the exception of some artificial grassland (e.g., alfalfa), roots in the temperate Loess Plateau grasslands are predominantly distributed in the 0–40 cm soil layer [38,39]. Furthermore, root index (length density, root weight, root surface area and root diame- ter) generally decrease with increasing soil depth. The maximum root index is for the 0–30 cm soil layer [39,40]. Decreasing root index with increasing soil depth reduces deep soil water uptake by plants, and thereby weakens plant-soil water relation. Thus MAP was the dominant factor driving spatial variations in ANPP for the entire temperate Loess Plateau grasslands. Also the effect of PSD on the spatial variations in ANPP of a given grassland type cannot be ignored, especially for meadow steppes. 12 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland Factors affecting variations in PUE Except for climatic factors, there was a significant correlation between LAI and PUE in the Loess Plateau. LAI explained 78% of the spatial variations in PUE, much higher than that of MAP and PSD. This is consistent with the findings of Hu et al. [22] where LAI was reported as the primary driving factor of spatiotemporal variations in water use efficiency. LAI significantly influences carbon assimilation and transpiration to evapotranspiration ratio [44,45]. Further- more, regions with relatively high LAI exhibit low runoff; which element conserves soil water for plant use [14] and thus causes high PUE. This study showed the feasibility of predicting PUE from LAI in grassland ecosystems. No significant correlation was noted between soil properties and PUE, except for SWS. Consistent with ANPP, the effects of SWS on PUE also decreased with increasing soil depth. This was ascribed to the pattern of distribution of plant roots along soil depth. It was therefore concluded that changes in both vegetation structure (and thus in LAI) and PSD could significantly influence ecosystem carbon and water cycle in grasslands. Furthermore, several studies have shown different spatial and temporal variations in PUE. This underscores the underlying differences in regional and local ecological processes [15,17]. Thus there is the need for further studies on the temporal patterns of PUE and the con- trolling factors, especially in China’s Loess Plateau region. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 41390461 and No. 51179180). The authors are indebted to the editors of the journal and the reviewers for their constructive comments and suggestions that greatly improve the earlier ver- sion of this manuscript. Conclusions In this study, we quantified the effects of climatic, biotic and soil factors on spatial variations in ANPP and PUE in Loess Plateau temperate grassland. We noted exponential correlation 13 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135490 August 21, 2015 Primary Production and Rain-Use Efficiency in Loess Plateau Grassland between MAP and ANPP for the Loess Plateau temperate grassland, suggested that PUE increased with increasing MAP. Our study suggested that LAI was the primary factor control- ling the spatial variations in PUE in the entire Loess Plateau temperate grassland. This showed the feasibility for easily predicting PUE from LAI in grassland ecosystems. Both ANPP and PUE decreased with increasing CVmp, suggesting that more even precipitation distribution favored higher ANPP and PUE in the Loess Plateau grasslands. We therefore concluded that changes in vegetation structure, LAI and in the patterns of seasonal distribution of rainfall due to global climate change could significantly influence ecosystem carbon and water cycle in grasslands. 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https://openalex.org/W4292546505	https://pure.tue.nl/ws/files/217122887/catalysts_12_00916.pdf	English	null	Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis	Catalysts	2,022	cc-by	17,823	Document status and date: Published: 10/08/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. p • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron- Based Catalysts for Fischer-Tropsch Synthesis Citation for published version (APA): Chang, H., Lin, Q., Cheng, M., Zhang, K., Feng, B., Chai, J., Lv, Y., & Men, Z. (2022). Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts, 12(8), Article 916. https://doi.org/10.3390/catal12080916 Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron- Based Catalysts for Fischer-Tropsch Synthesis Citation for published version (APA): Chang, H., Lin, Q., Cheng, M., Zhang, K., Feng, B., Chai, J., Lv, Y., & Men, Z. (2022). Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts, 12(8), Article 916. https://doi.org/10.3390/catal12080916 Citation for published version (APA): Chang, H., Lin, Q., Cheng, M., Zhang, K., Feng, B., Chai, J., Lv, Y., & Men, Z. (2022). Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts, 12(8), Article 916. https://doi.org/10.3390/catal12080916 Citation for published version (APA): Chang, H., Lin, Q., Cheng, M., Zhang, K., Feng, B., Chai, J., Lv, Y., & Men, Z. (2022). Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts, 12(8), Article 916. https://doi.org/10.3390/catal12080916 Citation for published version (APA): Chang, H., Lin, Q., Cheng, M., Zhang, K., Feng, B., Chai, J., Lv, Y., & Men, Z. (2022). Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts, 12(8), Article 916. https://doi.org/10.3390/catal12080916 Document license: CC BY DOI: 10.3390/catal12080916 Document status and date: Published: 10/08/2022 Document license: CC BY DOI: 10.3390/catal12080916 Document status and date: Published: 10/08/2022 General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. You may not further distribute the material or use it for any profit making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Tav follow below link for the End User Agreement: Citation: Chang, H.; Lin, Q.; Cheng, M.; Zhang, K.; Feng, B.; Chai, J.; Lv, Y.; Men, Z. Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis. Catalysts 2022, 12, 916. https://doi.org/10.3390/ catal12080916 Academic Editors: Federico Galli, Nicolas Abatzoglou, Gregory Patience and Ajay K. Dalai Received: 6 July 2022 Accepted: 17 August 2022 Published: 19 August 2022 Keywords: potassium (K); loading effects; precipitated iron-based catalyst; iron–silica interaction; Fischer-Tropsch synthesis catalysts catalysts Effects of Potassium Loading over Iron–Silica Interaction, Phase Evolution and Catalytic Behavior of Precipitated Iron-Based Catalysts for Fischer-Tropsch Synthesis 1,, Meng Cheng 1, Kui Zhang 1, Bo Feng 1, Jiachun Chai 2, Yijun Lv 1 and Zhuowu Men 1, 1 National Institute of Clean-and-Low-Carbon Energy, Beijing 102211, China 2 Laboratory of Inorganic Materials and Catalysis, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5600 Eindhoven, The Netherlands * Correspondence: hai.chang@chnenergy.com.cn (H.C.); quan.lin@chnenergy.com.cn (Q.L.); zhuowu.men@chnenergy.com.cn (Z.M.); Tel.: +86-10-57595623 (H.C.); +86-10-57339389 (Q.L.); +86-10-57339358 (Z.M.) Abstract: Potassium (K) promoter and its loading contents were shown to have remarkable effects on the Fe–O–Si interaction of precipitated Fe/Cu/K/SiO2 catalysts for low-temperature Fischer- Tropsch synthesis (FTS). With the increase in K content from 2.3% (100 g Fe based) up to 7% in the calcined precursors, Fe–O–Si interaction was weakened, as reﬂected by ATR/FTIR, H2-TPR and XPS investigations. XRD results conﬁrmed that the diffraction peak intensity from (510) facet of χ-Fe5C2 phase strengthened with increasing K loading, which indicates the crystallite size of χ-Fe5C2 increased with the increase in K contents either during the syngas reduction/carburization procedure or after FTS reaction. H2-TPH results indicated that more reactive surface carbon (alpha-carbon) was obtained over the higher K samples pre-carburized by syngas. Raman spectra illustrated that a greater proportion of graphitic carbon was accumulated over the surface of spent samples with higher K loading. At the same time, ATR-FTIR, XRD and Mössbauer spectra (MES) characterization results showed that a relatively higher level of bulk phase Fayalite (Fe2SiO4) species was observed discernibly in the lowest K loading sample (2.3 K%) in this work. The catalytic evaluation results showed that the CO conversion, CO2 selectivity and O/P (C2–C4) ratio increased progressively with the increasing K loading, whereas a monotonic decline in both CO conversion and O/P (C2–C4) ratio was observed on the highest K loading sample during c.a. 280 h of TOS. www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl idi d t il d ill i ti t l i providing details and we will investigate your claim. Download date: 24. Oct. 2024 catalysts catalysts 1. Introduction Fischer-Tropsch synthesis (FTS) has widely been considered a viable route for con- verting syngas (H2+CO) derived from carbon-containing non-petroleum resources (e.g., coal, natural gas, biomass, etc.) into transportation fuels and/or other useful chemicals [1]. Precipitated iron-based catalysts are preferable for FTS reactions, which shows the charac- teristics of high activity, feed ﬂexibility (H2/CO = 0.5~2.5), a wider temperature window for process operation (230~350 ◦C) and cheaper precursor materials [2,3]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Iron-based FTS catalyst is well known as an industrially applicable multi-component embodiment, among which potassium is the most important electronic promoter [4,5]. Many researchers have been exploring its effects on physical/textural properties, reduction and carburization degree, and the catalytic performance of the precipitated iron-based catalyst [6–11]. Potassium (K) causes an increase in crystallite size and a decrease in the BET surface area. With the increase in the potassium content, the reduction of the Fe/Mn catalyst was retarded due to the strong interaction of iron oxide with potassium oxide [6]. 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/catalysts Catalysts 2022, 12, 916. https://doi.org/10.3390/catal12080916 Catalysts 2022, 12, 916 2 of 22 In addition, the effects of potassium on facilitating CO-dissociative adsorption [6,8], weak- ening/suppressing H2 adsorption and dissociation [8,12,13], and strengthening Fe–C bond have well been interpreted by electronic effects. C.H. Bartholomew et al. [11] investigated the FT reaction and deactivation kinetics based on unpromoted and potassium-promoted iron catalysts. The author proposed a deactivation model involving parallel paths, i.e., si- multaneous conversion of (a) atomic carbon to polymeric to graphitic carbon and (b) active carbon-rich carbides to inactive carbon-poor carbides. The activation energy for the de- activation of Fe/K was consistent with the atomic-to-polymeric conversion being rate- determining, while that for Fe may be involved in the conversion of either polymeric to graphitic carbon and/or of active carbides to inactive carbides as the rate-determining step. 1. Introduction It was also reported that [9], in the case of a catalyst that is unpromoted or not K-promoted enough, the deactivation is attributable to the oxidation of iron carbide (χ-Fe5C2) into Fe3O4, while the deactivation rate was related to carbon deposition in terms of the catalysts with excessive K loading, which could embed iron carbide particles and cause the site blocking. Nevertheless, most of the above-mentioned research as executed over the iron catalyst without silica (SiO2) incorporation. y ( ) p SiO2 has been considered as a proper structural promoter for iron-based catalysts [4]. The formula 100Fe/5Cu/4.2K/25SiO2 from Ruhrchemie A. G. in Germany [14] was one of the classical recipes, which acts as the basic formula of precipitated iron-based FTS catalysts, has been widely used as a benchmark for basic research and industrial catalyst development [15–18]. In the research works related to SiO2-incorporation effects, some works have indicated that its promotion of the attrition property, FTS activity/stability and selectivity/hydrocarbon productivity of Fe catalyst seemed to some extent to exhibit contrary correlation [19,20]. These complicated effects of silica over catalyst performance have usually been attributed to the interaction between iron and SiO2 or potassium and the structural promoter. Actually, the iron hydroxide/oxyhydroxide formed during the precipitation usually possesses a large number of coordinated unsaturated sites (CUSs), which may interact with SiO2 via a hydrolysis reaction of surface hydroxyl groups between them to induce Fe–O–Si bonds [3,21,22]. This kind of interaction of Fe–O–Si was reported to result in the retardation of iron oxide reduction/carburization [23], strengthening of C, O adsorption on iron sites [3,22], lowering of CO converting activity [21] and so on. In addition, several works have proposed that there may also be an interaction between K and structural promoter SiO2 besides Fe–SiO2 interaction [20,23,24]. The authors observed decreased FTS activity with increasing SiO2 contents in Fe/Cu/K/SiO2 catalysts and pos- tulated that the increased support suppressed potassium basicity or its promotional effects. However, which kind of impacts potassium shows in turn toward Fe–SiO2 interaction, as well as how the potassium loading level affects the Fe–O–Si phase evolution and catalytic performance of silica incorporated in Fe/Cu/K catalysts, have scarcely been reported. 1. Introduction p p y y p In this paper, precipitated Fe/Cu/K/SiO2 FTS catalysts, the bulk structure of which shows the characteristic of ferrihydrite (FH), were prepared with different potassium loadings at an identical content of other components and subjected to an FTS performance test in a ﬁxed-bed reactor (FBR). Physico-chemical properties, bulk and surface structures and phase evolution of as-prepared/used catalysts, as well as the reduction/carburization behavior of fresh samples, were characterized by multiple techniques, including BET, IR(ATR), ex and in situ XRD, XPS, Raman spectra, Mössbauer spectroscopy (MES) and H2- TPR, H2-TPH. The obtained results are intended to provide new insight into potassium’s effects on SiO2-incorporated iron-based catalysts, the structure–performance relationship of which has been reported to be governed mainly by the iron–silica interaction. 2.1. Effect of K Loading on Fe–O–Si Interaction There are several weak bands observed in the 400–800 cm−1 spectral range, and one broad and stronger ab- sorption peak in the ranges 800–1200 cm−1 over each sample, while the IR spectrum above 1200 cm−1 is almost featureless. The bands in the lower wave-number range (less than 750 cm−1) were attributed to Fe-O and Fe-OH stretching or lattice vibrations [29–31]. Two weak peaks here, one at 551 cm−1 and another at c.a. 690 cm−1, are therefore characteristic ad- sorption bands of low crystalline FH [30]. It can be further observed, interestingly, that the position of the maximum of the most intense absorption peak shifts from 978 cm−1 toward 945 cm−1 (red shift: 978-->966-->951-->945 cm−1) in parallel with the increasing K loading, whereas the other bands at 400–750 cm−1 remain unaffected (Figure 2b). The po- sitions of the weak peaks below 750 cm−1 are identical among four samples, which indi- t th t F O b di ib ti [26] b l ff t d b th i ti f K l di The ATR-FTIR spectra of fresh samples are depicted in Figure 2. There are several weak bands observed in the 400–800 cm−1 spectral range, and one broad and stronger absorption peak in the ranges 800–1200 cm−1 over each sample, while the IR spectrum above 1200 cm−1 is almost featureless. The bands in the lower wave-number range (less than 750 cm−1) were attributed to Fe-O and Fe-OH stretching or lattice vibrations [29–31]. Two weak peaks here, one at 551 cm−1 and another at c.a. 690 cm−1, are therefore characteristic adsorption bands of low crystalline FH [30]. It can be further observed, interestingly, that the position of the maximum of the most intense absorption peak shifts from 978 cm−1 toward 945 cm−1 (red shift: 978–>966–>951–>945 cm−1) in parallel with the increasing K loading, whereas the other bands at 400–750 cm−1 remain unaffected (Figure 2b). The positions of the weak peaks below 750 cm−1 are identical among four samples, which indicates that Fe-O bonding vibration [26] was barely affected by the variation of K loading. cates that Fe-O bonding vibration [26] was barely affected by the variation of K loading. The band with the absorption peak at approx. 970 cm−1 can be indicative of a silica- rich Fe–O–Si or Fe–O–Si group in a local-composition environment with a lower Fe/Si ratio [22,32–34]. Eric van Steen et al. 2.1. Effect of K Loading on Fe–O–Si Interaction 2.1. Effect of K Loading on Fe–O–Si Interaction BET surface area, as well as pore volume of the four fresh precursors, decreased from 221.7 (Lab-1) to 200.4 (Lab-4) m2/g, and from 0.51 to 0.44 cm3/g as the K loading level increased from 2.3% to 7%, while the average pore diameter remained essentially Catalysts 2022, 12, 916 3 of 22 from vel in unchanged. These results are similar to those reported in the literature without or with SiO2 incorporation [6,25]. changed. These results are similar to those reported in the literature without or with SiO2 incorporation [6,25]. XRD illustrated that all samples show a broadened diffraction peak in the 2θ range unchanged. These results are similar to those reported in the literature without or with SiO2 incorporation [6,25]. changed. These results are similar to those reported in the literature without or with SiO2 incorporation [6,25]. XRD illustrated that all samples show a broadened diffraction peak in the 2θ range p XRD illustrated that all samples show a broadened diffraction peak in the 2θ range at around 35◦and 63◦, corresponding to a characteristic pattern of an FH diffraction [26,27] (see Figure 1). The diffraction pattern is so broad and the intensities of all four samples are basically identical to each other, which indicates that the crystallite of those iron oxides in all samples was so highly dispersed that they were hard to differentiate precisely by the powder-diffraction technique. The size diameter of a synthetic FH crystallite was reported to be very small [27,28]. XRD illustrated that all samples show a broadened diffraction peak in the 2θ range at around 35° and 63°, corresponding to a characteristic pattern of an FH diffraction [26,27] (see Figure 1). The diffraction pattern is so broad and the intensities of all four samples are basically identical to each other, which indicates that the crystallite of those iron oxides in all samples was so highly dispersed that they were hard to differentiate precisely by the powder-diffraction technique. The size diameter of a synthetic FH crystallite was re- ported to be very small [27,28]. Figure 1. XRD pattern of the calcined precursor samples. Figure 1. XRD pattern of the calcined precursor samples. Figure 1. XRD pattern of the calcined precursor samples. Figure 1. XRD pattern of the calcined precursor samples. The ATR-FTIR spectra of fresh samples are depicted in Figure 2. 2.1. Effect of K Loading on Fe–O–Si Interaction [22] studied nano-sized iron oxides with different levels of contents of surface silicate groups with an inverse approach and reported the absorption bands in the region of 870–1020 cm−1 to be typically ascribed to the Fe–O–Si stretching frequency, while the one at 940–952 cm−1 was ascribed to a bidentate ((=FeO)2- Si(OH)2) [35]. The author observed a blue shift in the band region that stepped across 940– y y g The band with the absorption peak at approx. 970 cm−1 can be indicative of a silica- rich Fe–O–Si or Fe–O–Si group in a local-composition environment with a lower Fe/Si ratio [22,32–34]. Eric van Steen et al. [22] studied nano-sized iron oxides with different levels of contents of surface silicate groups with an inverse approach and reported the absorption bands in the region of 870–1020 cm−1 to be typically ascribed to the Fe–O–Si stretching frequency, while the one at 940–952 cm−1 was ascribed to a bidentate ((=FeO)2-Si(OH)2) [35]. The author observed a blue shift in the band region that stepped across 940–952 cm−1, and attributed it to a change from tridentate to bidentate to monodentate bonding of the surface silanol group. M.S. Seehra et al. [27] investigated synthetic ferrihydrite nanoparticles doped with Si and observed a considerable IR band shift in frequency from the position at 870 cm−1 for 2Si% (FH based) to its location at ~1000 cm−1 for 50Si% (FH based) in parallel with an increasing trend of silicate contents. Furthermore, according to the study of Peter J. Swedlund’s group [35], the maximum IR absorbance at the lowest Si surface concentrations for H2SiO4 in FH suspension occurred at 945 cm−1, and this spectral feature Catalysts 2022, 12, 916 4 of 22 g to st Si has been attributed to a monomeric silicate surface species. Based on the above literature summary, those peaks, located at 945–978 cm−1, are securely ascribed to the Fe–O–Si stretching frequency, and the IR absorbance band with higher frequency is indicative of silica-rich Fe–O–Si. The red shift observed here could be attributed to the gradual decrease in both the surface silicate concentration and the Fe–O–Si interaction [36,37] in parallel with the increasing trend of K loading, even though the IR band shift is in a narrow range. 2.1. Effect of K Loading on Fe–O–Si Interaction That is, the higher the K loading in the sample, the lower the probability and extent of iron–silica interaction, which can be considered the consequence of the strengthened interruption of potassium towards the overall interaction. Therefore, the introduction of potassium and its loading content play important roles in Fe–O–Si. eature has been attributed to a monomeric silicate surface species. Based on the above terature summary, those peaks, located at 945–978 cm−1, are securely ascribed to the Fe– O–Si stretching frequency, and the IR absorbance band with higher frequency is indicative f silica-rich Fe–O–Si. The red shift observed here could be attributed to the gradual de- rease in both the surface silicate concentration and the Fe–O–Si interaction [36,37] in par- llel with the increasing trend of K loading, even though the IR band shift is in a narrow ange. That is, the higher the K loading in the sample, the lower the probability and extent f iron–silica interaction, which can be considered the consequence of the strengthened nterruption of potassium towards the overall interaction. Therefore, the introduction of otassium and its loading content play important roles in Fe–O–Si. Figure 2. ATR-FTIR spectra of fresh calcined samples: (a) full spectra range with 400–4000 cm−1; (b) spectra range with 400–1200 cm−1). Figure 2. ATR-FTIR spectra of fresh calcined samples: (a) full spectra range with 400–4000 cm−1; (b) spectra range with 400–1200 cm−1). igure 2. ATR-FTIR spectra of fresh calcined samples: (a) full spectra range with 400–4000 cm−1; (b) pectra range with 400–1200 cm−1). Figure 2. ATR-FTIR spectra of fresh calcined samples: (a) full spectra range with 400–4000 cm−1; (b) spectra range with 400–1200 cm−1). The reduction behavior of fresh samples in an H2 atmosphere was investigated by PR (see Figure 3). H2-TPR profiles roughly present two reduction curves [38]: a distinct The reduction behavior of fresh samples in an H2 atmosphere was investigated by TPR (see Figure 3). H2-TPR proﬁles roughly present two reduction curves [38]: a distinct sharp peak in the 185~285 ◦C temperature range represents the superposition of the reduction peak from iron (Fe3+) oxide toward lower-valanced iron species, mainly magnetite (Fe3O4), which also overlaps with that from CuO to Cu [10,25], and a prolonged weaker peak situ- ated at 340~740 ◦C corresponds to the reduction process of Fe3O4 to α-Fe [16,21,25,39,40]. 2.1. Effect of K Loading on Fe–O–Si Interaction This is indicative that the higher K level inhibits the reduction process of catalyst from Fe3+ oxide towards lower-valanced iron species (first step of reduction), whereas the end tem- perature of the second reduction peak moves apparently toward a lower-temperature zone with increased K loading, which seemed to facilitate the reduction of Fe3O4 (or per- haps through other types of Fe2+ species as an intermediate to metallic iron). Figure 3. Temperature programmed reduction profiles of the calcined samples. Figure 3. Temperature programmed reduction proﬁles of the calcined samples. Figure 3. Temperature programmed reduction profiles of the calcined samples. Figure 3. Temperature programmed reduction proﬁles of the calcined samples. As the K content increased, the first step of reduction was restrained, which can easily be understood by the inhibitory effects of strongly alkaline potassium towards the disso- ciation and adsorption of H2 and the strengthened Fe-O bond. This was evidenced by the H2 consumption in the TPR procedure (see Table 1 and Figure 4). It can be seen from the table that the H2 consumed in the first peak lowered from 0.27 mol H2/mol Fe (Lab-1) down to 0.218 mol H2/mol Fe (Lab-4). All of these H2 uptakes in the first peak (a little bit higher than the theoretical value of Fe3+ oxide to Fe3O4: 0.16–0.17 mol H2/mol Fe; lower than that of Fe3+ oxide to FeO: 0.5 mol H2/mol Fe) implies that (1) the first reduction pattern is safely attributable to the reduction of Fe3+ oxide toward the lower-valanced iron species (mainly Fe3O4); (2) the first reduction process also involves the parts of an in-depth reduc- ti f F 3 hi h i t d t bili d b th i ili i t ti [36] As the K content increased, the ﬁrst step of reduction was restrained, which can easily be understood by the inhibitory effects of strongly alkaline potassium towards the dissociation and adsorption of H2 and the strengthened Fe-O bond. This was evidenced by the H2 consumption in the TPR procedure (see Table 1 and Figure 4). It can be seen from the table that the H2 consumed in the ﬁrst peak lowered from 0.27 mol H2/mol Fe (Lab-1) down to 0.218 mol H2/mol Fe (Lab-4). 2.1. Effect of K Loading on Fe–O–Si Interaction According to the literature, the reduction in pure Fe2O3 [25] or Fe/SiO2 [3,22] in the ab- sence of CuO will occur at a temperature higher than about 300 ◦C, but it will markedly shift down to a lower reduction temperature range (215–280 ◦C) with the co-existence of CuO [25]. The latter process can also be divided into two parts: one with easier reduction Catalysts 2022, 12, 916 5 of 22 but it h the of Fe3O4 to α-Fe and the other with a harder reduction of Fe3O4 to metallic iron possibly via FeO or non-stoichiometric Fe2+ to α-Fe, which is mainly caused by the interference of iron–silica interaction over Fe–O–Si [21]. It is obvious (see Figure 3) that the ﬁrst reduc- tion peak shifts toward a higher temperature (227–>245–>252–>254 ◦C). This is indicative that the higher K level inhibits the reduction process of catalyst from Fe3+ oxide towards lower-valanced iron species (ﬁrst step of reduction), whereas the end temperature of the second reduction peak moves apparently toward a lower-temperature zone with increased K loading, which seemed to facilitate the reduction of Fe3O4 (or perhaps through other types of Fe2+ species as an intermediate to metallic iron). easier reduction of Fe3O4 to α-Fe and the other with a harder reduction of Fe3O4 to metallic iron possibly via FeO or non-stoichiometric Fe2+ to α-Fe, which is mainly caused by the interference of iron–silica interaction over Fe–O–Si [21]. It is obvious (see Figure 3) that the first reduction peak shifts toward a higher temperature (227-->245-->252-->254 °C). This is indicative that the higher K level inhibits the reduction process of catalyst from Fe3+ oxide towards lower-valanced iron species (first step of reduction), whereas the end tem- perature of the second reduction peak moves apparently toward a lower-temperature zone with increased K loading, which seemed to facilitate the reduction of Fe3O4 (or per- haps through other types of Fe2+ species as an intermediate to metallic iron). p g p ( ) that the higher K level inhibits the reduction process of catalyst from Fe3+ oxide towards lower-valanced iron species (ﬁrst step of reduction), whereas the end temperature of the second reduction peak moves apparently toward a lower-temperature zone with increased K loading, which seemed to facilitate the reduction of Fe3O4 (or perhaps through other types of Fe2+ species as an intermediate to metallic iron). 2.1. Effect of K Loading on Fe–O–Si Interaction Total H2 consumption during TPR for the fresh samples with different K loadings. Figure 4. Total H2 consumption during TPR for the fresh samples with different K loadings. Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. Sample Peak Peak Cen- t °C H2 Consumption, mol H2/mol Fe Fe% in P Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. Sample Peak Peak Centre, ◦C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Sample Peak Peak Cen- tre, °C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Figure 4. Total H2 consumption during TPR for the fresh samples with different K loadings. Figure 4. Total H2 consumption during TPR for the fresh samples with different K loadings. XPS data revealed further that the distinct interaction between iron and silica is af- fected apparently by potassium loading (Figure 5). As can be seen from the figure, the Fe2p binding energy of Lab-1 sample (the lowest K content) is 711.6 eV, and then the bind- ing energy gradually decreases with the increase in K loading. This is due to the Fe–O–Si interaction in the sample being weakened by increasing K content, which is consistent XPS data revealed further that the distinct interaction between iron and silica is affected apparently by potassium loading (Figure 5). As can be seen from the ﬁgure, the Fe2p binding energy of Lab-1 sample (the lowest K content) is 711.6 eV, and then the binding energy gradually decreases with the increase in K loading. This is due to the Fe–O– Si interaction in the sample being weakened by increasing K content, which is consistent with the results obtained by ATR-FTIR and H2-TPR. 2.1. Effect of K Loading on Fe–O–Si Interaction Thus, the increase in K loading in the Fe/Cu/K/SiO2 catalyst induced the weakening of Fe–O–Si interaction. g y g Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. bonding or coordination status between iron and silica. Thus, the increase in K loading in the Fe/Cu/K/SiO2 catalyst induced the weakening of Fe–O–Si interaction. Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. the Fe/Cu/K/SiO2 catalyst induced the weakening of Fe O Si interaction. Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. Sample Peak Peak Centre, ◦C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Sample Peak Peak Cen- tre, °C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Figure 4. Total H2 consumption during TPR for the fresh samples with different K loadings. Figure 4. Total H2 consumption during TPR for the fresh samples with different K loadings. Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. Sample Peak Peak Centre, ◦C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Sample Peak Peak Cen- tre, °C H2 Consumption, mol H2/mol Fe Fe% in First Peak Single Total Lab-1 I 226.8 0.27 1.42 19.11 II 589.9 0.6 / III 629.4 0.55 / Lab-2 I 245.3 0.24 1.23 19.83 II 579.1 0.858 / III 635.3 0.125 / Lab-3 I 252.3 0.237 1.1 21.42 II 558.3 0.868 / Lab-4 I 253.8 0.218 1.01 21.54 II 526.0 0.792 / Figure 4. 2.1. Effect of K Loading on Fe–O–Si Interaction All of these H2 uptakes in the ﬁrst peak (a little bit higher than the theoretical value of Fe3+ oxide to Fe3O4: 0.16–0.17 mol H2/mol Fe; lower than that of Fe3+ oxide to FeO: 0.5 mol H2/mol Fe) implies that (1) the ﬁrst reduction pattern is safely attributable to the reduction of Fe3+ oxide toward the lower-valanced iron species (mainly Fe3O4); (2) the ﬁrst reduction process also involves the parts of an in-depth reduction of Fe3+, which is prevented or stabilized by the iron–silica interaction [36]. tion of Fe3+, which is prevented or stabilized by the iron–silica interaction [36]. However, the classical electron-donating theory can hardly explain the case of the second step of reduction, which appeared to be accelerated. It is clear from Figure 3 and Table 1 that there is relatively higher H2 consumption, which requires a high temperature above 570 °C in the Lab-1 sample, less in Lab-2 and Lab-3, and negligible in the highest K- p y However, the classical electron-donating theory can hardly explain the case of the second step of reduction, which appeared to be accelerated. It is clear from Figure 3 and Table 1 that there is relatively higher H2 consumption, which requires a high temperature above 570 ◦C in the Lab-1 sample, less in Lab-2 and Lab-3, and negligible in the highest K- loading samples. It is reasonable to assume the higher K loading should degrade the intact interaction between iron and silica, so as to decrease or eliminate the induction of the hardly reducible iron species. Furthermore, the lower-valanced iron species besides magnetite with non-reducibility at temperatures below 300 ◦C, together with all of the residual iron species in higher K-loading samples, were reduced at a relatively lower temperature (less than 570 ◦C) in this study. Furthermore, the infrared spectrum mentioned above showed that the corresponding absorption band of Fe–O–Si vibration showed a “red shift” with increased K content, which can be attributed to the increased potassium weakening the Catalysts 2022, 12, 916 6 of 22 weak- bonding or coordination status between iron and silica. Thus, the increase in K loading in the Fe/Cu/K/SiO2 catalyst induced the weakening of Fe–O–Si interaction. g y g Table 1. Quantitative results of H2 consumption for the catalysts in the H2-TPR procedure. bonding or coordination status between iron and silica. 2.1. Effect of K Loading on Fe–O–Si Interaction The electronic structure of the surface from the XPS result shows that the Fe–O–Si interaction in the low K loading (e.g., Lab-1) sample is stronger, indicating that its electrons are difﬁcult to excite, while the binding energy of Fe2p on the sample with higher K is lower. It is noticeable that the binding energy of the catalyst with even the highest K loading is still higher than the typical binding energy of silica-free Fe2O3 (710.5 eV) reported in the literature [3], showing the silica species on the surface of all four samples [3,41], and it may further implied that the coating of an iron-based Fe/Cu/SiO2 catalyst with potassium will just weaken the iron–silica interaction instead of eradicate it. 7 of 22 with th Catalysts 2022, 12, 916 Figure 5. Fe2p spectra of as-prepared catalysts. 2.2. Effects of K Loading on Carburization of Catalysts In situ XRD measurements were carried out to study the phase transf fresh samples during syngas reduction. XRD patterns at different reduction d shown in Figure 6. With the progress of reduction, the crystal phase of the sa Figure 5. Fe2p spectra of as-prepared catalysts. Indeed, the iron–silica interaction in the precipitated iron-based catalyst is subtle. The participation of silica in precipitated iron catalyst is usually small (25% by mass in the typical recipe), so the silica species could not possibly interact directly with each of the iron atoms in the entire catalyst to induce the Fe-O-Si covalent bonding. However, it was documented that there should be a remote interaction between iron and silica, which was reﬂected by the continuously increasing electron-deﬁcient state of iron caused by the increasing content of SiO2 in the Fe/SiO2 binary model catalyst [3]. In the author’s work, with increasing content of SiO2 from 0 to 50%, the binding energy of Fe3p increased in a stepwise manner. However, the electrons in the Fe2p core level closer to the nuclei should hardly be more excited. Thus, the decreasing binding energy of Fe2p with the increase in K loading should imply a weakened iron–silica interaction. Figure 5. Fe2p spectra of as-prepared catalysts Figure 5 Fe2p spectra of as prepared cata Figure 5. Fe2p spectra of as-prepared catalysts. g p p p p y 2.2. Effects of K Loading on Carburization of Catalysts In situ XRD measurements were carried out to study the phase trans fresh samples during syngas reduction. 2.1. Effect of K Loading on Fe–O–Si Interaction XRD patterns at different reduction d shown in Figure 6. With the progress of reduction, the crystal phase of the sa Indeed, the iron–silica interaction in the precipitated iron-based catalyst is subtle. The participation of silica in precipitated iron catalyst is usually small (25% by mass in the typical recipe), so the silica species could not possibly interact directly with each of the iron atoms in the entire catalyst to induce the Fe-O-Si covalent bonding. However, it was documented that there should be a remote interaction between iron and silica, which was reﬂected by the continuously increasing electron-deﬁcient state of iron caused by the increasing content of SiO2 in the Fe/SiO2 binary model catalyst [3]. In the author’s work, with increasing content of SiO2 from 0 to 50%, the binding energy of Fe3p increased in a stepwise manner. However, the electrons in the Fe2p core level closer to the nuclei should hardly be more excited. Thus, the decreasing binding energy of Fe2p with the increase in K loading should imply a weakened iron–silica interaction. 2.2. Effects of K Loading on Carburization of Catalysts In situ XRD measurements were carried out to study the phase transformation of fresh samples during syngas reduction. XRD patterns at different reduction durations are shown in Figure 6. With the progress of reduction, the crystal phase of the samples gradually changed from FH phase to iron-carbide phase (χ-Fe5C2, the only detectable iron carbide species from XRD), and the crystallite size of χ-Fe5C2 calculated by (510) facet was remarkable with an increased K content. 8 of 22 facet wa Catalysts 2022, 12, 916 Figure 6. In situ XRD pattern of calcined samples during syngas reduction procedure (H2/CO = 20 from an ambient temperature up to 260 °C, and maintained at 260 °C). Figure 6. In situ XRD pattern of calcined samples during syngas reduction procedure (H2/CO = 20, from an ambient temperature up to 260 ◦C, and maintained at 260 ◦C). Figure 6. In situ XRD pattern of calcined samples during syngas reduction procedure (H2/CO = 2 rom an ambient temperature up to 260 °C, and maintained at 260 °C). Figure 6. In situ XRD pattern of calcined samples during syngas reduction procedure (H2/CO = 20, from an ambient temperature up to 260 ◦C, and maintained at 260 ◦C). The reactivity of iron carbide as well as other carbonaceous species induced from syngas reduction was further investigated by temperature-programmed hydrogenatio TPH). Gaussian fitting results of the TPH spectral line are given in Figure 7, and the a ribution temperature range of carbon species is listed in Table 2. The carbon species wer assigned to carbidic carbon (atomic C), amorphous carbon, β carbon (amorphous surfac methyl chains or films) and bulk carbide carbon based on the previous literature [42,43 The first two kinds of carbon (carbidic and amorphous), the hydrogenation temperatur of which is below 390 °C, can also be recognized as alpha-carbon (alpha-C) [17]. None o graphite carbon, the hydrogenation temperature of which should be above 700 °C [42–44 was detected in the TPH profiles of the pre-carburized samples. The figure shows that th hydrogenation end temperature of the bulk carbide carbon in the Lab-1 sample reache c.a. 700 °C, which is comparable to that of other samples, decrease with the increase in oading. 2.2. Effects of K Loading on Carburization of Catalysts This may indicate that the bulk carbide carbon in the samples with higher K con ent can be hydrogenated at a lower temperature, as the peak temperature increases in th order of decreasing reactivity with H2 [43]. It has been reported that the addition of silic argely changes the adsorption sites of the iron-based catalysts which can improve th The reactivity of iron carbide as well as other carbonaceous species induced from syngas reduction was further investigated by temperature-programmed hydrogenation (TPH). Gaussian ﬁtting results of the TPH spectral line are given in Figure 7, and the attribution temperature range of carbon species is listed in Table 2. The carbon species were assigned to carbidic carbon (atomic C), amorphous carbon, β carbon (amorphous surface methyl chains or ﬁlms) and bulk carbide carbon based on the previous literature [42,43]. The ﬁrst two kinds of carbon (carbidic and amorphous), the hydrogenation temperature of which is below 390 ◦C, can also be recognized as alpha-carbon (alpha-C) [17]. None of graphite carbon, the hydrogenation temperature of which should be above 700 ◦C [42–44], was detected in the TPH proﬁles of the pre-carburized samples. The ﬁgure shows that the hydrogenation end temperature of the bulk carbide carbon in the Lab-1 sample reaches c.a. 700 ◦C, which is comparable to that of other samples, decrease with the increase in K loading. This may indicate that the bulk carbide carbon in the samples with higher K content can be hydrogenated at a lower temperature, as the peak temperature increases in the order of decreasing reactivity with H2 [43]. It has been reported that the addition of silica largely changes the adsorption sites of the iron-based catalysts, which can improve the adsorption strengths of H, C, and O on reduced or carburized catalysts [3]. argely changes the adsorption sites of the iron-based catalysts, which can improve th adsorption strengths of H, C, and O on reduced or carburized catalysts [3]. It is noteworthy that the more K content in the sample, the more carbidic carbon (atomic carbon) is formed over it (see Table 3). It was reported that alpha-carbon is highly reactive and essential to FTS reactions to some extent [42]. Thus, the relationship of alpha- carbon and bulk carbide carbon with K loading is plotted in Figure 8. The relative content of alpha-carbon increased proportionally with the increasing K loading, while that of bulk carbide carbon was decreased. It is noteworthy that the more K content in the sample, the more carbidic carbon atomic carbon) is formed over it (see Table 3). It was reported that alpha-carbon is highly i d i l FTS i [42] Th h l i hi f l h Table 3. Relative contents of carbonaceous species calculated from the corresponding peak area of Gaussian ﬁtting of H2-TPH patterns over the catalysts with a different potassium loading. 2.2. Effects of K Loading on Carburization of Catalysts Catalysts 2022, 12, 916 Catalysts 2022, 12, x FOR 9 of 22 9 of 22 Figure 7. Peak splitting and fitting of H2-TPH profiles of the four catalyst samples (color corre- spondance by, red: carbidic carbon; black: amorphous carbon; green: β carbon; blue: bulk carbon 1; purple: bulk carbon 2; brown: bulk carbon 3). Figure 7. Peak splitting and ﬁtting of H2-TPH proﬁles of the four catalyst samples (color correspon- dance by, red: carbidic carbon; black: amorphous carbon; green: β carbon; blue: bulk carbon 1; purple: bulk carbon 2; brown: bulk carbon 3). Figure 7. Peak splitting and fitting of H2-TPH profiles of the four catalyst samples (color corre- spondance by, red: carbidic carbon; black: amorphous carbon; green: β carbon; blue: bulk carbon 1; purple: bulk carbon 2; brown: bulk carbon 3). Figure 7. Peak splitting and ﬁtting of H2-TPH proﬁles of the four catalyst samples (color correspon- dance by, red: carbidic carbon; black: amorphous carbon; green: β carbon; blue: bulk carbon 1; purple: bulk carbon 2; brown: bulk carbon 3). Table 2. Attributed temperature of carbon species corresponding to the surface reaction of the H2- TPH process over catalysts with different potassium loading. Table 2. Attributed temperature of carbon species corresponding to the surface reaction of the H2-TPH process over catalysts with different potassium loading. Table 2. Attributed temperature of carbon species corresponding to the surface reaction of the H2- TPH process over catalysts with different potassium loading. Table 2. Attributed temperature of carbon species corresponding to the surface reaction of the H2-TPH process over catalysts with different potassium loading. Sample TPH Peak Temperature, °C Carbidic C Amorphous C β- C Bulk C1 Bulk C2 Bulk C3 Lab-1 324 385 446 504 552 625 Lab-2 312 365 / 523 562 624 Lab-3 319 385 / 473 544 / Lab-4 319 373 / 470 540 / Sample TPH Peak Temperature, ◦C Carbidic C Amorphous C β-C Bulk C1 Bulk C2 Bulk C3 Lab-1 324 385 446 504 552 625 Lab-2 312 365 / 523 562 624 Lab-3 319 385 / 473 544 / Lab-4 319 373 / 470 540 / It is noteworthy that the more K content in the sample, the more carbidic carbon (atomic carbon) is formed over it (see Table 3). It was reported that alpha-carbon is highly Table 3. 2.2. Effects of K Loading on Carburization of Catalysts Relative contents of carbonaceous species calculated from the corresponding peak area of Gaussian ﬁtting of H2-TPH patterns over the catalysts with a different potassium loading. reactive and essential to FTS reactions to some extent [42]. Thus, the relationship of alpha- carbon and bulk carbide carbon with K loading is plotted in Figure 8. The relative content of alpha-carbon increased proportionally with the increasing K loading, while that of bulk carbide carbon was decreased. Table 3. Relative contents of carbonaceous species calculated from the corresponding peak area of Gaussian fitting of H2-TPH patterns over the catalysts with a different potassium loading. Sample Carbon Species Assignment and Relative Percentage, % Sample Carbon Species Assignment and Relative Percentage, % Carbidic C Amorphous C β-C Bulk C1 Bulk C2 Bulk C3 Total Bulk C Lab-1 4.53 5.44 16.33 5.07 27.89 40.74 73.63 Lab-2 25.99 5.12 / 29.61 32.94 6.34 68.89 Lab-3 41.67 11.67 / 16.02 30.64 / 46.66 Lab-4 49.91 11.2 / 29.25 9.64 / 38.89 10 of 22 10 of 22 10 of 22 10 of 22 Catalysts 2022, 12, 916 Catalysts 2022, 12, x FOR Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. 2 3 Fischer-Tropsch Synthesis Performance Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. 2.3. Fischer-Tropsch Synthesis Performance Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. 2 3 Fi h T h S th i P f Figure 8. Relationship between reactive carbon and bulk carbide carbon contents with K loading. 2.3. Fischer-Tropsch Synthesis Performance 2 3 Fischer Tropsch Synthesis Performance 2.3. Fischer-Tropsch Synthesis Performance 2.3. Fischer-Tropsch Synthesis Performance Th FTS f f th f t l 2.3. Fischer-Tropsch Synthesis Performance The FTS performance of the four catalysts is illustrated in Figure 9. The initial activity increased progressively with increasing K loading. 2.2. Effects of K Loading on Carburization of Catalysts This might be attributed to the fact that increased reactive carbon can be achieved over the higher K loading samples after syngas reduction (see Figure 8). However, the deactivation of high K loading (Lab-4) was espe- cially evident after approx. 150 h of TOS, and its activity decreased monotonically there- after, dropping evenly to a lower level than that of Lab-3. In contrast, the activity of the remaining three samples was rather stable with TOS The FTS performance of the four catalysts is illustrated in Figure 9. The initial activity increased progressively with increasing K loading. This might be attributed to the fact that increased reactive carbon can be achieved over the higher K loading samples after syngas reduction (see Figure 8). However, the deactivation of high K loading (Lab-4) was especially evident after approx. 150 h of TOS, and its activity decreased monotonically thereafter, dropping evenly to a lower level than that of Lab-3. In contrast, the activity of the remaining three samples was rather stable with TOS. The FTS performance of the four catalysts is illustrated in Figure 9. The initial activity increased progressively with increasing K loading. This might be attributed to the fact that increased reactive carbon can be achieved over the higher K loading samples after syngas reduction (see Figure 8). However, the deactivation of high K loading (Lab-4) was espe- cially evident after approx. 150 h of TOS, and its activity decreased monotonically there- after, dropping evenly to a lower level than that of Lab-3. In contrast, the activity of the remaining three samples was rather stable with TOS. remaining three samples was rather stable with TOS. remaining three samples was rather stable with TOS. Figure 9. Variation of CO conversion with time on stream over the samples (Reaction conditions: FBR, H2/CO = 1.5, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 9. Variation of CO conversion with time on stream over the samples (Reaction conditions: FBR, H2/CO = 1.5, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 9. Variation of CO conversion with time on stream over the samples (Reaction conditions: FBR, H2/CO = 1.5, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). Carbon deposition over spent samples was characterized by Raman spectroscopy, and Figure 9. 2.2. Effects of K Loading on Carburization of Catalysts The calculated ratio of I(D)/I(G) was in the range of 0.51–0.56, which is closer to the value of standard graphite materials re- ported in the literature [45]. Even though the I(D)/I(G) ratio is a little bit higher than the standard graphite material (0.37), the relatively higher I(G) to I(D) in the higher K loading may imply that the deposition of graphitic carbon was higher for the higher K loading samples. It has been reported that the deposition of graphite-like inert carbon over the catalyst surface gives rise to the deactivation of iron catalysts, especially the K-promoted one [9,11]. However, the results from the Raman spectra may be insufficiently responsible for the faster deactivation rate observed over the Lab-4 sample, which cannot explain the best stability in Lab-3. Figure 10. Raman spectra of four spent catalysts. The inset pattern is the intensity of the D band (I(D)) and G band (I(G)) plotted with potassium loading. Figure 10. Raman spectra of four spent catalysts. The inset pattern is the intensity of the D band (I(D)) and G band (I(G)) plotted with potassium loading. Figure 10. Raman spectra of four spent catalysts. The inset pattern is the intensity of the D band I(D)) and G band (I(G)) plotted with potassium loading. Figure 10. Raman spectra of four spent catalysts. The inset pattern is the intensity of the D band (I(D)) and G band (I(G)) plotted with potassium loading. The CO2 and CH4 selectivity of the catalysts are shown in Figure 11. Similar to the CO conversion activity, the corresponding CO2 selectivity of the sample with higher K is also higher, which seems likely to coincide with the theory of the electron-donating effect of the K promoter. However, it is noteworthy that the CO2 selectivity did not decrease proportionally with the decline in the CO-conversion activity of Lab-4 (7.0 K%), which indicates that the declining activity of Lab-4 (after approx. 150 h) is mainly due to the loss of FTS reaction performance. This was further manifested by the olefin-to-paraffin ratio in the hydrocarbon products The CO2 and CH4 selectivity of the catalysts are shown in Figure 11. Similar to the CO conversion activity, the corresponding CO2 selectivity of the sample with higher K is also higher, which seems likely to coincide with the theory of the electron-donating effect of the K promoter. 2.2. Effects of K Loading on Carburization of Catalysts Variation of CO conversion with time on stream over the samples (Reaction conditions: Figure 9. Variation of CO conversion with time on stream over the samples (Reaction conditions: FBR, H2/CO = 1.5, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 9. Variation of CO conversion with time on stream over the samples (Reaction conditions: FBR, H2/CO = 1.5, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). FBR, H2/CO = 1.5, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Carbon deposition over spent samples was characterized by Raman spectroscopy, and the results are given in Figure 10. The Raman spectrum of the graphite shows, respectively, a sharp band (G-band) and a broad band (D-band) at 1580 and 1360 cm−1. It is shown that FBR, H2/CO = 1.5, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Carbon deposition over spent samples was characterized by Raman spectroscopy, and the results are given in Figure 10. The Raman spectrum of the graphite shows, respectively, a sharp band (G-band) and a broad band (D-band) at 1580 and 1360 cm−1. It is shown that Catalysts 2022, 12, 916 11 of 22 scopy, espec 11 of 22 scopy, espec the intensities of both the G-band and the D-band were strengthened with the increasing K contents (see inset pattern in Figure 10). The calculated ratio of I(D)/I(G) was in the range of 0.51–0.56, which is closer to the value of standard graphite materials reported in the literature [45]. Even though the I(D)/I(G) ratio is a little bit higher than the standard graphite material (0.37), the relatively higher I(G) to I(D) in the higher K loading may imply that the deposition of graphitic carbon was higher for the higher K loading samples. It has been reported that the deposition of graphite-like inert carbon over the catalyst surface gives rise to the deactivation of iron catalysts, especially the K-promoted one [9,11]. However, the results from the Raman spectra may be insufﬁciently responsible for the faster deactivation rate observed over the Lab-4 sample, which cannot explain the best stability in Lab-3. shown that the intensities of both the G-band and the D-band were strengthened with the increasing K contents (see inset pattern in Figure 10). 2.2. Effects of K Loading on Carburization of Catalysts Combined with the methane selectivity, the lower K loading samples exhibited a higher hydrogenation activity, which easily acquires the chain terminals of the hydrocar- bon products during FTS. The effects of K hindering H2 dissociation and adsorption on iron-based catalysts have been reported [8]. This evidence implies that, even though the increase in K content is beneficial to increasing the olefin/paraffin ratio in the early stages of FTS reaction, a steady-state conversion activity is more important. Figure 12 shows the olefin-to-paraffin ratio (O/P) in the C2–C4 section. It is clear from the figure that, with the increase in K loading, the O/P ratio increased in a stepwise man- ner during the initial periods of the FTS reaction, where the maximum value approached approximately 3.5 for the highest loading (Lab-4). However, the O/P ratio over this sample decreased obviously after approx. 150 h TOS, which coincides with the decline in CO con- version. Combined with the methane selectivity, the lower K loading samples exhibited a higher hydrogenation activity, which easily acquires the chain terminals of the hydrocar- bon products during FTS. The effects of K hindering H2 dissociation and adsorption on iron-based catalysts have been reported [8]. This evidence implies that, even though the increase in K content is beneficial to increasing the olefin/paraffin ratio in the early stages of FTS reaction, a steady-state conversion activity is more important. Figure 12. Olefin-to-paraffin ratio in the C2–C4 section with time in-stream over the samples (reac- tion conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 12. Oleﬁn-to-parafﬁn ratio in the C2–C4 section with time in-stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). Figure 12. Olefin-to-paraffin ratio in the C2–C4 section with time in-stream over the samples (reac- tion conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 12. Oleﬁn-to-parafﬁn ratio in the C2–C4 section with time in-stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). Figure 12. Olefin-to-paraffin ratio in the C2–C4 section with time in-stream over the samples (reac- tion conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). The chemical structures of the four spent samples was also investigated by ATR-FTIR (see Figure 13). 2.2. Effects of K Loading on Carburization of Catalysts However, it is noteworthy that the CO2 selectivity did not decrease proportionally with the decline in the CO-conversion activity of Lab-4 (7.0 K%), which indicates that the declining activity of Lab-4 (after approx. 150 h) is mainly due to the loss of FTS reaction performance. This was further manifested by the oleﬁn-to-parafﬁn ratio in the hydrocarbon products. in the hydrocarbon products. y p Figure 12 shows the oleﬁn-to-parafﬁn ratio (O/P) in the C2–C4 section. It is clear from the ﬁgure that, with the increase in K loading, the O/P ratio increased in a stepwise manner during the initial periods of the FTS reaction, where the maximum value approached approximately 3.5 for the highest loading (Lab-4). However, the O/P ratio over this sample decreased obviously after approx. 150 h TOS, which coincides with the decline in CO conversion. Combined with the methane selectivity, the lower K loading samples exhibited a higher hydrogenation activity, which easily acquires the chain terminals of the hydrocarbon products during FTS. The effects of K hindering H2 dissociation and adsorption on iron-based catalysts have been reported [8]. This evidence implies that, even though the increase in K content is beneﬁcial to increasing the oleﬁn/parafﬁn ratio in the early stages of FTS reaction, a steady-state conversion activity is more important. 12 of 22 12 of 22 12 of 22 12 of 22 Catalysts 2022, 12, 916 Catalysts 2022, 12, x FOR Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 12 shows the olefin-to-paraffin ratio (O/P) in the C2–C4 section. It is clear from the figure that, with the increase in K loading, the O/P ratio increased in a stepwise man- ner during the initial periods of the FTS reaction, where the maximum value approached approximately 3.5 for the highest loading (Lab-4). However, the O/P ratio over this sample decreased obviously after approx. 150 h TOS, which coincides with the decline in CO con- version. 2.2. Effects of K Loading on Carburization of Catalysts Combined with the methane selectivity, the lower K loading samples exhibited a higher hydrogenation activity, which easily acquires the chain terminals of the hydrocar- bon products during FTS. The effects of K hindering H2 dissociation and adsorption on iron-based catalysts have been reported [8]. This evidence implies that, even though the increase in K content is beneficial to increasing the olefin/paraffin ratio in the early stages of FTS reaction, a steady-state conversion activity is more important. Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 12 shows the olefin-to-paraffin ratio (O/P) in the C2–C4 section. It is clear from the figure that, with the increase in K loading, the O/P ratio increased in a stepwise man ner during the initial periods of the FTS reaction, where the maximum value approache approximately 3.5 for the highest loading (Lab-4). However, the O/P ratio over this sampl decreased obviously after approx. 150 h TOS, which coincides with the decline in CO con version. Combined with the methane selectivity, the lower K loading samples exhibited higher hydrogenation activity, which easily acquires the chain terminals of the hydroca bon products during FTS. The effects of K hindering H2 dissociation and adsorption o iron-based catalysts have been reported [8] This evidence implies that even though th Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 °C; Activation: H2/CO = 20, 260 °C, 24 h). Figure 11. CO2 and CH4 selectivity with time on stream over the samples (reaction conditions: FBR, 2.3 MPa, 235 ◦C; Activation: H2/CO = 20, 260 ◦C, 24 h). increase in K content is beneficial to increasing the olefin/paraffin ratio in the early stages of FTS reaction, a steady-state conversion activity is more important. Figure 12 shows the olefin-to-paraffin ratio (O/P) in the C2–C4 section. It is clear from the figure that, with the increase in K loading, the O/P ratio increased in a stepwise man- ner during the initial periods of the FTS reaction, where the maximum value approached approximately 3.5 for the highest loading (Lab-4). However, the O/P ratio over this sample decreased obviously after approx. 150 h TOS, which coincides with the decline in CO con- version. 2.2. Effects of K Loading on Carburization of Catalysts Evidently, a crystal-like Fayalite compound was formed in the Lab-1 catalyst during the period of FTS reaction, but it did not show up in the other three samples. It was reported that the formation of the ferrous silicate compound could only be brought about when an intimate contact between the support and the iron precursor was achieved [47]. This supports the IR and H2-TPR results from fresh precursors, where the iron–silica interaction between Fe–O–Si is considered stronger for the sample with lower K loading. The result is further in good accordance with the infrared spectrum results of the spent catalysts. Figure 13. FTIR spectra of the spent catalysts following FTS reaction. Figure 13. FTIR spectra of the spent catalysts following FTS reaction. XRD patterns of the spent catalysts are given in Figure 14. The strong diffraction peaks at 2θ = 36.5°, 39.5°, 40.3°, 45.8, and 60.0° are ascribed to the diffraction of quartz beads (SiO2) (JCPDS card 46-1045). The peaks at 40.8, 41.1, 43.4°, 44.2°, 45.1°, 45.8°, 47.3° and 58.3° can be attributed to the characteristic diffraction of Hägg carbide (χ-Fe5C2) (JCPDS card 89-2544). As the result of searching the JCPDS databases, we found that χ- Fe5C2 is the single iron carbide phase of four spent samples. The peaks at 30.1°, 35.5°, 37.1°, 43.1°, 53.5°, 57.0° and 62.6° were attributed to the characteristic diffraction of Fe3O4 (JCPDS card 65-3107). Furthermore, the XRD profile of the spent Lab-1 catalyst is quite different from the others, which shows clear characteristic diffraction at 31.7°, 34.1°, 34.2°, 35.0°, XRD patterns of the spent catalysts are given in Figure 14. The strong diffraction peaks at 2θ = 36.5◦, 39.5◦, 40.3◦, 45.8, and 60.0◦are ascribed to the diffraction of quartz beads (SiO2) (JCPDS card 46-1045). The peaks at 40.8, 41.1, 43.4◦, 44.2◦, 45.1◦, 45.8◦, 47.3◦and 58.3◦can be attributed to the characteristic diffraction of Hägg carbide (χ-Fe5C2) (JCPDS card 89-2544). As the result of searching the JCPDS databases, we found that χ-Fe5C2 is the single iron carbide phase of four spent samples. The peaks at 30.1◦, 35.5◦, 37.1◦, 43.1◦, 53.5◦, 57.0◦and 62.6◦were attributed to the characteristic diffraction of Fe3O4 (JCPDS card 65-3107). Furthermore, the XRD proﬁle of the spent Lab-1 catalyst is quite different from the others, which shows clear characteristic diffraction at 31.7◦, 34.1◦, 34.2◦, 35.0◦, 35.9◦, 37.3◦, 39.1◦, 51.5◦and 61.2◦. These peaks are attributed to the characteristic diffraction of Fayalite (JCPDS card 09-0307). 2.2. Effects of K Loading on Carburization of Catalysts The infrared spectra of the samples changed noticeably compared with those of the fresh precursors (see Figure 2). The common point is that the absorption peak of the Fe-O bonding with the characteristic of low-crystalline FH located at 400–750 cm−1 becomes more obvious. One visible shoulder peak at ~717 cm−1 emerged, which is the absorption band of the Fe-O in the FH [26], at the expense of the shoulder peak located at 690 cm−1 in fresh precursors. The intensity of the strong absorption peak corresponding to Fe–O–Si in the FH structure at 800–1200 cm−1 is relatively weak, and all of the wave- number of the main peak maxima are centered at ~945 cm−1. The difference is that in the spectrum of Lab-1, the peak intensity (551 cm−1) caused by Fe-O absorption is signiﬁcantly Catalysts 2022, 12, 916 13 of 22 -num- e spec 13 of 22 -num- e e stronger than that of the other three samples, and a new strong band was observed at ~866 cm−1. The strengthened peak intensity of Lab-1 at 551 cm−1 implies that a greater Fe-O bond emerged in the chemical structure of the spent catalyst sample. Meanwhile, according to the literature, an absorption band with the characteristic of a pure Fayalite (Fe2SiO4) occurs at an IR frequency of 870 cm−1 [33,46]. Therefore, the adsorption band here at 866 cm−1 can tentatively be assigned to Fe2SiO4. It can be envisaged that after c.a. 280 h of FTS reaction, the infrared spectrum shows a characteristic peak of the SiO32−group (in the range of 700–750 cm−1) in all four spent samples, while the obvious absorption peak with the characteristic of Fe2SiO4 appears in the sample with the lowest K content, over which the Fe2+ ion should be stabilized by the strong Fe–O–Si interaction. stronger than that of the other three samples, and a new strong band was observed at ~866 cm−1. The strengthened peak intensity of Lab-1 at 551 cm−1 implies that a greater Fe-O bond emerged in the chemical structure of the spent catalyst sample. Meanwhile, according to the literature, an absorption band with the characteristic of a pure Fayalite (Fe2SiO4) occurs at an IR frequency of 870 cm−1 [33,46]. Therefore, the adsorption band here at 866 cm−1 can tentatively be assigned to Fe2SiO4. It can be envisaged that after c.a. 2.2. Effects of K Loading on Carburization of Catalysts 280 h of FTS reaction, the infrared spectrum shows a characteristic peak of the SiO32− group (in the range of 700– 750 cm−1) in all four spent samples, while the obvious absorption peak with the character- istic of Fe2SiO4 appears in the sample with the lowest K content, over which the Fe2+ ion should be stabilized by the strong Fe–O–Si interaction. Figure 13. FTIR spectra of the spent catalysts following FTS reaction. Figure 13. FTIR spectra of the spent catalysts following FTS reaction. Figure 13. FTIR spectra of the spent catalysts following FTS reaction. XRD patterns of the spent catalysts are given in Figure 14. The strong diffraction peaks at 2θ = 36.5°, 39.5°, 40.3°, 45.8, and 60.0° are ascribed to the diffraction of quartz beads (SiO2) (JCPDS card 46-1045). The peaks at 40.8, 41.1, 43.4°, 44.2°, 45.1°, 45.8°, 47.3° and 58.3° can be attributed to the characteristic diffraction of Hägg carbide (χ-Fe5C2) (JCPDS card 89-2544). As the result of searching the JCPDS databases, we found that χ- Fe5C2 is the single iron carbide phase of four spent samples. The peaks at 30.1°, 35.5°, 37.1°, 43.1°, 53.5°, 57.0° and 62.6° were attributed to the characteristic diffraction of Fe3O4 (JCPDS card 65-3107). Furthermore, the XRD profile of the spent Lab-1 catalyst is quite different from the others, which shows clear characteristic diffraction at 31.7°, 34.1°, 34.2°, 35.0°, Figure 13. FTIR spectra of the spent catalysts following FTS reaction. XRD patterns of the spent catalysts are given in Figure 14. The strong diffraction peaks at 2θ = 36.5◦, 39.5◦, 40.3◦, 45.8, and 60.0◦are ascribed to the diffraction of quartz beads (SiO2) (JCPDS card 46-1045). The peaks at 40.8, 41.1, 43.4◦, 44.2◦, 45.1◦, 45.8◦, 47.3◦and 58.3◦can be attributed to the characteristic diffraction of Hägg carbide (χ-Fe5C2) (JCPDS card 89-2544). As the result of searching the JCPDS databases, we found that χ-Fe5C2 is the single iron carbide phase of four spent samples. The peaks at 30.1◦, 35.5◦, 37.1◦, 43.1◦, 53.5◦, 57.0◦and 62.6◦were attributed to the characteristic diffraction of Fe3O4 (JCPDS card 65-3107). Furthermore, the XRD proﬁle of the spent Lab-1 catalyst is quite different from the others, which shows clear characteristic diffraction at 31.7◦, 34.1◦, 34.2◦, 35.0◦, 35.9◦, 37.3◦, 39.1◦, 51.5◦and 61.2◦. These peaks are attributed to the characteristic diffraction of Fayalite (JCPDS card 09-0307). 2.2. Effects of K Loading on Carburization of Catalysts Crystallite Size, nm Lab-1 Lab-2 Lab-3 Lab-4 a Fresh samples <8 <8 <8 <8 b χ-Fe5C2 12 15 20 41 a: confirmed by TEM; b: calculated by an XRD signal of (510) facet. The iron-phase composition of each of the spent catalysts was further determined by Table 4. The crystallite size of the fresh samples (oxide) and χ-Fe5C2 over the spent samples after FTS reaction (c.a. 280 h). Crystallite Size, nm Lab-1 Lab-2 Lab-3 Lab-4 a Fresh samples <8 <8 <8 <8 b χ-Fe5C2 12 15 20 41 a: conﬁrmed by TEM; b: calculated by an XRD signal of (510) facet. Table 4. The crystallite size of the fresh samples (oxide) and χ-Fe5C2 over the spent samples after TS reaction (c.a. 280 h). Table 4. The crystallite size of the fresh samples (oxide) and χ-Fe5C2 over the spent samples after FTS reaction (c.a. 280 h). p p p y y MES at an ambient temperature. The Mössbauer spectra are shown in Figure 15. MES parameters of various phases are deduced from the figure, and their relative contributions to spectral absorption areas are listed in Table 5 and Figure 16. As revealed in Figure 15 and Table 5, the MES line of each sample is the result of the superposition of at least six The iron-phase composition of each of the spent catalysts was further determined by MES at an ambient temperature. The Mössbauer spectra are shown in Figure 15. MES parameters of various phases are deduced from the ﬁgure, and their relative contributions to spectral absorption areas are listed in Table 5 and Figure 16. As revealed in Figure 15 and Table 5, the MES line of each sample is the result of the superposition of at least six sub- spectra including three doublets associated with the different iron sites. The two doublets with 0.339 mm/s < IS < 0.3598 mm/s, 0.602 mm/s < QS < 1.147 mm/s and without magnetic hyperﬁne splitting contribute to the Fe3+ sites (superparamagnetic, spm) [21,48] and the FH phase [23,49]. More precisely, the higher and lower QS values with near-identical IS values can be attributed to the Fe3+ ions located on the surface and in the bulk of the crystallites, respectively [48]. 2.2. Effects of K Loading on Carburization of Catalysts Evidently, a crystal-like Fayalite compound was formed in the Lab-1 catalyst during the period of FTS reaction, but it did not show up in the other three samples. It was reported that the formation of the ferrous silicate compound could only be brought about when an intimate contact between the support and the iron precursor was achieved [47]. This supports the IR and H2-TPR results from fresh precursors, where the iron–silica interaction between Fe–O–Si is considered stronger for the sample with lower K loading. The result is further in good accordance with the infrared spectrum results of the spent catalysts. 14 of 22 ger for e infra- 14 of 22 ger for e infra- Catalysts 2022, 12, 916 Figure 14. XRD pattern of the spent catalyst samples following FTS reaction (c.a. 280 h). Figure 14. XRD pattern of the spent catalyst samples following FTS reaction (c.a. 280 h). Figure 14. XRD pattern of the spent catalyst samples following FTS reaction (c.a. 280 h). Figure 14. XRD pattern of the spent catalyst samples following FTS reaction (c.a. 280 h). In addition, the crystallite size of χ-Fe5C2 over the spent samples in accordance with the (510) plane and that of the fresh samples are depicted in Table 4. Apparently, the crys- tallite growth of the iron carbide calculated by (510) facet was obvious in accordance with the increasing K loading. This is consistent with the result from in situ XRD in the syngas reduction in the fresh samples. Thus, the iron–silica interaction is evident in the size di- ameter and shows that for the highest K loading sample (7 K%), the crystallite sizes in- creased by a factor of four In addition, the crystallite size of χ-Fe5C2 over the spent samples in accordance with the (510) plane and that of the fresh samples are depicted in Table 4. Apparently, the crystallite growth of the iron carbide calculated by (510) facet was obvious in accordance with the increasing K loading. This is consistent with the result from in situ XRD in the syngas reduction in the fresh samples. Thus, the iron–silica interaction is evident in the size diameter and shows that for the highest K loading sample (7 K%), the crystallite sizes increased by a factor of four. Table 4. The crystallite size of the fresh samples (oxide) and χ-Fe5C2 over the spent samples after FTS reaction (c.a. 280 h). 2.2. Effects of K Loading on Carburization of Catalysts The subspectra with both magnetic hyperﬁne splitting and quardrple splitting, where Hhf in the range 45.59~50.23 T, contribute to the A (tetra)/B (hexa) of two different sites of the ferrimagnetic Fe3O4 phase [3,48]. Those with 0.20 mm/s < IS < 0.40 mm/s and 18.54 T < Hhf < 20.579 T correspond to the two different sites of the stoichiometric iron carbide (χ-Fe5C2) [3,48,50]. The results in Table 5 and Figure 16 show Catalysts 2022, 12, 916 Catalysts 2022, 12, x FOR 15 of 22 16 of 22 15 of 22 16 of 22 that the relative content of the iron carbide phase (χ-Fe5C2) increased from 13.38% (Lab-1) to 25.03% (Lab-4) with increasing K content. degree of crystallite sintering was the smallest after a long-term FTS reaction in four sam- ples. that the relative content of the iron carbide phase (χ-Fe5C2) increased from 13.38% (Lab-1) to 25.03% (Lab-4) with increasing K content. degree of crystallite sintering was the smallest after a long-term FTS reaction in four sam- ples. Figure 15. MES spectra of samples after FTS reaction. Table 5. MES spectrum analysis results of four spent catalysts with different potassium contents after the FTS reaction Figure 15. MES spectra of samples after FTS reaction. Table 5. MES spectrum analysis results of four spent catalysts with different potassium contents after the FTS reaction. Figure 15. MES spectra of samples after FTS reaction. Figure 15. MES spectra of samples after FTS reaction. Table 5. MES spectrum analysis results of four spent catalysts with different potassium contents after the FTS reaction Table 5. MES spectrum analysis results of four spent catalysts with different potassium contents after the FTS reaction. Cat. 2.2. Effects of K Loading on Carburization of Catalysts Phases Mossbauer Parameters Phase Percentage (%) IS (mm·s−1) QS (mm·s−1) Hhf (T) Area (%) Mixed M+Q (1) Fe3O4 (A) 0.3089 −0.0238 50.235 21.49 Fe3O4: 37.06 Mixed M+Q (2) Fe3O4 (B) 0.6825 0.01489 46.83 15.57 / Mixed M+Q (3) χ-Fe5C2 (I) 0.5039 0.241 20.579 7.87 χ-Fe5C2: 18.82 Mixed M+Q (4) χ-Fe5C2 (II) 0.0586 −0.1324 19.926 10.95 / Lab-4 Doublet1 Fe3+ (spm) 0.339 0.6685 / 5.49 FH: 34.06 Doublet2 Fe3+ (spm) 0.3594 0.9918 / 28.57 / Doublet3 Fe2SiO4 1.0144 2.2969 / 9.21 Fe2SiO4: 9.21 Mixed M+Q (1) Fe3O4 (A) 0.271 −0.06778 49.089 17.98 Fe3O4: 31.7 Mixed M+Q (2) Fe3O4 (B) 0.738 0.0879 46.539 13.72 / Mixed M+Q (3) χ-Fe5C2 (I) 0.411 0.2115 20.525 11.91 χ-Fe5C2: 25.03 Mixed M+Q (4) χ-Fe5C2 (II) 0.04487 −0.05465 19.749 13.12 / Catalysts 2022, 12, x FOR PEER REVIEW 17 of 22 Mixed M+Q (3) χ-Fe5C2 (I) 0.5039 0.241 20.579 7.87 χ-Fe5C2: 18.82 Mixed M+Q (4) χ-Fe5C2 (II) 0.0586 −0.1324 19.926 10.95 / Lab-4 Doublet1 Fe3+ (spm) 0.339 0.6685 / 5.49 FH: 34.06 Doublet2 Fe3+ (spm) 0.3594 0.9918 / 28.57 / Doublet3 Fe2SiO4 1.0144 2.2969 / 9.21 Fe2SiO4: 9.21 Mixed M+Q (1) Fe3O4 (A) 0.271 −0.06778 49.089 17.98 Fe3O4: 31.7 Mixed M+Q (2) Fe3O4 (B) 0.738 0.0879 46.539 13.72 / Mixed M+Q (3) χ-Fe5C2 (I) 0.411 0.2115 20.525 11.91 χ-Fe5C2: 25.03 Mixed M+Q (4) χ-Fe5C2 (II) 0.04487 −0.05465 19.749 13.12 / Figure 16. Relative contents of each phase acquired from MES over four spent catalyst samples. Figure 16. Relative contents of each phase acquired from MES over four spent catalyst samples. Figure 16. Relative contents of each phase acquired from MES over four spent catalyst samples. Figure 16. Relative contents of each phase acquired from MES over four spent catalyst samples. 3. Materials and Methods 3.1. Materials H2 (99.9%) and CO (99.9%) were purchased from Beijing AP BAIF gases industry Co., Ltd. (Beijing, China) and were desulfurized (up to <0.05 μg/g), deoxidized and dehydrated before usage. Fe(NO3)3·9H2O, Cu(NO3)2·3H2O, Na2CO3 and K2SiO3 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were used as re- ceived. 3.2. Catalyst Preparation The oxide catalyst precursors were prepared by the co-precipitation method, fol- lowed by promoter/binder addition, drying and calcination. In brief, the mixed ferric ni- trate and copper nitrate solutions with the desired Fe3+/Cu2+ ratio were co-precipitated with sodium carbonate solution under a temperature of 70 °C and maintaining a pH at ~7. 2.2. Effects of K Loading on Carburization of Catalysts Phases Mossbauer Parameters Phase Percentage (%) IS (mm·s−1) QS (mm·s−1) Hhf (T) Area (%) Lab-1 Doublet1 Fe3+ (spm) 0.3378 0.602 / 6.16 FH: 38.45 Doublet2 Fe3+ (spm) 0.3356 1.025 / 32.29 / Doublet3 Fe2SiO4 1.138 2.806 / 15.07 Fe2SiO4: 15.07 Mixed M+Q (1) Fe3O4 (A) 0.294 0.0075 49.98 15.78 Fe3O4: 33.1 Mixed M+Q (2) Fe3O4 (B) 0.617 −0.064 46.61 17.32 / Mixed M+Q (3) χ-Fe5C2 0.109 −0.0656 19.009 13.38 χ-Fe5C2: 13.38 Lab-2 Doublet1 Fe3+ (spm) 0.363 0.628 / 9.19 FH: 46.86 Doublet2 Fe3+ (spm) 0.3565 1.014 / 37.67 / Doublet3 Fe2SiO4 0.988 2.392 / 4.15 Fe2SiO4: 4.15 Mixed M+Q (1) Fe3O4 (A) 0.314 −0.0368 49.015 12.73 Fe3O4: 31.13 Mixed M+Q (2) Fe3O4 (B) 0.671 −0.0274 45.599 18.39 / Mixed M+Q (3) χ-Fe5C2 0.1936 0.1155 18.54 17.86 χ-Fe5C2: 17.86 Lab-3 Doublet1 Fe3+ (spm) 0.345 0.695 / 14.88 FH: 35.97 Doublet2 Fe3+ (spm) 0.3598 1.147 / 21.09 / Cat. Phases Mossbauer Parameters Phase Percentage (%) IS (mm·s−1) QS (mm·s−1) Hhf (T) Area (%) Lab-1 Doublet1 Fe3+ (spm) 0.3378 0.602 / 6.16 FH: 38.45 Doublet2 Fe3+ (spm) 0.3356 1.025 / 32.29 / Doublet3 Fe2SiO4 1.138 2.806 / 15.07 Fe2SiO4: 15.07 Mixed M+Q (1) Fe3O4 (A) 0.294 0.0075 49.98 15.78 Fe3O4: 33.1 Mixed M+Q (2) Fe3O4 (B) 0.617 −0.064 46.61 17.32 / Mixed M+Q (3) χ-Fe5C2 0.109 −0.0656 19.009 13.38 χ-Fe5C2: 13.38 Lab-2 Doublet1 Fe3+ (spm) 0.363 0.628 / 9.19 FH: 46.86 Doublet2 Fe3+ (spm) 0.3565 1.014 / 37.67 / Doublet3 Fe2SiO4 0.988 2.392 / 4.15 Fe2SiO4: 4.15 Mixed M+Q (1) Fe3O4 (A) 0.314 −0.0368 49.015 12.73 Fe3O4: 31.13 Mixed M+Q (2) Fe3O4 (B) 0.671 −0.0274 45.599 18.39 / Mixed M+Q (3) χ-Fe5C2 0.1936 0.1155 18.54 17.86 χ-Fe5C2: 17.86 Lab-3 Doublet1 Fe3+ (spm) 0.345 0.695 / 14.88 FH: 35.97 Doublet2 Fe3+ (spm) 0.3598 1.147 / 21.09 / Doublet3 Fe2SiO4 0.9594 2.354 / 8.16 Fe2SiO4: 8.15 Catalysts 2022, 12, 916 16 of 22 Table 5. Cont. Table 5. Cont. Cat. 2.2. Effects of K Loading on Carburization of Catalysts The generated precursor precipitation was followed by filtering and washing. The filter cakes were re-slurried in DI water, followed by adding a certain stoichiometric aqueous solution of potassium silicate. The obtained slurry was divided equally into four parts and Interestingly but unexpectedly, the subspectra of the remaining doublet with the char- acteristics of Fayalie are observed in the MES spectrum of all spent samples. According to the literature, the crystal-like structure of Fayalie (Fe2SiO4) is orthorhombic and consists of an approximately hexagonal close-packed arrangement of the oxygen atoms, silicon occur- ring at the tetrahedrally coordinated position, and iron located at two octahedral coordi- nated positions and shows relatively higher IS and QS values in MES measurements [21,51]. Generally, the isomer shift for iron cations with lower oxidation states is higher, while their different spin states could be differentiated with the help of quadruple splitting values. There have been several research works observing these kinds of higher values. Haiyun Suo et al. associated the subspectrum with IS = 1.33 mm/s and QS = 2.93 mm/s with crys- talline fayalite (Fe2SiO4) [3]. In an earlier work, A.F.H. Wielers et al. reported that a direct formation of a Fe2+ compound with MES parameters (IS = 1.22 mm/s; QS = 2.30 mm/s) is essentially different from Fe1−xO, and it was attributed to a ferrous silicate but not a ferrous oxide [47]. Furthermore, Yuen et al. observed, during the reduction of 1 wt% Fe/SiO2 catalyst reduced at increasingly severe conditions, two Fe2+ doublets. An inner doublet (IS = 1.06 mm/s; QS = 0.93 ± 0.03 mm/s) was attributed to the cations of low coordination (denoted as iron surface silicate), and an outer doublet (IS = 1.28 mm/s; QS = 1.84 mm/s) Catalysts 2022, 12, 916 17 of 22 17 of 22 was ascribed to the Fe2+ cations of high coordination (present in highly dispersed iron(II) oxide particles) [52]. Taking the overall summary of the literature into consideration, the phase with the higher QS value (2.806 mm/s) for high spin states exists in the bulk region, while that with lower values (2.29–2.39 mm/s) is located at or near the surface region, thus explaining the reason why the bulk-phase Fe2+ silicate was detected only in the X-ray diffraction over the spent sample of the Lab-1, with the exception of none of the others. 3.1. Materials H2 (99.9%) and CO (99.9%) were purchased from Beijing AP BAIF gases industry Co., Ltd. (Beijing, China) and were desulfurized (up to <0.05 µg/g), deoxidized and dehydrated before usage. Fe(NO3)3·9H2O, Cu(NO3)2·3H2O, Na2CO3 and K2SiO3 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were used as received. 2.2. Effects of K Loading on Carburization of Catalysts MES results revealed that in the bulk phase of the spent samples, the relative content of iron carbide (χ-Fe5C2) was increased with increasing K loading, while a relatively higher level of Fayalite species was observed on the lowest K-loading sample. The higher content of the χ-Fe5C2 phase may rationalize the higher FTS activity [23]. However, it can hardly explain the decreased activity (Figure 9) or the O/P ratio (Figure 12) of the Lab-4 sample at the end of FTS run, since the χ-Fe5C2 phase was the only carbide phase out of all the samples according to XRD and MES characterization. There were no other types of carbon- poor carbides; e.g., θ-Fe3C was observed, which was proposed to be less reactive than the χ-Fe5C2 phase, thus causing the catalyst deactivation [11]. MES results also indicate that the magnetite phase in the spent Lab-4 was not the highest one. Instead, it is revealed by XRD results (Figure 14 and Table 4) on spent samples that the most severe crystallite growth was obtained for the highest K loading sample (Lab-4), which increased by a factor of four. This should explain the highest deactivation rate observed on the highest K loading, which could have severely decreased the active surface. Considering the results and discussion in the foregoing section, it should originate from the K-loading effects on the Fe–O–Si interaction. ATR, H2-TPR, and XPS results indicated that Fe–O–Si interaction was the weakest in the Lab-4 (7 K%) sample, while it was strongest in Lab-1 (2.3 K%), whose degree of crystallite sintering was the smallest after a long-term FTS reaction in four samples. 3.2. Catalyst Preparation The oxide catalyst precursors were prepared by the co-precipitation method, followed by promoter/binder addition, drying and calcination. In brief, the mixed ferric nitrate and copper nitrate solutions with the desired Fe3+/Cu2+ ratio were co-precipitated with sodium carbonate solution under a temperature of 70 ◦C and maintaining a pH at ~7. The generated precursor precipitation was followed by ﬁltering and washing. The ﬁlter cakes were re-slurried in DI water, followed by adding a certain stoichiometric aqueous solution of potassium silicate. The obtained slurry was divided equally into four parts and ﬁltered, respectively. The ﬁnal ﬁlter cakes were dried at 120 ◦C overnight and then calcined in a mufﬂe with air at 500 ◦C for 6 h. The resulting powders were crushed and sieved, respectively, into a diameter range of 80~150 µm before activity tests and other characterizations. The ﬁnal oxide precursor samples, of which XRF (X-ray ﬂuorescence, Rigaku ZSX Primus II (Tokyo, Japan)) analysis shows x K/100Fe, x = 2.3, 3.8, 5.6 and 7.0, respectively, were denoted as Lab-1, -2, -3 and -4. The contents of Cu, and SiO2 were kept identical in each sample. 3.3. Catalyst Characterization 3.3.7. X-ray Photoelectron Spectroscopy (XPS) 3.3.7. X-ray Photoelectron Spectroscopy (XPS) The sXPS experiment was recorded with a Thermo Fischer ESCALAB 250 Xi spectrom- eter at a base pressure of 1 × 10−9 mbar. The spectra were obtained at 20 eV pass energy with monochromatic Al Kα (1486.6 eV) radiation. The C1s peak of adventitious carbon (284.6 eV) was used as a reference for estimating the binding energy. The binding energies were given with an accuracy of ±0.1 eV. 3.3.3. In Situ XRD In situ XRD was carried out in a continuous mode on a D/max-2600/PC apparatus (Rigaku, Tokyo, Japan) equipped with a D/teX ultra-high-speed detector. The X-ray generator consisted of a Cu rotating anode target with a maximum power of 9 kW. All the tests were operated at 150 mA and 40 kV. In situ XRD patterns were recorded in an Anton Paar XRK-900 cell equipped with an H2/CO = 20 gas system at 260 ◦C at different duration up to 24 h. 3.3.1. Nitrogen Adsorption/Desorption 3.3.1. Nitrogen Adsorption/Desorption BET surface area and pore size distribution of catalysts were determined by nitrogen physisorption using an Autosorb IQ (Quantachrome instrument, Boynton Beach, FL, USA). Catalysts 2022, 12, 916 18 of 22 18 of 22 Each sample was degassed under vacuum at 1.43 × 10−6 MPa at 350 ◦C for 3 h prior to each measurement. 3.3.2. X-ray Diffraction X-ray diffractometer (Bruker D8 Advance, Karlsruhe, German) was used to determine the phase composition of the catalysts. Cu Kα radiation (λ = 1.54056 Å) was used as the X-ray source, and an electric current of 40 mA, a voltage of 40 kV, and a scanning speed of 0.30◦/min were used. 3.3.6. Temperature-Programmed Hydrogenation (TPH) Temperature-Programed Hydrogenation (TPH) was conducted in a quartz tube reactor coupled with a mass spectrometer. Typically, 50 mg of the sample was reduced in situ and carburized by a ﬂow rate of 30 mL (STP)/min with H2/CO = 5 at atmospheric pressure and 280 ◦C before the TPH experiment. During the TPH, the temperature was ramped from room temperature to 1000 ◦C at a rate of 10 ◦C/min in 10 vol% H2/He ﬂow (50 mL (STP)/min in total). 3.3.4. FTIR/ATR Spectra The IR (ATR, Attenuated Total Reﬂectance) spectra of the samples (fresh and spent) were acquired on an IRPrestige-21 spectrometer operating with Diamond crystal ATR (Specac Ltd., Orpington, UK). Thirty-two scans were taken over the wave-number range of 400~4000 cm−1 for each sample with a resolution of 4 cm−1. 3.3.5. Temperature-Programmed Reduction (TPR) 3.3.5. Temperature-Programmed Reduction (TPR) 3.3.5. Temperature-Programmed Reduction (TPR) Temperature-programmed reduction (H2-TPR) proﬁles of calcined catalysts were recorded using an Autochem II 2920 unit (Micromeritics, Norcross, GA, USA) equipped with a thermal conductivity detector (TCD). A sample of approx. 100 mg of catalyst was loaded into a U-type quartz tube reactor and pre-treated by purging with argon (50 mL (STP)/min for 40 min at 120 ◦C, after which the system was cooled down in Ar to about 40 ◦C. The gas supply was then switched to a mixture of H2/90% Ar (50 mL (STP)/min), and the temperature was ramped to 800 ◦C with a heating rate of 10 ◦C/min. 3.3.6. Temperature-Programmed Hydrogenation (TPH) 3.3.6. Temperature-Programmed Hydrogenation (TPH) 3.3.8. Raman Spectrum LRS was obtained on LabRAM HR800 (Horiba Jobin Yvon, Palaiseau, France) us- ing Synapse CCD as the detector and an air-cooled frequency-doubled Nd:Yag laser (λ = 532 nm) as the laser source. 19 of 22 Catalysts 2022, 12, 916 19 of 22 3.4.1. FTS Test in Fixed-Bed Reactor (FBR) FTS performance of catalysts was evaluated in a ﬁxed bed reactor and its matching product analysis systems. A 1.5 g sample of fresh precursor catalyst (80~150 µm), mixed with a similar size of quartz beads in a certain proportion was charged into the reactor. The outlet of the reactor was connected with a hot trap and a cold trap at the system pressure. After the product collectors, the pressure of the tail gas was released through a backpressure regulator. The ﬂow rate of the tail gas was monitored by a wet-gas ﬂow meter. The catalysts were pre-treated in situ at 260 ◦C for 24 h in a syngas stream (1 bar, H2/CO = 20, 1800 mL/(g-cat·h) GHSV). The ﬂow rate of the puriﬁed syngas was controlled using a mass ﬂow meter. Subsequently, the bed temperature was lowered down to about 150 ◦C, the system was pressurized to 2.3 MPa, and the syngas was adjusted H2/CO = 1.5, 3000 mL/(g-cat·h) GHSV. The reaction allowed for more than 280 h at 235 ◦C on-stream to reach a steady state before the analysis of the efﬂuents. 3.4.2. Product Analysis Reactants and gas-phase products were analyzed using an online Agilent GC (6890N) equipped with a thermo conductivity detector (TCD) and a Chromosorb column. The heavy hydrocarbon products were analyzed using an ofﬂine GC (Agilent 1260) with a ﬂame-ionization detector (FID) and a fused silica capillary column. 3.3.9. Mössbauer Spectra (MES) 57Fe Mössbauer spectra were recorded at room temperature using a Topologic 500A spectrometer with a proportional counter. 57Co (Rh) moving in constant-acceleration mode was used as the radioactive source. The Doppler velocity of the spectrometer was calibrated with respect to a standard α-Fe foil. The absorption thickness of the sample was adjusted to 10 mg Fe/cm2. The analysis of the Mössbauer spectrum is based on the Lorentz absorption curve and ﬁtted by a computer with moss winn 4.0 software (http: //www.mosswinn.com/english/index.html). The spectral components were identiﬁed based on their isomer shift (IS), quadruple splitting (QS), absorption linewidth (LW) and magnetic hyperﬁne ﬁelds (Hhf). The relative content of various iron phases in the catalyst was determined by integrating the absorption peak area of each phase. 3.4. Fischer-Tropsch Synthesis Performance Test 4. Conclusions The physical-chemical structural evolution, phase transformation and FTS catalytic behavior of precipitated Fe/Cu/K/SiO2 catalysts with four different potassium loading levels were investigated. ATR-FTIR results showed the formation of the Fe–O–Si structure in fresh precursors. This kind of iron–silica interaction was weakened with an increase in K loading, which was evidenced by an apparent “red shift” over the peak maxima of Fe–O–Si infrared spectra, a step-wise lowering of a peak temperature corresponding to Fe3O4 reduction to α-Fe, and a clear decreasing Fe2p binding energy observed, respectively, as a consequence of ATR-FTIR, H2-TPR and XPS investigation. The obvious retardation of the second reduction process observed on the lower K sample indicated that a stronger Fe–O–Si interaction was formed, and it appeared to stabilize the Fe2+. The marked shift toward the lower-end temperature of the second reduction peak occurred on the samples with a rising K loading indicating the acceleration of the Fe3O4 reduction to the metallic iron (α-Fe). Obviously, this was the result of weakened iron–silica interaction affected by higher K content. The H2-TPH experiment revealed that a greater proportion of reactive surface carbon (atomic and amorphous) was obtained with increasing K loading, while the iron carbide (χ-Fe5C2) phase in the samples with lower K loading was restrained and stabilized by the stronger iron–silica interaction between iron and silica from hydrogenation. In situ XRD results showed that the crystallite size of χ-Fe5C2 calculated by (510) facet was remarkable with the increased K content. Catalysts 2022, 12, 916 20 of 22 20 of 22 In the ﬁxed-bed reactor, the higher K loading sample is intended to expose a higher CO conversion activity and a higher O/P (C2–C4) ratio, which might be attributed to the fact that the increased reactive surface carbon (alpha-carbon) can be achieved over the higher K loading samples after syngas reduction. After the FTS reaction, the Raman spectra showed a greater proportion of graphitic carbon intended to accumulate on the higher K sample, which may be insufﬁciently responsible for the high deactivation observed over the Lab-4 (7 K%) sample. MES results revealed that the relative content of the iron carbide (χ-Fe5C2) phase was increased with increasing K loading, while highly dispersed/stabilized Fe2+ ions; structurally Fayalite (Fe2SiO4) compound, which was also detected by ATR-FTIR; and XRD characterizations were observed both in the bulk and surface region of the lowest K loading (2.3 K%) sample. 4. Conclusions The iron–silica interaction was evident in the size diameter and showed that the crystallite sizes increased by a factor of four for the 7 K% sample after long-term FTS reaction, which rationalized the high deactivation obtained. Author Contributions: Conceptualization, H.C., Q.L. and M.C.; investigation, H.C., M.C. and K.Z.; Characterization, B.F. and J.C.; writing—original draft, H.C., Q.L., M.C. and K.Z.; writing—review and editing, H.C., Q.L., M.C. and K.Z.; project administration, Q.L., Y.L. and Z.M.; funding acquisition, Q.L., Y.L. and Z.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Technical Innovation project of China Energy Investment Corporation (Project No. GJNY-21-72). Funding: This research was funded by the Technical Innovation project of China Energy Investment Corporation (Project No. GJNY-21-72). 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 1. Dry, M.E. The Fischer-Tropsch process: 1950–2000. Catal. 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Paalanen, P.P.; van Vreeswijk, S.H.; Weckhuysen, B.M. Combined in situ x-ray diffractrometry/raman spectroscopy of iron carbide and carbon species evolution in Fe(-Na-S)/a-Al2O3 catalysts during Fisch-Tropsch synthesis. ACS Catal. 2020, 10, 9837–9855. 45. Paalanen, P.P.; van Vreeswijk, S.H.; Weckhuysen, B.M. Combined in situ x-ray diffractrometry/raman spectroscopy of iron carbide and carbon species evolution in Fe(-Na-S)/a-Al2O3 catalysts during Fisch-Tropsch synthesis. ACS Catal. 2020, 10, 9837–9855. [CrossRef] [CrossRef] 22 of 22 22 of 22 Catalysts 2022, 12, 916 p y , , p py , , [ ] 47. Wielers, A.F.H.; Kock, A.J.H.M.; Hop, C.E.C.A.; Geus, J.W.; van der Kran, A.M. The Reduction Behavior of Silica-Supported and Alumina-Supported Iron Catalysts: A Mossbauer and Infrared Spectroscopic Study. J. Catal. 1989, 117, 1–18. [CrossRef] pp y p p y J [ ] 48. Hou, W.J.; Wu, B.S.; An, X.; Li, T.Z.; Tao, Z.C.; Zheng, H.Y.; Xiang, H.W.; Li, Y.W. 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https://openalex.org/W4287394518	https://zenodo.org/records/4415052/files/3213ijbb01.pdf	English	null	A COMPARATIVE IN SILICO STUDY FINDS A FUNCTIONAL CO-RELATION BETWEEN HUMAN HSP7C AND PLANT PROTEIN	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,439	International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Animesh Sarker1, and Al Amin2 Animesh Sarker1, and Al Amin2 1Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 animesh_du_geb@yahoo.com 2Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 niloygenetics@gmail.com Animesh Sarker1, and Al Amin2 1Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 animesh_du_geb@yahoo.com 2Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 Animesh Sarker1, and Al Amin2 1Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 animesh_du_geb@yahoo.com Telephone:+088-092162407 animesh_du_geb@yahoo.com ment of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. 2Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902.Bangladesh. Telephone:+088-092162407 niloygenetics@gmail.com ABSTRACT A bioinformatics finding shows that numerous human heat shock proteins (HSPs) homologues are common in plants. Human heat shock proteins (HSPs), which are expressed to higher temperature or other stress, have chaperone activity belong to four conserved classes: HSP60, HSP70, HSP90 and HSP100. Bioinformatics blast search reveals that each of the human HSP classes possess a number of plant homologues. The closest identified plant homologue of human HSP_7C is a protein of unknown function (NCBI Accession XP_002332067) derived from Populus trichocarpa. In silico comparative studies have showed invigorating similarity of human HSP_7C and the designated protein of Populus trichocarpa. Secondary and three-dimensional (3D) structure analysis of the predicted plant protein strongly supports its functional relationship to the class of human HSP70. 1. INTRODUCTION Heat shock proteins (HSPs) are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperature or other stresses [1]. HSPs are found in virtually all living organisms, from bacteria to human. Heat shock proteins, as a class, are among the most highly expressed cellular proteins across all species. As their name implies, heat shock proteins protect cells when stressed by elevated temperatures. They account for 1–2% of total protein in unstressed cells. However when cells are heated, the fraction of heat shock proteins increases to 4–6% of cellular proteins [2, 3]. In eukaryotic organisms, the principal class of HSPs in human are HSP60, HSP70, HSP90 and HSP100 [4]. Another novel class HSP33 is also found exclusively, which interestingly absence in archaea or other eukaryotes [5]. In plants, Hsps are greatly varied in level of expression as well as their type [6]. The most prominent types are HSP20, HSP70, HSP90 and HSP100 according to well studied plant species Arabidopsis thaliana. Higher plants are characterized by the presence of at least 20 types of sHsps, but one species could contain 40 types of these sHsps[7]. 1 1 DOI: 10.5121/ijbb.2013.3201 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 In plants, most common class protein is Hsp70 has been characterised in diverse species [7,8]. The simplest flowering plant genome Arabidopsis thaliana includes at least 18 genes encoding members of the Hsp70 family, of which 14 belong to the DnaK subfamily and four to the Hsp110/SSE subfamily [3,9,10]. At least 12 Hsp70 members have been found in the spinach genome [11].Expression profile analysis of the Arabidopsis and spinach Hsp70 genes demonstrated that members of Hsp70 chaperones are expressed in response to environmental stress conditions such as heat, cold and drought, as well as osmotic, salinity, oxidative, desiccation, high intensity irradiations, wounding, and heavy metals stresses [9-11,12]. On the basis of sequence and structure similarities in silico comparative studies have been performed between human HSPs and homologous plant protein. Most of the computer aided bioinformatics findings reveal that proteins for HSP70 family are mostly analogous to its plant homologues. BLASTp search of the entire Angiosperm plantae kingdom against all of the human HSP classes discover a protein of unknown function from Populus trichocarpa closely similar to human HSP70. 1. INTRODUCTION By using different computational tools and software the sequence and structure of this unknown protein was intensely compared with human HSP70 which discovered a functional co-relation between these two proteins. The goal of the present work was to identify plant homologues of human HSPs that could play significant role in protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize proteins and membranes, and could assist in protein refolding under stress conditions. They could also play a crucial role in protecting plants against stress by re- establishing normal protein conformation and thus cellular homeostasis[13]. It seems that the synthesis of these proteins require enormous energy that impact on the yield of the organism [14]. 2. MATERIALS AND METHODS The full-length protein sequences of all the four types heat shock proteins (HSPs) were retrieved from NCBI protein Database (http://www.ncbi.nlm.nih.gov/), with accession numbers, HSP60 (P10809), HSP70 (P11142), HSP90 (P07900), HSP100 (Q92598). The homologous sequences of human HSPs were identified from position specific iteration BLASTp (NCBI). Percent similarities and identities were computed by NCBI blast2seq program. Global alignment of the coding sequences was performed with the program AVID using a window size of 100 bp and a conservation level of 70%. The results were viewed with the program VISTA. To map sequence conservation, human HSP7c were aligned with several other plant proteins using multiple sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/tools/clustalw2). This program uses a progressive method to build its alignments using the BLOSUM 62 substitution matrix for proteins. Phylogenetic tree was also constructed for HSP7C_HUMAN and its plant homologues by using ClastalW. Secondary structure of the predicted HSP_Populus1 and HSP7C_Human were computed using the program Hierarchical Neural Network (HNN: http://www.expasy.org/tools/). The 3D structure of predicted protein and HSP7C were constructed by DeepView/Swiss-PdbViewer v4.0.1(spdbv) (http://www.expasy.org/spdbv/). Swiss-PdbViewer is tightly linked to SWISS-MODEL, an automated homology-modelling server developed by the Structural Bioinformatics Group of the Swiss Institute of Bioinformatics (SIB) at the Biozentrum in Basel. To comprehend the functional relationship between these two proteins, SVMProt was used to predict protein function. 3.1 Homology study: The primary knowledge of Human heat shock proteins, whose expression rises when cells are unprotected to higher temperatures or other stress, were collected from public databases and published research papers in order to homology study [10]. NCBI protein blast (BLASTp) was 2 2 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 carried out intended for scanning Human Heat Shock Proteins arranged in (Table 1), against the kingdom of flowering plants. A number of plant homologues (Angiosperm) of human heat shock protein 60, 70, 90 and 100 were identified on the basis of better E-value and higher percentage of similarity-identity (Table 2). Most significant members of each family are tabulated here, however there are some species which may express additional chaperones, co-chaperones, and heat shock proteins. In addition, many of these proteins may have multiple splice variants (Hsp90α and Hsp90β, for instance) or conflicts of nomenclature (Hsp72 is sometimes called Hsp70) [17]. Since UniProt and NCBI database carries enormous sequence of human heats hock proteins, so prior to blast search, one candidate from each of the four classes were selected (Table 1). These are: Heat shock protein 105 kDa (GI:97536358), Heat shock protein 90A kDa (GI:92090606), Heat shock protein 70C kDa (GI: 123648), Heat shock protein 60 kDa (GI:41399285). Table 1. Selected candidates of human heat shock protein (HSPs). Table 1. Selected candidates of human heat shock protein (HSPs). HSPs NCBI (GI) UniProt (Accession) HSP60 GI: 41399285 P10809 (HSP60_Human) HSP70 GI: 123648 P11142 (HSP7C_HUMAN) HSP90 GI: 92090606 P07900 (HS90A_HUMAN) HSP100 GI:97536358 Q92598 (HSP105_Human) Position specific iterated BlAST scanning of each the candidate HSPs hits a lot of predicted plant homologous proteins (those function is not identified yet). Two of those hypothetical members from each class were selected by filtering proteins with better E-values and higher percentage of similarities-identities (Table 2). 3.2 Phylogenetic study According to BLAST result it is clear that two members of Populus trichocarpa have higher sequence similarity to human HSP7C as one (Gb\|EEE71404.1\|) hits with 88% similarity and 78- 79% identity and other (Gb\|EEE71404.1\|) hits with 88% similarity and 78% identity. In order to identify the closest homologues, these two potential candidates are allowed for pair wise sequence alignments with several other flowering plant proteins (Table 3), which were selected from BLAST result of HSP7C . The members of higher percentage of sequence similarity and identity were allowed for MSA and finally a phylogenetic tree was constructed. According to this cladistic data, a common clade with human heat shock protein(HSP7C) is formed by protein gb\|EEE70430.1\| (gi\|224115828\|HSP_ Populus1)(Figure 1). 3 Table 2. Plant homologues of each the human HSP class. HSPs Predicted Protein Description E value Similarity Identity HSP 105_Human gb\|EFH64112.1\| hypothetical protein ARALYDRAFT_896005 [Arabidopsis lyrata subsp. lyrata] 1e-165 59% 40% gb\|EES19148.1\| hypothetical protein SORBIDRAFT_09g005570 [Sorghum bicolor] 4e-162 58% 39% Table 2. Plant homologues of each the human HSP class. 3 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 HS90A_HUMAN gb\|EEE74469.1\| predicted protein [Populus trichocarpa] 0 83% 71% gb\|EEE85774.1\| predicted protein [Populus trichocarpa] 0 84% 71% HSP7C_HUMAN gb\|EEE71404.1\| predicted protein [Populus trichocarpa] 0 88% 79% gb\|EEE70430.1\| predicted protein [Populus trichocarpa] 0 88% 78% HSP60_Human gb\|EEE82364.1\| predicted protein [Populus trichocarpa] 0 79% 60% gb\|EER94121.1\| hypotheticalprotein SORBIDRAFT_01g020010[Sor ghum bicolor] 0 79% 60 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Figure 1 Phylogenetic tree of human HSP7C with its plant homologues Figure 1 Phylogenetic tree of human HSP7C with its plant homologues Figure 1. Phylogenetic tree of human HSP7C with its plant homologues. GI No. Accession No. Organism Length (aa) Identity (%) Similarity (%) E- value 38325815 AAR17080.1 Nicotiana tabacum 648 78 89 0 115464309 ABF95267.1 Oryza sativa Japonica Group 653 79 88 0 211906498 ACJ11745.1 Gossypium hirsutum 648 78 88 0 45331281 AAS57912.1 Vigna radiata 649 78 88 0 145203152 ABP35942.1 Cyclamen persicum 650 77 87 0 6911551 CAB72129.1 Cucumis sativus 653 77 87 0 224115828 XP_002332067.1 Populus trichocarpa 655 78 88 0 224113495 XP_002332589.1 Populus trichocarpa 648 78 88 0 3.3 Sequence conservation study Global alignment of the coding sequence of Human HSP7c and predicted protein gene sequence was performed with the program AVID using a window size of 100 bp and a conservation level 4 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 of 70%. Results were viewed with the program VISTA (Figure 2). From here it can be observe that these two sequences are highly conserved with each other. of 70%. Results were viewed with the program VISTA (Figure 2). From here it can be observed that these two sequences are highly conserved with each other. of 70%. Results were viewed with the program VISTA (Figure 2). From here it can be observed that these two sequences are highly conserved with each other. Figure 2. Conservation level of human HSP7C and predicted populus1 protein. VISTA plot of the AVID alignment (Figure 2) indicated that major part in predicted gene is highly conserved (more than 70%) in human HSP7C. Pair wise sequence alignment has also showed that the predicted protein is 77.3% identical and 86.7% similar with human HSP7C. The score obtained from such alignment is 2581 with 1.4% gaps. Global sequence alignment of human HSP7C and other related flowering plant proteins including Populus HSP70 was also performed and it showed strong full length sequence conservation. But sequence conservation result only for peptide binding domain was displayed here (Figure 3). Figure 2. Conservation level of human HSP7C and predicted populus1 protein. Figure 2. Conservation level of human HSP7C and predicted populus1 protein. Figure 2. Conservation level of human HSP7C and predicted populus1 protein. VISTA plot of the AVID alignment (Figure 2) indicated that major part in predicted gene is highly conserved (more than 70%) in human HSP7C. Pair wise sequence alignment has also showed that the predicted protein is 77.3% identical and 86.7% similar with human HSP7C. The score obtained from such alignment is 2581 with 1.4% gaps. Global sequence alignment of human HSP7C and other related flowering plant proteins including Populus HSP70 was also performed and it showed strong full length sequence conservation. But sequence conservation result only for peptide binding domain was displayed here (Figure 3). VISTA plot of the AVID alignment (Figure 2) indicated that major part in predicted gene is highly conserved (more than 70%) in human HSP7C. 3.3 Sequence conservation study Pair wise sequence alignment has also showed that the predicted protein is 77.3% identical and 86.7% similar with human HSP7C. The score obtained from such alignment is 2581 with 1.4% gaps. Global sequence alignment of human HSP7C and other related flowering plant proteins including Populus HSP70 was also performed and it showed strong full length sequence conservation. But sequence conservation result only for peptide binding domain was displayed here (Figure 3). Figure 3. Global alignment of peptide binding domain of Human HSP7c protein with plant homologous. Figure 3. Global alignment of peptide binding domain of Human HSP7c protein with plant homologous. Figure 3. Global alignment of peptide binding domain of Human HSP7c protein with plant homologous 5 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 3.4 Secondary structure study: The Human HSP_7C includes two core functional domains: ATPase domain which located at N- terminal region and the peptide binding domain which consists of C-terminal region [18]. Helix and coil structure play strong role to form the architecture of these functional domains. It has already been established that structure determines corresponding function [19]. Here, secondary structure of this predicted protein was obtained by bioinformatics secondary structure prediction tool (HNN model) and successive comparative studies with Human_7C were carried on (Figure 4). HNN model demonstrates that the hypothetical protein almost carries equal percentage of alpha helix, extended strands and random coils (Table 4). Figure 4. Secondary structure of human HSP7C and predicted populus1 prote Figure 4. Secondary structure of human HSP7C and predicted populus1 protein. Figure 4. Secondary structure of human HSP7C and predicted populus1 protein. Table 4: Secondary structure of human HSP7C and predicted populus1 protein in details. Table 4: Secondary structure of human HSP7C and predicted populus1 protein in details. Table 4: Secondary structure of human HSP7C and predicted populus1 protein in details. Features HSP7C_Human Predicted Protein Sequence length : 646 655 Alpha helix (Hh) : 228 is 35.29% 253 is 38.63% 310 helix (Gg) : 0 is 0.00% 0 is 0.00% Pi helix (Ii) : 0 is 0.00% 0 is 0.00% Beta bridge (Bb) : 0 is 0.00% 0 is 0.00% 6 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Extended strand (Ee) : 129 is 19.97% 108 is 16.49% Beta turn (Tt) : 0 is 0.00% 0 is 0.00% Bend region (Ss) : 0 is 0.00% 0 is 0.00% Random coil (Cc) : 289 is 44.74% 294 is 44.89% Ambigous states (?) : 0 is 0.00% 0 is 0.00% Other states : 0 is 0.00% 0 is 0.00% International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 3.5 3D structure study Three dimensional structure of predicted populus hsp70 and human HSP7c were constructed by using Swiss Pdb Viewer (SPDBV) (Figure 5). 3D structures of these two proteins are quite similar and they have formed same functional clefts. Eletrostatic potential of their active sites are also alike that firmly indicates their equivalent function. D Figure 5: 3D structure and electrostatic potential of HSP7C_Human and Populus1 protein. A and B represent for 3D structure of human HSP7C and predicted populus1 protein respectively. C and D represent for electrostatic potential human HSP7C and predicted populus1 protein respectively. A C B D C D B B A C Figure 5: 3D structure and electrostatic potential of HSP7C_Human and Populus1 protein. A and B represent for 3D structure of human HSP7C and predicted populus1 protein respectively. C and D represent for electrostatic potential human HSP7C and predicted populus1 protein respectively. Table 5. Hypothetical function of HSP7C_Human and HSP_Populus1. Table 5. Hypothetical function of HSP7C_Human and HSP_Populus1. HSP7C_Human HSP_POPULUS1(predicted protein) Function P Value (%) Function P Value (%) All lipid-binding proteins All DNA-binding Zinc-binding TC 1.A. Channels/Pores - Alpha-Type channels TC 3.A. Primary Active Transporters - P-P-bond- hydrolysis-driven transporters Actin binding Copper-binding 97.0 91.3 76.2 73.8 65.4 58.6 58.6 All lipid-binding proteins All DNA-binding Zinc-binding TC 1.A. Channels/Pores - Alpha-Type channels TC 3.A. Primary Active Transporters - P-P-bond- hydrolysis-driven transporters Actin binding Copper-binding 93.6 94.7 82.2 97.5 58.6 62.2 58.6 7 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 4. CONCLUSION Based on all the findings it can be concluded that the predicted protein may comprise human HSP_7C like function. Since plant HSP70 show higher sequence and structure homology with Human HSP70 family, it can further be concluded that the predicted protein may take part in protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize proteins and membranes, and assist in protein refolding under stress conditions like plant hsp70[20]. Even though these hypothetical functions of this predicted protein (HSP_Populus1) should be further characterized by laboratory means before their existence can be conclusively affirmed, the results presented in this study suggested and identified structurally similar protein of heat shock protein family. The findings can provide new insight in plant protein characterization. 3.6 Hypothetical protein function To comprehend the functional relationship between these proteins, function of predicted protein was retrieved by SVMProt. The results have revealed that all of the functional domain of Human HSP_7C exist in the predicted populus1 protein and showed similar modular function with almost equal p value (Table 5). REFERENCES [1] De Maio A (January 1999). “Heat shock proteins: facts, thoughts and dreams. Shock (Augusta, G 11 (1): 1-12 [2] Crevel G, Bates H, Huikeshoven H, Cotterill S (1 June 2001). "The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase alpha". J. Cell. Sci. 114 (Pt 11): 2015–25. PMID 11493638. [3] Schlesinger, MJ (1990-07-25). "Heat shock proteins". The Journal of Biological Chemistry 265 (21): 12111–12114. PMID 2197269. [4] Kregel, K.C., 2002. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J.Appl. Physiol. 92, 2177–2186. [5] Schlesinger, M .J., 1990. Heat shock proteins. J. Biol. Chem. 265, 12111–12114. [6] Hamilton, E.W., Heckathorn, S.A., Downs, C.A., Schwarz, T.E.,Coleman, J.S., Hallberg, R.L., 1996. Heat shock proteins are produced by ﬁeld-grown naturally occurring plants in the summer in the temperate northeast US Bulletin of the Ecologic. Soc. Am.77, Suppl. Part 2: 180 (Abstr.) [7] Vierling, E. (1991) The roles of heat shock proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 579–620 [8] Boston, R.S. et al. (1996) Molecular chaperones and protein folding inplants. Plant Mol. Biol. 32, 191–222 [9] Lin, B.L. et al. (2001) Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress Chaperones 6, 201–208 [10] Sung, D.Y. et al. (2001) Comprehensive expression profile analysis ofthe Arabidopsis Hsp70 gene family. Plant Physiol. 126, 789–800 [11] Guy, C.L. and Li, Q.B. (1998) The organization and evolution of the spinach stress 70 molecular chaperone gene family. Plant Cell 10, 539–556 [12] Swindell, W.R., Huebner, M., Weber, A.P., 2007. Transcriptional proﬁling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8, 125 <http://www.biomed-central.com/1471- 2164/8/125> [13] Wangxia Wang1,3, Basia Vinocur1, Oded Shoseyov1,2 and Arie Altman1,2 Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. TRENDS in 8 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 International Journal on Bioinformatics & Biosciences (IJBB) Vol.3, No.2, June 2013 Pl t S i V l 9 N 5 M 2004 Plant Science Vol.9 No.5 May 2004 Plant Science Vol.9 No.5 May 2004 [14] Al-Whaibi M.H (June 2010). Plant heat-shock proteins: A mini review. doi: 10.1016/j.jksus [15] Vigh, L. et al. (2007) , Membrane Associated Stress Response:a Theoretical and Practical Approach [16] Flaherty KM, DeLuca-Flaherty C, McKay DB (August 1990). REFERENCES "Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein". Nature 346 (6285): 623–8. [17] Tavaria M, Gabriele T, Kola I, Anderson RL (April 1996). "A hitchhiker's guide to the human Hsp70 family". Cell Stress Chaperones 1 (1): 23–8. [18] Takayama S., Bimston D.N., Matsuzawa S.-I.”BAG-1 modulates the chaperone activity of Hsp70/Hsc70.", [PubMed: 9305631] [19] Gabaldon, T; M.A. Huynen (2004). "Prediction of protein function and pathways in the genome era". Cellular and Molecular Life Sciences 61: 930–944. [20] Lindquist, S. (1986) The heat-shock response. Annu. Rev. Biochem. 55,1151–1191 AUTHOR: Animesh Sarker, Lecturer Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh,Tangail- 1902.Bangladesh. Telephone:+088-092162407 Email: animesh_du_geb@yahoo.com Email: animesh_du_geb@yahoo.com 9
https://openalex.org/W2076055777	https://orca.cardiff.ac.uk/id/eprint/63838/1/P-2014-5.pdf	English	null	Theoretical Analysis and Computational Simulation of Advanced Structured Materials	Advances in condensed matter physics	2,014	cc-by	1,283	Hanxing Zhu,1 Daniel Balint,2 and Zaoyang Guo3 Hanxing Zhu,1 Daniel Balint,2 and Zaoyang Guo3 1 School of Engineering, Cardiff University, Cardiff CF24 3AA, UK 2 Department of Mechanical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK 3 Department of Engineering Mechanics, Chongqing University, Chongqing 400044, China chool of Engineering, Cardiff University, Cardiff CF24 3AA, UK Department of Mechanical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U Department of Engineering Mechanics, Chongqing University, Chongqing 400044, China Correspondence should be addressed to Hanxing Zhu; zhuh3@cf.ac.uk Received 11 August 2014; Accepted 11 August 2014 Copyright © 2014 Hanxing Zhu 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. 1 Structured materials may be broadly classified as comprising a single phase with an underlying topology or multiple phases in a mixture [1]. Examples include porous materials [2–5], multilayers [6, 7], and functionally graded [7–9] and compos- ite materials [10]. Structures can vary in size, typically from macro- [2] down to meso-, micro-, and nanoscales [3, 11–13], and may also be hierarchical [12, 13]. Some structured mate- rials comprise both topology and multiple phase mixtures at different length scales [3, 10], for example, metal matrix composite foams. In all cases, advanced structured materials are designed to have exceptional properties [7–9, 12, 13] that cannot be achieved by conventional materials, for example, very high toughness, specific stiffness, multifunction [3, 4], and so forth. The knowledge and understanding of these supermaterials rely not only on advanced characterization and processing techniques, but also on progress in theoretical analyses and computational simulations. research of advanced materials and structures. Compared to real experimental measurements, computer simulations are not only faster, but also more economic. Moreover, computer simulations could help us understand in detail how each of the parameters and factors affects the properties and performance of advanced materials and hence which of these are most important.h p The same applies to the design of advanced materials. For example, theoretical analyses and computational simu- lations could guide the design of mechanical and geometrical properties of nanostructured hierarchical honeycombs and open-cell foams so that they are size dependent, tunable, and controllable [3, 12, 13]. Multilayer materials [6–9] can be designed to have extremely good thermal resistance, in addition to good mechanical strength, stiffness, and ductility. Hindawi Publishing Corporation Advances in Condensed Matter Physics Volume 2014, Article ID 367916, 2 pages http://dx.doi.org/10.1155/2014/367916 Hindawi Publishing Corporation Advances in Condensed Matter Physics Volume 2014, Article ID 367916, 2 pages http://dx.doi.org/10.1155/2014/367916 References [1] D. S. Balint, V. S. Deshpande, A. Needleman, and E. van der Giessen, “Discrete dislocation plasticity analysis of the grain size dependence of the flow strength of polycrystals,” International Journal of Plasticity, vol. 24, no. 12, pp. 2149–2172, 2008. 1. Please check and conﬁrm the author(s) ﬁrst and last names and their order which exist in the last page. [2] H. X. Zhu, J. F. Knott, and N. J. Mills, “Analysis of the elastic properties of open-cell foams with tetrakaidecahedral cells,” Journal of the Mechanics and Physics of Solids, vol. 45, no. 3, pp. 319–343, 1997. [3] H. X. Zhu, “Size-dependent elastic properties of micro- and nano-honeycombs,” Journal of the Mechanics and Physics of Solids, vol. 58, no. 5, pp. 696–709, 2010. [4] Z. y. Guo and F. C. Caner, “Mechanical behaviour of transversely isotropic porous neo-Hookean solids,” International Journal of Applied Mechanics, vol. 2, no. 1, pp. 11–39, 2010. [5] Z. Guo, F. Caner, X. Peng, and B. Moran, “On constitutive modelling of porous neo-Hookean composites,” Journal of the Mechanics and Physics of Solids, vol. 56, no. 6, pp. 2338–2357, 2008. [6] D. S. Balint and J. W. Hutchinson, “Undulation instability of a compressed elastic film on a nonlinear creeping substrate,” Acta Materialia, vol. 51, no. 13, pp. 3965–3983, 2003. [7] H. X. Zhu, N. A. Fleck, A. C. F. Cocks, and A. G. Evans, “Numer- ical simulations of crack formation from pegs in thermal barrier systems with NiCoCrAlY bond coats,” Materials Science and Engineering A, vol. 404, no. 1-2, pp. 26–32, 2005. [8] D. S. Balint and J. W. Hutchinson, “An analytical model of rumpling in thermal barrier coatings,” Journal of the Mechanics and Physics of Solids, vol. 53, no. 4, pp. 949–973, 2005. [9] D. S. Balint, S.-S. Kim, Y. F. Liu, R. Kitazawa, Y. Kagawa, and A. G. Evans, “Anisotropic TGO rumpling in EB-PVD thermal barrier coatings under in-phase thermomechanical loading,” Acta Materialia, vol. 59, no. 6, pp. 2544–2555, 2011. [10] Z. Y. Guo, X. Q. Peng, and B. Moran, “A composites-based hyperelastic constitutive model for soft tissue with application to the human annulus fibrosus,” Journal of the Mechanics and Physics of Solids, vol. 54, no. 9, pp. 1952–1971, 2006. [11] Z. P. Baˇzant and Z. Guo, “Size effect and asymptotic matching approximations in strain-gradient theories of micro-scale plas- ticity,” International Journal of Solids and Structures, vol. 39, no. 21-22, pp. 5633–5657, 2002. [12] H. Hanxing Zhu,1 Daniel Balint,2 and Zaoyang Guo3 It may also be possible to design and synthesize biological materials that have mechanical properties similar to those of natural materials [5, 10].h y p Experimental measurements are truly important in devel- oping, characterizing, and understanding advanced mate- rials. However, they are usually very expensive and time consuming. As the material properties and performance usually depend on the interplay of many parameters and factors, it is quite difficult to understand how each of them affects the properties and performance and which one is most important. Consequently, it is difficult to obtain the global picture about how each of the parameters and factors affects the material performance only from experimental measurements. With the advancement of computer hardware and computational software, simulations (i.e., numerical experiments) play an increasingly important role in scientific This special issue focuses mainly on theoretical analyses and computational simulations of advanced structured mate- rials including high-performance cements, shape memory alloys, nanostructured composites, and multilayered materi- als. We hope that readers will find in this special issue not only accurate data/results and up-to-date reviews on the relevant research areas, but also useful guidance on important areas for further research work. Hanxing Zhu Daniel Balint Zaoyang Guo Advances in Condensed Matter Physics Advances in Condensed Matter Physics 2 Composition Comments References X. Zhu, L. B. Yan, R. Zhang, and X. M. Qiu, “Size-dependent and tunable elastic properties of hierarchical honeycombs with regular square and equilateral triangular cells,” Acta Materialia, vol. 60, no. 12, pp. 4927–4939, 2012. [13] H. X. Zhu and Z. B. Wang, “Size-dependent and tunable elastic and geometric properties of hierarchical nano-porous materials,” Science of Advanced Materials, vol. 5, no. 6, pp. 677– 686, 2013. Author(s) Name(s) It is very important to conﬁrm the author(s) last and ﬁrst names in order to be displayed correctly on our website as well as in the indexing databases: Last Name: Balint Author 3 Given Names: Zaoyang Last Name: Guo Author 1 Given Names: Hanxing Last Name: Zhu Author 2 Given Names: Daniel It is also very important for each author to provide an ORCID (Open Researcher and Contributor ID). ORCID aims to solve the name ambiguity problem in scholarly communications by creating a registry of persistent unique identiﬁers for individual researchers. To register an ORCID, please go to the Account Update page (http://mts.hindawi.com/update/) in our Manuscript Tracking System and aer you have logged in click on the ORCID link at the top of the page. is link will take you to the ORCID website where you will be able to create an account for yourself. Once you have done so, your new ORCID will be saved in our Manuscript Tracking System automatically.
https://openalex.org/W3093651295	http://www.jurnal.stie-aas.ac.id/index.php/jei/article/download/1403/758	Indonesian	null	Analisis Kesehatan Keuangan Bank Syariah Mandiri Ditinjau dari Faktor Permodalan (CAR), Pembiayaan (FDR), Profitabilitas (ROA & ROE) Serta Risiko Kredit (NPF)	Jurnal ilmiah ekonomi Islam	2,020	cc-by	3,364	Abstract The aims of the research is to find out the financial soundness of Bank Syariah Mandiri (BSM) seen from the capital factor (CAR), financing (FDR), profitability (ROA & ROE), and credit risk (NPF). This research uses quantitative descriptive methods. The data used are the 2014-2018 financial statements. The results of this study indicate that BSM in terms of capital using the CAR ratio shows an average CAR ratio of 14.75%. This means that BSM in terms of capital is ranked very well. This means that BSM's ability to generate profits is ranked quite well. Bank Syariah Mandiri in terms of financing using the FDR ratio shows an average FDR ratio of 79.81%. This means that BSM's ability to repay short-term loans and meet agreed financing is at a healthy rating. Bank Syariah Mandiri in terms of profitability using the ROA and ROE ratio shows an average ROA of 0.53% and ROE of 6%. Bank Syariah Mandiri in terms of credit risk using the NPF ratio shows an average NPF ratio of 3.18%. This means that there is very little credit provided by Bank Syariah Mandiri and the bank is viewed from the aspect of credit risk, including in a good rating. From the results of these research, indicate that the the soundness level of Bank Syariah Mandiri (BSM) is in a good level of soundness. Keywords: financial soundness; capital; financing; profitability; credit risk Saran Sitasi: Diharto, A. K. (2020). Analisis Kesehatan Keuangan Bank Syariah Mandiri Ditinjau dari Faktor Permodalan (CAR), Pembiayaan (FDR), Profitabilitas (ROA & ROE) Serta Risiko Kredit (NPF). Jurnal Ilmiah Ekonomi Islam, 6(03), 396-402. doi: http://dx.doi.org/10.29040/jiei.v6i3.1403 Saran Sitasi: Diharto, A. K. (2020). Analisis Kesehatan Keuangan Bank Syariah Mandiri Ditinjau dari Faktor Permodalan (CAR), Pembiayaan (FDR), Profitabilitas (ROA & ROE) Serta Risiko Kredit (NPF). Jurnal Ilmiah Ekonomi Islam, 6(03), 396-402. doi: http://dx.doi.org/10.29040/jiei.v6i3.1403 DOI: http://dx.doi.org/10.29040/jiei.v6i3.1403 Available at http://jurnal.stie-aas.ac.id/index.php/jie Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 396-402 Available at http://jurnal.stie-aas.ac.id/index.php/jie Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 396-402 Available at http://jurnal.stie-aas.ac.id/index.php/jie Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 396-402 Analisis Kesehatan Keuangan Bank Syariah Mandiri Ditinjau dari Faktor Permodalan (CAR), Pembiayaan (FDR), Profitabilitas (ROA & ROE) Serta Risiko Kredit (NPF) Awan Kostrad Diharto Fakultas Ekonomi dan Bisnis Islam (FEBI) IAIN Surakarta *Email korespondensi: awan_kostrad@yahoo.com 1. PENDAHULUAN (Al-Hares, dkk, 2013); (Rozzani and Rahman, 2013); (Amelia, 2015). Kegiatan perekonomian yang ada di masyarakat hampir tidak dapat terlepaskan dari lembaga keuangan saat ini terutama lembaga keuangan perbankan, baik bank syariah maupun bank konvensional. Kondisi yang umum terjadi dimana surplus sektor keuangan yang ada pada masyarakat untuk menyimpan uang yang mereka miliki maupun kondisi defisit sektor untuk melakukan pengajuan permodalan atau pinjaman. Masyarakat sebelum memutuskan tempat penyimpanan uangnya, tentunya mereka akan memilih lembaga keuangan yang memiliki kesehatan keuangan yang baik dan bahkan tingkat kesehatan keuangannya sangat baik. Hal tersebut mereka lakukan dalam rangka memberikan ketenangan dan rasa aman ketika mereka menyimpan uangnya di bank tersebut. Kinerja perbankan terutama kinerja keuangan akan menjadi perhatian para nasabahnya (Farooq & Zaheer, 2015); Ditinjau dari sudut pandang syariah bank ada dua jenis, yaitu Bank Konvensional dan Bank Syariah (bank umum syariah (BUS) & unit usaha syariah (UUS). Perbedaan dari bank tersebut terletak pada akad serta prinsip yang akan digunakan dalam melakukan transaksi dengan nasabahnya. Bank konvensional berlandaskan bunga dari pokok pinjaman serta dalam beroperasi berprinsip meraih tingkat keuntungan sebesar-besarnya tanpa melihat manajemen serta kondisi hasil usaha dari nasabahnya. Berdasarkan syariat Islam dengan adanya pengenaan bunga menurut sebagian besar ulama merupakan riba yang sangat dilarang dan dibenci Allah dan Rasul serta merupakan perbuatan dosa besar (Gilani, 2015); (Alamgir, 2016). Disisi yang lain Bank syariah dalam beroperasi menggunakan bagi hasil sebagai salah satu prinsipnya serta menghindari adanya berbagai praktek Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 397 laporan dalam pembuatan keputusan ekonomi (Iatridis, 2010); (Minnis, 2011). Melalui laporan keuangan akan dapat dinilai kemampuan perusahaan untuk memenuhi kewajiban-kewajiban jangka pendeknya, struktur modal perusahaan, distribusi dan keefektifan pengggunaan aktiva, hasil usaha/pendapatan yang telah dicapai, beban-beban yang harus dibayar serta nilai-nilai buku tiap lembar saham perusahaan yang bersangkutan. Peraturan Bank sentral (BI) perihal penilaian tingkat kesehatan bank umum adalah Peraturan Bank Indonesia nomor 13/ 1/PBI/2011 yang dikeluarkan tanggal 5 Januari 2011. Kemudian lebih detail tentang tata cara penilaiannya diterangkan dalam Surat Edaran Bank Indonesia nomor 13/ 24 /DPNP tanggal 25 Oktober 2011. Penilaian Tingkat Kesehatan Bank berdasarkan peraturan tersebut mencakup penilaian terhadap faktor-faktor berikut: Profil Risiko, Good Corporate Governance, Earning, dan Capital Permodalan. riba. Bank syariah menggunakan prinsip-prinsip syariat Islam dengan senantiasa mengacu kepada ketentuan-ketentuan yang ada pada kitab Allah SWT (Qur’an) maupun Hadits Nabi SAW (Wilardjo, 2005); (Jalil & Rahman, 2010); (Iqbal & Molyneux, 2016); (Wulandari dan Subagio, 2015). 1. PENDAHULUAN Bank syariah sebagai intermediasi masyarakat diharapkan mampu tampil memberikan yang terbaik jika dibandingkan dengan bank konvensional. Keberadaan bank syariah ini dengan mengunakan konsep pelarangan penerapan bunga dalam berbagai transaksi karena termasuk riba (Uusmani and Taqī ʻUs̲ mānī, 2002); (Chong & Liu, 2009); (Farooq, 2011); (Tho’in, Irawati & Lee, 2018); (Nasim & Mediawati, 2019). Umat Islam dan seluruh masyarakat tentunya sangat mengharapkan bank syariah ini dapat berkembang semakin baik serta tingkat kesehatan keuangannya juga bagus. Pengukuran tingkat kesehatan bank yang dilakukan pemerintah tentunya melalui Bank Indonesia yaitu dengan menerbitkan peraturan BI nomor 13/ 1 /PBI/2011 tentang penilaian tingkat kesehatan bank umum pada tanggal 5 Januari 2011. Kemudian lebih detail tentang tata cara penilaiannya diterangkan dalam Surat Edaran Bank Indonesia nomor 13/ 24 /DPNP tanggal 25 Oktober 2011. Bank Syariah Mandiri merupakan salah satu bank syariah yang berkembang dengan baik, hal ini dibuktikan dengan semakin eksisnya lembaga keuangan ini serta laporan keuangan yang dirilisnya menjadikan masyarakat semakin percaya dengan adanya lembaga keuangan ini. Oleh sebab itu ketertarikan untuk melihat kinerja keuangannya selama ini sangat diperlukan. Bank adalah badan usaha di bidang keuangan yang menarik dan mengeluarkan uang dalam masyarakat, terutama memberikan kredit dan jasa dalam lalu lintas pembayaran dan peredaran uang. Syariah menurut Kamus Besar Bahasa Indonesia adalah hukum agama yang menetapkan peraturan hidup manusia, hubungan manusia dengan Allah, hubungan manusia dengan manusia, dan alam sekitar berdasarkan Al-Quran dan hadis (Indonesia, K. B. B., & Bahasa, T. P. K. P., 2015). 2. METODOLOGI PENELITIAN Penelitian ini menggunakan metode deskriptif kuantitatif. Data yang digunakan adalah laporan keuangan yang dipublikasikan oleh Bank Syariah Mandiri (BSM) periode tahun 2014 sampai tahun 2018 atau selama 5 tahun. Metode analisis dalam penelitian ini adalah menggunakan rumus penghitungan faktor permodalan dalam hal ini yaitu dengan menggunakan rasio CAR, faktor pembiayaan dengan menggunakan rasio FDR, faktor profitabilitas dengan menggunakan rasio ROA & rasio ROE, serta dari faktor risiko kredit yaitu dengan menggunakan rasio NPF. Laporan keuangan dalam akuntansi bank syariah adalah laporan keuangan yang memberikan sebuah gambaran fungsi bank Islam sebagai investor, hak dan kewajiban, dengan tidak memandang tujuan bank Islam dari masalah investasinya, apakah ekonomi atau sosial (Hassan dan Harahap, 2010). Tujuan pembuatan laporan keuangan adalah untuk memberikan informasi mengenai posisi keuangan, kinerja keuangan dan arus kas yang bermanfaat bagi berbagai pihak pengguna 3. HASIL PENELITIAN Secara garis besar, selama lima tahun Laporan Keuangan Bank Syariah Mandiri (BSM) digambarkan pada tabel di bawah ini: Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 398 Tabel 1. Ikhtisar Keuangan BSM Tahun 2014-2018 Ikhtias Keuangan (RP miliar) Tahun 2014 2015 2016 2017 2018 Neraca Modal 4.937 5.613 6.392 7.314 8.039 Modal Inti, Pelengkap, Tambahan 5.570 6.187 6.942 7.844 8.566 Total Aktiva 66.955 70.369 78.831 87.915 98.341 Dana Pihak Ketiga 59.821 62.113 69.950 77.903 87.471 ATMR 37.746 48.146 49.555 49.350 52.670 Pembiayaan yang diberikan 32.654 33.443 34.787 34.739 37.007 Pembiayaan bermasalah Dalam perhatian khusus 2.637 3.095 2.479 1.880 1.154 Kurang lancar 601 530 355 470 206 Diragukan 208 201 215 347 101 Macet 601 509 701 89 289 Laba/rugi Laba sebelum pajak 110 374 434 487 815 Laba usaha 99 369 442 470 839 Beban bagi hasil 2.451 2.438 2.339 2.541 2.659 Laba bersih 72 289 325 365 605 Sumber: Laporan Tahunan 2014-2018 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 398 Berdasarkan laporan tahunan pada tabel di atas, menunjukkan bahwa secara keseluruhan posisi atau tingkat kesehatan Bank Syariah Mandiri selama periode 2014-2018 dalam keadaan yang sangat baik. 3.1 Penilaian Kesehatan Bank Syariah Mandiri (BSM) dari Faktor Permodalan dengan Menggunakan Rasio CAR Rasio CAR Tabel 2. Perhitungan Permodalan (CAR) dan Penilaian Peringkat BSM 2014-2018 Tahun Modal (Rp) ATMR (Rp) CAR Peringkat Predikat 2014 5.570.000.000.000 37.746 .000.000.000 14,76% 1 Sangat baik 2015 6.187.000.000.000 48.146.000.000.000 12,85% 1 Sangat baik 2016 6.942.000.000.000 49.555.000.000.000 14,01% 1 Sangat baik 2017 7.844.000.000.000 49.350.000.000.000 15,89% 1 Sangat baik 2018 8.566 .000.000.000 52.670.000.000.000 16,26% 1 Sangat baik Sumber: data diolah Tabel 2. Perhitungan Permodalan (CAR) dan Penilaian Peringkat BSM 2014-2018 dan tahun 2018 terdapat kenaikan sebesar 0,37% menjadi 16,26%. Pada tabel di atas dapat diketahui bahwa nilai CAR tertinggi dicapai pada tahun 2018. Artinya faktor permodalan selama periode tersebut sangat baik. Hasil perhitungan rasio CAR menggunakan rumus yang telah ditetapkan diketahui bahwa CAR 2014 sebesar 14,76%. Pada tahun 2015 mengalami penurunan sebesar 1,91% menjadi 12,85%. Mulai tahun 2016 sampai tahun 2018 selalu terjadi kenaikan. Tahun 2016 kenaikan sebesar 1,16% menjadi 14,01%. Tahun 2017 kenaikan sebesar 1,88% menjadi 15,89% Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 399 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 399 3.2 Penilaian Kesehatan Bank Syariah Mandiri dari Faktor Pembiayaan dengan Menggunakan Rasio FDR Tabel 3. 3. HASIL PENELITIAN Perhitungan Pembiayaan (FDR) dan Penilaian Peringkat BSM 2014-2018 Tahun Kredit yang diberikan (Rp) Dana pihak ketiga (Rp) FDR Peringkat Predikat 2014 49.133.000.000.000 59.821.000.000.000 82,13 2 Sehat 2015 51.090.000.000.000 62.113.000.000.000 82,25 2 Sehat 2016 55.580.000.000.000 69.950.000.000.000 79,46 2 Sehat 2017 60.584.000.000.000 77.903.000.000.000 77,77 2 Sehat 2018 67.752.000.000.000 87.471.000.000.000 77,46 2 Sehat Sumber: data diolah Kesehatan Bank Syariah Mandiri dari Faktor Pembiayaan dengan Menggunakan Rasio Bank Syariah Mandiri dari Faktor Pembiayaan dengan Menggunakan Rasio .2 Penilaian Kesehatan Bank Syariah Mandiri dari Faktor Pembiayaan dengan Meng FDR bahwa dari tahun 2014 sampai tahun 2018 peringkat FDR selalu berada pada peringkat dua. Artinya kesehatan Bank Syariah Mandiri berdasarkan rasio pembiayaan (FDR) selama periode tersebut pada ketegori sehat. Hasil perhitungan rasio FDR menggunakan rumus yang telah ditetapkan diketahui bahwa FDR 2014 sebesar 82,13%. Pada tahun 2015 sebesar 82,25%. Perkembangan selalu lebih baik sejak tahun 2016 sebesar 79,46%, kemudian tahun 2017 sebesar 77,77%. FDR paling baik dicapai pada tahun 2018 sebesar 77,46%. Dari data tersebut dapat diketahui 3.3 Penilaian Kesehatan Bank Syariah Mandiri (BSM) dari Faktor Profitabilitas dengan Menggunakan Rasio ROA dan ROE Tabel 4. Perhitungan Profitabilitas (ROA) dan Penilaian Peringkat BSM 2014-2018 Tahun Laba sebelum pajak (Rp) Total Asset (Rp) ROA Peringkat Predikat 2014 110.000.000.000 66.955.000.000.000 0,16% 4 Kurang baik 2015 374.000.000.000 70.369.000.000.000 0,53% 3 Cukup baik 2016 434.000.000.000 78.831.000.000.000 0,55% 3 Cukup baik 2017 487.000.000.000 87.915.000.000.000 0,55% 3 Cukup baik 2018 815.000.000.000 98.341.000.000.000 0,83% 3 Cukup baik Sumber: data diolah Kesehatan Bank Syariah Mandiri (BSM) dari Faktor Profitabilitas dengan Menggunakan A dan ROE 3.3 Penilaian Kesehatan Bank Syariah Mandiri (BSM) dari Faktor Profitabilitas deng Rasio ROA dan ROE bahwa tahun 2014 rasio ROA masih kurang baik. Namum demikian selanjutnya terjadi peningkatan, di mana sejak tahun 2015 sampai dengan tahun 2018 peringkat ROA selalu pada posisi 3, artinya faktor kamampuan bank dalam menghasilkan laba dari sisi asset yang dimiliki cukup baik. Hasil perhitungan rasio ROA menggunakan rumus yang telah ditetapkan diketahui bahwa ROA 2014 sebesar 0,16%. Pada tahun 2015 sampai dengan tahun 2018 selalu terjadi kenaikan. Tahun 2015 ROA menjadi 0,53%, tahun 2016 dan 2017 ROA menjadi 0,55% dan tahun 2018 ROA pada posisi tertinggi sebesar 0,83%. Dari data tersebut dapat diketahui Tabel 5. Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 3. HASIL PENELITIAN Perhitungan Profitabilitas (ROE) dan Penilaian Peringkat BSM 2014- g ( ) g Tahun Laba setelah pajak (Rp) Rata-rata modal disetor (Rp) ROE Peringkat Predikat 2014 72.000.000.000 4.937.000.000.000 1,46% 4 Kurang baik 2015 289.000.000.000 6.187.000.000.000 5,15% 3 Cukup baik 2016 325.000.000.000 6.942.000.000.000 5,08% 3 Cukup baik 2017 365.000.000.000 7.844.000.000.000 4,99% 4 Kurang baik 2018 605.000.000.000 8.566.000.000.000 7,53% 3 Cukup baik Sumber: data diolah Sumber: data diolah tahun 2016 sebesar 5,08%, tahun 2017 terjadi penurunan menjadi 4,19% dan tahun 2018 ROE kembali meningkat dan berada pada posisi tertinggi sebesar 7,53%. Dari data tersebut dapat diketahui Hasil perhitungan rasio ROE menggunakan rumus yang telah ditetapkan diketahui bahwa ROE 2014 sebesar 1,46%. Terjadi peningkatan signifikan pada tahun 2015 menjadi sebesar 5,15%. Selanjutnya Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 400 faktor kamampuan bank dalam menghasilkan laba bersih dari sisi setoran modal cukup baik. bahwa tahun 2014 dan 2017 rasio ROE masih kurang baik. Namum demikian selanjutnya terjadi peningkatan signifikan pada tahun 2015 dan 2018. Peringkat ROE secara umum ada pada posisi 3, artinya bahwa tahun 2014 dan 2017 rasio ROE masih kurang baik. Namum demikian selanjutnya terjadi peningkatan signifikan pada tahun 2015 dan 2018. Peringkat ROE secara umum ada pada posisi 3, artinya bahwa tahun 2014 dan 2017 rasio ROE masih kurang baik. Namum demikian selanjutnya terjadi peningkatan signifikan pada tahun 2015 dan 2018. Peringkat ROE secara umum ada pada posisi 3, artinya 3.4 Penilaian Kesehatan Bank Syariah Mandiri dari Faktor Risiko Kredit dengan Menggunakan Rasio NPF Tabel 6. Perhitungan NPF dan Penilaian Peringkat BSM 2014-2018 Tahun Pembiayaan (KL, D, M) (Rp) Total Pembiayaan (Rp) NPF Peringkat Predikat 2014 1.410.000.000.000 32.654.000.000.000 4,32 2 Baik 2015 1.240.000.000.000 33.443.000.000.000 3,71 2 Baik 2016 1.271.000.000.000 34.787.000.000.000 3,65 2 Baik 2017 906.000.000.000 34.739.000.000.000 2,61 2 Baik 2018 596.000.000.000 37.007.000.000.000 1,61 1 Sangat Baik Sumber: data diolah Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 401 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 401 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 401 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 401 Berdasarkan tabel 7 pada rata-rata peringkat dapat diketahui bahwa hasil penilaian kesehatan selama lima tahun terakhir Bank Syariah Mandiri berada pada predikat sehat. Dari tahun 2014 hingga tahun 2018 aspek permodalan menunjukkan rasio CAR selalu berada pada peringkat satu. Artinya faktor permodalan Bank Syariah Mandiri selama periode tersebut sangat baik. Kemudian dari aspek profitabilitas, rasio ROA tahun 2014 menunjukkan predikat kurang baik. Namun demikian tahun 2015 sampai 2018 rasio ROA meningkat menjadi cukup baik. Demikian pula rasio ROE, predikat kurang baik terjadi pada tahun 2014 dan 2017. Akan tetapi terjadi perbaikan di tahun 2015, 2016 dan 2018 menjadi cukup baik, sehingga secara rata-rata predikat selama lima tahun adalah cukup baik. Dari rasio tersebut dapat diketahui bahwa kemampuan menghasilkan laba Bank Syariah Mandiri baik dari sisi aset yang dimiliki maupun dari sisi modal yang disetor cukup baik. Tingkat likuiditas yang dinilai dari rasio FDR tahun 2014-2018 berada pada peringkat 2 atau predikat sehat. Dari sini dapat disimpulkan bahwa tingkat kemampuan Bank Syariah Mandiri untuk melunasi kewajiban jangka pendeknya tepat pada waktunya dan kemampuan memenuhi semua permohonan kredit yang telah disetujui sangat baik. Kualitas pembiayaan Bank Syariah Mandiri dilihat dari rasio NPF berpredikat baik pada tahun 2014-2017, sedangakan tahun 2018 meningkat sampai pada predikat sangat baik. Alamgir, M. (2016). An analysis of islamic banking activities in Bangladesh: Issues and challenges. Thoughts on Economics, 25(03), 29- 58. Al-Hares, O. M., AbuGhazaleh, N. M., & El-Galfy, A. M. (2013). Financial performance and compliance with Basel III capital standards: Conventional vs. Islamic banks. Journal of Applied Business Research (JABR), 29(4), 1031- 1048. Amelia, E. (2015). Financial Ratio and Its Influence to Profitability in Islamic Banks. Al-Iqtishad: Jurnal Ilmu Ekonomi Syariah, 7(2), 229-240. Chong, B. S., & Liu, M. H. (2009). Islamic banking: interest-free or interest-based?. Pacific-Basin finance journal, 17(1), 125-144. Farooq, M. O. (2011). Qarḍ Ḥasan, Wadī ʾah/Amānah and Bank Deposits: Applications and Misapplications of Some Concepts in Islamic Banking. Arab Law Quarterly, 25(2), 115-146. Farooq, M., & Zaheer, S. (2015). Are Islamic banks more resilient during financial panics?. Pacific Economic Review, 20(1), 101-124. Gilani, H. (2015). Exploring the ethical aspects of Islamic banking. International Journal of Islamic and Middle Eastern Finance and Management. Hassan, A., & Harahap, S. S. (2010). Exploring corporate social responsibility disclosure: the case of Islamic banks. Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 401 International Journal of Islamic and Middle Eastern Finance and Management. 4. KESIMPULAN Iatridis, G. (2010). International Financial Reporting Standards and the quality of financial statement information. International review of financial analysis, 19(3), 193-204. Berdasarkan hasil analisis penelitian pada bab sebelumnya, maka dapat disimpulkan bahwa tingkat kesehatan Bank Syariah Mandiri (BSM) berdasarkan rata-rata peringkat dengan menggunakan rasio permodalan, rasio profitabilitas, rasio pembiayaan, dan rasio risiko kredit selama lima tahun (2014-2018) termasuk bank syariah yang sehat. Indonesia, K. B. B., & Bahasa, T. P. K. P. (2015). Jakarta: Balai Pustaka, 2005. Cetakan Pertama Edisi Ketiga. Iqbal, M., & Molyneux, P. (2016). Thirty years of Islamic banking: History, performance and prospects. Springer. 3.4 Penilaian Kesehatan Bank Syariah Mandiri dari Faktor Risiko Kredit dengan Menggunakan Rasio 3.4 Penilaian Kesehatan Bank Syariah Mandiri dari Faktor Risiko Kredit dengan Menggunakan Rasio Tabel 6. Perhitungan NPF dan Penilaian Peringkat BSM 2014-2018 Sumber: data diolah tersebut dapat diketahui bahwa dari tahun 2014 sampai tahun 2017 peringkat NPF selalu berada pada peringkat dua, yaitu predikat baik. Kemudian tahun 2018 terjadi peningkatan ke predikat sangat baik. Hasil perhitungan rasio NPF menggunakan rumus yang telah ditetapkan diketahui bahwa NPF 2014 sebesar 4,32%. %. Tahun-tahun berikutnya selalu terjadi perkembangan lebih baik, dimulai tahun 2015 menjadi sebesar 3,71%, kemudian tahun 2016 sebesar 3,65%, tahun 2017 sebesar 2,61%. NPF paling baik dicapai pada tahun 2018 sebesar 1,61%. Dari data Adapun rata-rata dari penilaian seluruh faktor dapat dilihat pada tabel berikut ini: Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 dicapai pada tahun 2018 sebesar 1,61%. Dari data Tabel 7. Rata-rata Peringkat BSM 2014-2018 Tahun Faktor Rasio Peringkat Predikat Rata-rata peringkat 2014 CAR 14,76% 1 Sangat baik 2,6 ROA 0,16% 4 Kurang baik ROE 1,46% 4 Kurang baik FDR 82,13% 2 Sehat NPF 4,32% 2 Baik 2015 CAR 12,85% 1 Sangat baik 2,2 ROA 0,53% 3 Cukup baik ROE 5,15% 3 Cukup baik FDR 82,25% 2 Sehat NPF 3,71% 2 Baik 2016 CAR 14,01% 1 Sangat baik 2,2 ROA 0,55% 3 Cukup baik ROE 5,08% 3 Cukup baik FDR 79,46% 2 Sehat NPF 3,65% 2 Baik 2017 CAR 15,89% 1 Sangat baik 2,4 ROA 0,55% 3 Cukup baik ROE 4,99% 3 Kurang baik FDR 77,77% 2 Sehat NPF 2,61% 2 Baik 2018 CAR 16,26% 1 Sangat baik 2 ROA 0,83% 3 Cukup baik ROE 7,53% 3 Cukup baik FDR 77,46% 2 Sehat NPF 1,61% 1 Sangat Baik Rata-rata Peringkat 2,28 Predikat Sehat Sumber: data diolah Tabel 7. Rata-rata Peringkat BSM 2014-2018 Tabel 7. Rata-rata Peringkat BSM 2014-2018 Tabel 7. Rata-rata Peringkat BSM 2014-2018 Tabel 7. Rata-rata Peringkat BSM 2014-201 5. UCAPAN TERIMA KASIH Terima kasih disampaikan kepada Bapak Dr. Rahmawan Arifin, SE., M.Si selaku Dekan Fakultas Ekonomi dan Bisnis Islam IAIN Surakarta beserta jajarannya yang telah memberikan dukungan serta berbagai fasilitas atas terlaksananya penelitian ini. Jalil, A., & Rahman, M. K. (2010). Financial transactions in Islamic Banking are viable alternatives to the conventional banking transactions. International Journal of Business and Social Science, 1(3). Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 402 Jurnal Ilmiah Ekonomi Islam, 6(03), 2020, 402 Minnis, M. (2011). The value of financial statement verification in debt financing: Evidence from private US firms. Journal of accounting research, 49(2), 457-506. Tho'in, M., Irawati, T., & Lee, M. (2018). Risk and Financial Health Level of Shariabanking. Journal Research and Analysis: Economy, 1(1), 19-26. Uusmani, M. T., & Taqī ʻUs̲ mānī, M. (2002). An introduction to Islamic finance (Vol. 20). Brill. Nasim, A., & Mediawati, E. (2019). Critical Study the Application of the Principle of Profit Sharing in Syirkah Mudharabah and Musyarakah in Islamic Banking. KnE Social Sciences, 1351-1359. Wilardjo, S. B. (2005). pengertian, peranan dan perkembangan bank syari’ah Di Indonesia. Value Added\| Majalah Ekonomi Dan Bisnis, 2(1). Wulandari, D., & Subagio, A. (2015). Consumer decision making in conventional banks and islamic bank based on quality of service perception. Procedia-Social and Behavioral Sciences, 211, 471-475. Rozzani, N., & Rahman, R. A. (2013). Camels and performance evaluation of banks in Malaysia: conventional versus Islamic. Journal of Islamic Finance and Business Research, 2(1), 36-45. Surat Edaran Bank Indonesia nomor 13/ 24 /DPNP tanggal 25 Oktober 2011. Penilaian Tingkat Kesehatan Bank mencakup penilaian terhadap faktor-faktor berikut: Profil Risiko, Good Corporate Governance, Earning, dan Capital Permodalan. Diakses pada tanggal 4 Maret 2020. Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534 Jurnal Ilmiah Ekonomi Islam, ISSN: 2477-6157; E-ISSN 2579-6534
https://openalex.org/W4392254800	https://ejournal.almaata.ac.id/index.php/IJMA/article/download/3871/2138	Indonesian	null	PENGARUH PEMBERDAYAAN DAN KNOWLEDGE MANAGEMENT TERHADAP KINERJA KARYAWAN PADA PT. HADJI KALLA TOYOTA KENDARI	Deleted Journal	2,023	cc-by-sa	2,334	Pengaruh Pemberdayaan dan Knowledge Management Terhadap Kinerja Karyawan Pada PT. Hadji Kalla Toyota Kendari Adwi1, Andika Dzulhaji Pratama Putra2, Dwi Zulkifar Mulyadi3, Asraf4, Arfin Bagea5 1,2,3,4,5, Sekolah Tinggi Ilmu Ekonomi Enam, Enam Kendari adwisarjanamanajemen@gmail.com adwisarjanamanajemen@gmail.com IJMA (Indonesian Journal of Management and Accounting) Volume 5 No. 1 \| 2024 https://ejournal.almaata.ac.id/index.php/IJMA/index Keyword: Pemberdayaan Management, Kinerja Karyawan IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index ABSTRAK Tujuan penelitian adalah untuk mengetahui dan menganalisis: (1) Pemberdayaan dan knowledge management terhadap kinerja karyawan pada PT.Hadji Kalla Toyota Kendari; (2) Pemberdayaan terhadap kinerja karyawan pada PT. Hadji Kalla Toyota Kendari; (3) Knowledge management terhadap kinerja karyawan pada PT. Hadji KallaToyota Kendari. Populasi dalam penelitian ini adalah karyawan PT. Hadji Kalla Toyota Kendari jumlah populasi dalam penelitian ini sebanyak 150 orang. Sampel penelitian ini adalah 60 responden dengan menggunakan metode slovin. Jenis data yang digunakan dalam penelitian ini adalah data primer. Data primer adalah data yang berasal langsung dari sumber data yang dikumpulkan secara khusus dan berhubungan langsung dengan masalah penelitian yang diteliti. Metode analisis data yang diguna kan adalah analisis deskriptif kuantitatif. Alat analisis yang digunakan adalah Regresi Linear berganda. Hasil penelitian menyatakan bahwa (1) Pemberdayaan dan knowledge management secara simultan berpengaruh positif dan signifikan terhadap kinerja pegawai; (2) Pemberdayaan berpengaruh positif dan signifikan terhadap kinerja karyawan; (3) Knowledge Management berpengaruh positif dan signifikan terhadap kinerja karyawan. Article History Received : November 23 th, 2023 Revised : Desember 1 th, 2023 Accepted : Desember 6rd, 2023 Keyword: Pemberdayaan Management, Kinerja Karyawan 9 9 PENDAHULUAN Kinerja menunjukkan keberhasilan pegawai dalam memenuhi tugas dan tanggung jawabnya. Semakin tinggi kinerja karyawan, semakin besar produktivitas organisasi secara keseluruhan cenderung meningkat. Kinerja karyawan sangat menentukan kinerja seluruh perusahaan. Penting bagi setiap organisasi untuk mencapai tujuan organisasi secara efektif dan efisien. Kinerja dapat digunakan untuk mengukur tingkat pencapaian atau praktik individu atau kelompok. Kinerja staf merupakan faktor terpenting dalam meningkatkan efisiensi dan efektifitas perusahaan, yang tercermin dari keuntungan atau profitabilitas perusahaan yang semakin berkembang. Kinerja karyawan sangat menentukan kinerja perusahaan secara keseluruhan. Hal ini penting bagi setiap organisasi untuk mencapai tujuan organisasi secara efektif dan efisien sehingga perusahaan lebih mampu melakukan hal-hal yang dapat meningkatkan kinerja karyawan. Dalam meningkatkan kinerja karyawan di perusahaan melalui motivasi, keterikatan dan produktivitas karyawan melalui pemberdayaan karyawan. Hal ini harus diperhatikan karena berdampak langsung pada karyawan. Pemberdayaan karyawan yang diterapkan dapat meningkatkan kinerja karyawan. Sebaliknya, jika pemberdayaan karyawan tidak dilaksanakan, maka produktivitas mereka akan menurun. Pemberdayaan karyawan dianggap baik ketika karyawan dapat terlibat dalam pengambilan keputusan di tempat kerja. Menurut Sedarmayanti (2014), pemberdayaan pegawai merupakan upaya untuk menciptakan sumber daya manusia yang berkualitas, kemampuan menggunakan, mengembangkan dan mengelola ilmu pengetahuan dan teknologi, serta kemampuan kepemimpinan. Menurut Arshad et al. (2021), pemberdayaan karyawan adalah pendekatan karyawan ke tempat kerja yang memfasilitasi pencapaian tujuan mereka. Faktor lain yang dapat mempengaruhi kinerja karyawan adalah manajemen pengetahuan. Menurut David Gurteen (2012), manajemen pengetahuan adalah perasaan manusia untuk melakukan proses berbagi informasi, pembelajaran dan kolaborasi secara lebih efektif. Pada dasarnya knowledge management mengacu pada teori knowledge management atau manajemen pengetahuan. Manajemen pengetahuan sangat penting untuk semua orang dalam bisnis terutama untuk pemula karena pengetahuan dan visi adalah hal penting dalam pengembangan bisnis. Sangat penting bahwa informasi yang diperoleh dikelola secara optimal sehingga dapat memberikan dampak positif bagi perkembangan bisnis. Manajemen informasi atau manajemen pengetahuan adalah 10 seperangkat alat, metode, dan strategi untuk menganalisis, menyimpan, mentransfer, meningkatkan, dan mengatur informasi perusahaan. Pada dasarnya, tujuan dari manajemen informasi adalah untuk meningkatkan efisiensi perusahaan dan menyimpan informasinya. Melalui pengelolaan informasi ini, seluruh bagian perusahaan dapat menerima informasi dan informasi mengenai aspek operasional dan bisnis. Dengan demikian, kegiatan tersebut dapat dilakukan secara lebih mendalam dan efektif. PT. Hadji Kalla Toyota (Kalla Toyota) sebagai salah satu pendiri diler resmi PT Toyota-Astra Motor (TAM) di Indonesia yang wilayah operasi dan pemasarannya adalah Provinsi Sulawesi Selatan, Provinsi Sulawesi Barat, Provinsi Sulawesi Tengah dan Provinsi Tenggara. Sulawesi termasuk. PENDAHULUAN Pencapaian target penjualan yang semakin besar setiap bulannya tentu saja di dukung oleh kinerja marketing yang handal dengan mengedepankan kinerja pegawai yang berkualitas. Dalam proses penjualan, perusahaan harus mampu mendekatkan diri dengan masyarakat, memahami keinginan dan memenuhi kebutuhan masyarakat yang sekaligus memiliki kemampuan dan keleluasaan dalam bekerja. Selain itu harus pula diperhatikan aspek profesionalisme, transparansi, non diskriminatif, ramah, kemudahan dan kecepatan masyarakat dalam mendapatkan informasi produk, serta bebas korupsi.Penelitian ini didasarkan pada penelitian sebelumnya oleh Meita Pragiwan, Mohammad Benny Alexandr dan R Chandra (2018) Analisis Pemberdayaan Pegawai dan Manajemen Pengetahuan Dalam Meningkatkan Kinerja Pegawai Sektor Pariwisata. Hal tersebut menyatakan bahwa pengembangan karyawan dan pengetahuan manajerial berpengaruh positif dan signifikan terhadap kinerja karyawan. Meliana Rifka Fitricia (2021) Hasil penelitian menunjukkan bahwa knowledge management dan empowerment tidak berpengaruh signifikan terhadap kinerja karyawan saat bekerja di rumah. METODOLOGI Dalam penelitian ini penulis menggunakan penelitian kuantitatif, karena data yang diperoleh nantinya berupa angka. Dari angka yang diperoleh akan dianalisis lebih lanjut dalam analisis data. Penelitian ini termasuk kategori penelitian surey karena dalam pengumpulan data menggunakan angket sebagai alat penelitian yang dilakukan pada populasi besar maupun kecil, data yang dipelajari adalah data dari sampel yang diambil dari populasi tersebut, sehingga ditemukan kejadian relative, distribusi dan hubungan antar variabel, sosiologis maupun psikologis, (Sugiyono, 2013). Sumber data primer atau utama dalam penelitian ini yakni data diambil berdasarkan hasil kuesioner yang akan didistribusikan nantinya. Instrumen atau alat yang digunakan dalam penelitian ini yakni kuesioner mengenai keseimbangan kehidupan kerja, keterlibatan kerja, dan Turnover 11 Pengaruh Pemberdayaan dan Knowledge Management terhadap Kinerja Karyawan Berdasarkan hasil analisis data menunjukkan bahwa Pemberdayaan dan Knowledge Management secara simultan berpengaruh positif dan signifikan terhadap kinerja karyawan, hal ini ditunjukan dengan nilai signifikansi lebih kecil dari nilai alpha. Hal ini mengindikasikan bahwa variabel Pemberdayaan dan Knowledge Management kerja secara bersama-sama dapat meningkatkan kinerja karyawan. Kajian ini berdasarkan teori, menurut Priansa (2019:45), pemberdayaan karyawan merupakan salah satu langkah yang harus dilakukan perusahaan untuk menghasilkan sumber daya manusia yang berkualitas dengan kemampuan menggunakan, mengembangkan dan mengelola ilmu pengetahuan dan teknologi, serta keterampilan manajemen dan kemampuan untuk memecahkan berbagai masalah nyata di Perusahaan. Menurut Turban dan lain-lain (2015), manajemen pengetahuan adalah proses membantu identitas perusahaan, proses memilih, mengatur, mendistribusikan dan mengkomunikasikan informasi dan keahlian penting, termasuk bagian dari memori organisasi yang secara unik terletak di area terstruktur organisasi. Selain itu, menurut Rigby (2016: 5-6), diterjemahkan oleh Dalkir, knowledge management mengembangkan sistem dan proses untuk memperoleh dan berbagi kekayaan intelektual. Menumbuhkan generasi berdasarkan pengetahuan yang berguna, bertanggung jawab dan bermakna dan bertujuan untuk meningkatkan pembelajaran baik individu maupun kelompok. Selain itu, dapat memaksimalkan nilai dasar kebijakan organisasi di semua fungsi dan lokasi. KM mensyaratkan bahwa kesuksesan bisnis bukanlah kumpulan produk, tetapi basis pengetahuan khusus. Kecerdasan adalah kunci yang memberikan daya saing Perusahaan Hasil penelitian ini juga didukung oleh penelitian sebelumnya oleh Meita Pragiwani, Mohammad Benny Alexandri dan R Chandra (2018) bahwa empowerment dan knowledge management berpengaruh signifikan terhadap kinerja karyawan. IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index Intention. Kuesioner yang digunakan dengan skala likert yang terdiri atas lima penilaian yakni Sangat Setuju, Setuju, Tidak. Teknik pengolahan data adalah suatu proses yang terdapat dalam memperoleh data yang dilakukan sistematis dalam pengumpulan dan penyajian fakta untuk tujuan tertentu. Setiap pertanyaan dilengkapi dengan lima alternatif jawaban, yaitu Sangat Tidak Setuju (STS) hingga Sangat Setuju (SS). Penentuan nilai skala likert untuk jawaban dilakukan dalam bentuk checklist. Jawaban setiap item instrumen akan dijawab dengan format: IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index Intention. Kuesioner yang digunakan dengan skala likert yang terdiri atas lima penilaian yakni Sangat Setuju, Setuju, Tidak. Teknik pengolahan data adalah suatu proses yang terdapat dalam memperoleh data yang dilakukan sistematis dalam pengumpulan dan penyajian fakta untuk tujuan tertentu. Setiap pertanyaan dilengkapi dengan lima alternatif jawaban, yaitu Sangat Tidak Setuju (STS) hingga Sangat Setuju (SS). Penentuan nilai skala likert untuk jawaban dilakukan dalam bentuk checklist. Jawaban setiap item instrumen akan dijawab dengan format: SS = Sangat Setuju Diberi Skor 5 S = Setuju Diberi Skor 4 N = Netral Diberi Skor 3 TS = Tidak Setuju Diberi Skor 2 STS = Sangat Tidak Setuju Diberi Skor 1 Angka 5 (lima) menunjukan bahwa responden memberikan tanggapan yang bersifat sangat tidak setuju (sangat negatif) terhadap pertanyaan-pertanyaan yang diajukan, sedangkan angka 1 (satu) menunjukan tanggapan yang bersifat sangat setuju (sangat positif). Jawaban dari setiap item instrumen yang menggunakan skala Likert mempunyai gradasi dari sangat positif sampai dengan sangat negatif. Adapun alternatif jawaban dengan menggunakan skala Likert, yaitu dengan memberikan skor pada masing- masing jawaban pertanyaan alternatif sebagai berikut: Tabel 1. Skala Nilai Alternatif Jawaban Kuesioner Interval Skor Tingkat Hubungan 4,22 – 5,00 Sangat Baik 3,40 – 4,19 Baik 2,60 – 3,39 Cukup Baik 1,80 – 2,59 Kurang Baik 1,00 – 1,79 Sangat Tidak Baik Sumber: (Sugiyono, 2017) Tabel 1. Skala Nilai Alternatif Jawaban Kuesioner 12 IJMA (Indonesian Journal of Management and Accounting) Volume 4 No. 2 \| SEPTEMBER 2023 https://ejournal.almaata.ac.id/index.php/IJMA/index tugas, tantangan tugas, dukungan sumberdaya, dukungan atasan langsung, dukungan rekan sekerja, pengakuan/penghargaan, persatuan dan kerjasama dan dukungan kreativitas berada dalam kategori baik. Hasil tersebut menunjukkan bahwa kondisi yang berkaitan dengan hubungan karyawan yang dapat mempengaruhi kinerja karyawan. Pemberdayaan dapat memengaruhi emosional karyawan. Jika karyawan menyenangi pemberdayaannya maka karyawan tersebut akan betah di tempat kerjanya, melakukan aktifitasnya sehingga waktu kerja dipergunakan secara efektif. Produktifitas akan tinggi dan prestasi kerja karyawan juga tinggi. Fakta empiris yang ditunjukan berdasarkan persepsi karyawan bahwa pemberdayaan berpengaruh signfikan terhadap kinerja karyawan mengindikasikan bahwa memiliki keinginan untuk berubahmenjadi lebih baik daripada sebelumnya, memiliki keinginan untuk berkembang menjadi lebih baik daripada sebelumnya, memiliki keinginan bekerja lebih giat tiap harinya, memiliki kepercayaan dalam hubungan antar manusia, percaya bahwa sebagian orang umumnyabertujuan baik, cenderung berasumsi baik tentang manusia, yakin bahwa saya akan sukses dalam pekerjaan, yakin ketika ada kesulitan dalam pekerjaan, mampu menyelesaikannya. Pengaruh Pemberdayaan Terhadap Kinerja Karyawan Berdasarkan hasil analisis menujukan bahwa pemberdayaan berpengaruh signifikan terhadap kinerja karyawan yang ditandai dengan nilai signifikansi lebih kecil dari nilai alpha. Dengan demikian dapat diinterpretasikan bahwa semakin baik pemberdayaan pada PT. Hadji Kalla Kendari, maka akan semakin tinggi kinerja karyawan. Berdasarkan hasil penelitian bahwa karakteristik karyawan dalam penelitian ini berada ada rentang umur produkif, pendidikan yang cukup serta masa kerja yang lama, sehingga pemberdayaan perusahaan kepada karyawan akan memberikan dukungan atas upaya pencapaian tujuan yang telah ditetapkan oleh organisasi. Berdasarkan hasil analisis data yang telah dilakukan maka dapat diketahui bahwa kebebasan atau otonomi pelaksanaan 13 Pengaruh Knowledge Management Terhadap Kinerja Karyawan Berdasarkan hasil analsisi data menunjukan bahwa Knowledge Management berpengaruh signifikan terhadap kinerja karyawan, yang ditandai dengan nilai signifikansi lebih kecil dari nilai alpha. Dengan demikian dapat diinterpretasikan bahwa semakin baik Knowledge Management pada PT. Hadji Kalla Kendari, maka akan semakin tinggi kinerja karyawan. Berdasarkan hasil penelitian, karyawan yang mengikuti penelitian ini dicirikan dengan masa kerja yang panjang, Knowledge management sebagai persiapan individu untuk bertanggung jawab, yang berkaitan dengan penanaman keterampilan intelektual atau emosional yang diperlukan untuk melakukan pekerjaan dengan lebih baik. Oleh karena itu, karyawan harus memiliki pengalaman kerja yang cukup untuk dapat berkembang dalam karirnya. Pengalaman kerja jangka panjang dapat meningkatkan potensi karyawan tersebut Hasil penelitian ini juga didukung dengan persepsi karyawan melalui fakta empiris bahwa Knowledge Management dikategorikan baik, dalam hai ini mengindikasikan bahwa dengan pengetahuan yang dimiliki dapat menyelesaikan pekerjaan dengan baik, dapat menyelesaikan pekerjaan dengan mengikuti metode kerja yang telah ditentukan, memiliki tingkat pengetahuan yang memadai dalam bidang 14 pekerjaan yang dilakukan, perusahaan berbagi informasi dan pengetahuan yang diperlukan untuk tugas, terdapat pertukaran informasi/ pengetahuan antar karyawan, aktif mengajak rekan kerja untuk berdiskusi mengenai masalah pekerjaan yangsaya kurang mengerti, terdapat kebijakan insentif untuk saran baru dalam memanfaatkan pengetahuan yang ada, perlu adanya alur kerja yang digunakan dalam melakukan tugas dan perusahaan secara efektif mengelola sumberdan jenis pengetahuan yang berbeda. KESIMPULAN Pemberdayaan dan pengelolaan pengetahuan secara bersama-sama berpengaruh positif signifikan terhadap kinerja karyawan, artinya semakin baik pemberdayaan dan pengelolaan pengetahuan maka kinerja karyawan akan meningkat secara signifikan. Pemberdayaan berpengaruh positif signifikan terhadap kinerja karyawan, artinya semakin baik pemberdayaan melalui keinginan, kepercayaan, keyakinan, kehandalan, tanggung jawab dan komunikasi, maka kinerja karyawan akan semakin baik. Knowledge Management berpengaruh positif signifikan terhadap kinerja karyawan, artinya semakin baik Knowledge Management melalui pengetahuan (creation), berbagi pengetahuan (sharing) dan pengguna akan pengetahuan (ultization) maka kinerja karyawan akan semakin meningkat secara signifikan. REFERENCES Achmadi, Umar F. 2010. Manajemen Penyakit Berbasis Wilayah. Jakarta: UI Press. Achmadi, Umar F. 2010. Manajemen Penyakit Berbasis Wilayah. Jakarta: UI Press. Afandi, P. 2018. Manajemen Sumber Daya Manusia (Teori, Konsep dan Indikator). Riau: Zanafa Publishing. Afandi, P. 2018. Manajemen Sumber Daya Manusia (Teori, Konsep dan Indikator). Riau: Zanafa Publishing. Alvin sholeh. Smart Knowledge Worker. KMplus, Jakarta 2015. Alvin sholeh. Smart Knowledge Worker. KMplus, Jakarta 2015. Arsyad. 2021 .Media pembelajaran. \| Online] dari https://edel.staff.unja.ac.id/blog (20 Febuari 2021) Atmo soeprapto, 2OO2.Menuju SDM Berdaya - Dengan Kepemimpinan Efektif dan Manajemen Efisien, PT. Elex Media Komputindo, Jakarta. Atmo soeprapto, 2OO2.Menuju SDM Berdaya - Dengan Kepemimpinan Efektif dan Manajemen Efisien, PT. Elex Media Komputindo, Jakarta. Bangun, W. 2012. Manajemen Sumber Daya Manusia. Jakarta: Erlangga angun, W. 2012. Manajemen Sumber Daya Manusia. Jakarta: Erlangga Dalkir, K. 2016. Knowledge Management in Theory and Practice. Burlington, MA, Elsevier Butterworth-Heinemann. Dialih bahasakan oleh Jimmy Sadeli dan Bayu Prawira Hie, Salemba Empat, Jakarta. Dalkir, K. 2016. Knowledge Management in Theory and Practice. Burlington, MA, Elsevier Butterworth-Heinemann. Dialih bahasakan oleh Jimmy Sadeli dan Bayu Prawira Hie, Salemba Empat, Jakarta. 15 IJMA (Indonesian Journal of Management and Accounting) https://ejournal.almaata.ac.id/index.php/IJMA/index Fernandez, Becerra, R., & Sabherwal, R. (2015). Knowledge Management Systems and Processes (2 ed.). New York and London: Routledge. Gibson et al., 2012, Organisasi dan Manajemen, Edisi keempat, Erlangga, Jakarta.Gurteen,D. 2012. Creating a KnowledgeSharing Culture, Knowledge Management Magazine, vol 2. No 2, Februari. Hasibuan. 2003. Manajemen Sumber Daya Manusia. Jakarta: Bumi Aksara Hasibuan. 2016. Manajemen Sumber Daya Manusia. Jakarta: Penerbit Bumi Aksara Hasibuan. 2016. Manajemen Sumber Daya Manusia. Jakarta: Penerbit Bumi Aksara Mangkunegara, A.A Anwar Prabu. (2012). Manajemen Sumber Daya Manusia. Bandung: PT. Remaja Rosda karya. Mangkunegara, A.A Anwar Prabu. (2012). Manajemen Sumber Daya Manusia. Bandung: PT. Remaja Rosda karya. 16
https://openalex.org/W4200299600	https://jurnal.univpgri-palembang.ac.id/index.php/dedikasi/article/download/6330/pdf_45	Malay	null	Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Di Sma Negeri 2 Kayuagung	Wahana Dedikasi	2,021	cc-by	5,159	Abstrak Pengabdian kepada masyarakat ini dilakukan sebagai perwujudan pelaksanaan Tri Dharma Perguruan Tinggi Dosen Universitas PGRI Palembang dan L2 Dikti Palembang. Sasaran yang ingin dicapai atas pelaksanaan Pengabdian Kepada Masyarakat (PKM) ini adalah para dosen dapat menerapkan dan mengembangkan kemampuan akademik yang dimilikinya, selain itu setelah mengikuti pelatihan ini para siswa diharapkan dapat menampilkan percakapan Bahasa Inggris secara langsung dan tulisan sehingga siswa dapat berbicara Bahasa inggris secara lancar, komunikatif dan cerdas dalam menggunakan bahasa Inggris dengan judul Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG. Kata Kunci: Model Pembelajaran Inkuiri, Writing Skill, Perangkat Multimedia Artikel disetujui tanggal:24-11-2021 Corresponden Author:Yuspar Uzer e-mail: yusparuzer@gmail.com DOI: http://dx.doi.org/10.31851/dedikasi.v4i2.6330 pelajaran yang penting dalam dunia pendidikan mulai dari tingkat dasar. Sedemikian pentingnya bahasa Inggris dalam kehidupan masyarakat saat ini sehingga dalam mempelajari bahasa Inggris kita dituntut untuk menguasainya secara lisan maupun secara tulisan, yang utama adalah secara lisan bahasa Inggris sebagai bahasa pengantar komunikasi antar 95 \| Wahana Dedikasi Copyright@Yuspar Uzer PENDAHULUAN Bahasa Inggris yang digunakan sebagai bahasa komunikasi antar bangsa telah menjadi sebuah bahasa yang dibutuhkan selain bahasa ibu di Indonesia saat ini. Tidak sulit kita temukan orang yang ingin menguasai bahasa Inggris, bahkan saat ini bahasa Inggris telah menjadi mata Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 96 \| Wahana Dedikasi Copyright@Yuspar Uzer Menulis Menurut Yusuf dan Suparno (2007), menulis didefinisikan sebagai suatu kegiatan penyampaian pesan atau komunikasi dengan menggunakan bahasa tulis sebagai sebagai alat atau medianya. cara penyusunan kalimat dalam bahasa Inggris serta cara pengucapannya dalam belajar percakapan, tetapi dalam pelatihan ini kami para dosen DTY Program Studi FKIP melalui kegiatan Pengabdian Kepada Masyarakat (PKM) ini akan memberikan pembelajaran Bahasa Inggris dengan judul Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG dalam satu hari kegiatan belajar (Durasi 4 jam pembelajaran). bangsa sering digunakan dalam bentuk lisan atau percakapan (conversation). bangsa sering digunakan dalam bentuk lisan atau percakapan (conversation). bangsa sering digunakan dalam bentuk lisan atau percakapan (conversation). Bahasa Inggris sebagai mata pelajaran yang penting di tingkat sekolah menengah pertama, siswa sudah dituntut untuk menguasai empat keahlian dalam berbahasa inggris yaitu keahlian membaca, keahlian berbicara, keahlian mendengar, dan keahlian menulis. Pada berbicara diyakini sebagai keahlian utama yang harus dimiliki siswa agar dapat berkomunikasi secara benar dan lancar. Dalam hal ini berbicara melibatkan interaksi satu atau lebih lawan bicara (Harmer, 2001). Pengabdian kepada masyarakat ini dilakukan sebagai perwujudan pelaksanaan Tri Dharma Perguruan Tinggi Dosen Universitas PGRI Palembang da L2 Dikti Palembang. Sasaran yang ingin dicapai atas pelaksanaan Pengabdian Kepada Masyarakat (PKM) ini adalah para dosen dapat menerapkan dan mengembangkan kemampuan akademik yang dimilikinya, selain itu setelah mengikuti pelatihan ini para siswa diharapkan dapat menampilkan percakapan Bahasa Dengan berdasarkan hal tersebut maka kami berusaha untuk membantu siswa dalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYUAGUNG serta memperlancar bahasa Inggris melalui pelatihan percakapan bahasa Inggris komunikatif dengan sistem 1 jam. Umumnya siswa hanya mempelajari bentuk–bentuk dasar kalimat dengan 96 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Inggris secara langsung dan tulisan sehingga siswa dapat berbicara Bahasa inggris secara lancar, komunikatif dan cerdas dalam menggunakan bahasa Inggris dengan judul Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG. penyampaian pesan (informasi) secara tertulis kepada pihak lain dengan menggunakan bahasa tulis sebaga alat atau medianya. Aktifitas menulis melibatkan melibatkan bebrapa unsur, yaitu: penulis sebagai penyampaian pesan, isi tulisan, saluran, atau media, dan pembaca. Sejalan dengan itu Tarigan (1994), menjelaskan bahwa menulis merupakan suatu keterampilan berbahasa yang dipergunakan untuk berkomunikasi secara tidak langsungg, tidak secara tatap muka dengan orang lain. Dasar pelaksanaan kegiatan Pengabdian Kepada Masyarakat ini adalah: Dasar pelaksanaan kegiatan Pengabdian Kepada Masyarakat ini adalah: 1. Pembukaan dan UUD 1945 2. Tri Dharma Perguruan Tinggi 3. Pedoman akademik FKIP Universitas PGRI Palembang 4. Program kerja Program Studi DTY FKIP dan program kerja jurusan bahasa dan seni 1. Pembukaan dan UUD 1945 2. Tri Dharma Perguruan Tinggi 3. Pedoman akademik FKIP Universitas PGRI Palembang 1. Pembukaan dan UUD 1945 2. Tri Dharma Perguruan Tinggi 3. Pedoman akademik FKIP Universitas PGRI Palembang Berdasarkan pendapat- pendapat tersebut dapat disimpulkan bahwa menulis adalah suatu kegiatan berbahasa yang melahirkan pikiran yang dilakukan secara tidak langsung untuk menyampaikan suatu pesan dengan media bahasa tulis. 4. Program kerja Program Studi DTY FKIP dan program kerja jurusan bahasa dan seni Teks Anekdot Teks anekdot merupakan teks yang berbentuk cerita, di dalamnya mengandung humor sekaligus kritik. Karena berisi kritik, anekdot sering kali bersumber dari kisah-kisah faktual dengan tokoh nyata yang Menurut Dalman (2015), mengemukakan menulis ialah suatu kegiatan komunikasi berupa 97 \| Wahana Dedikasi Copyright@Yuspar Uzer 97 \| Wahana Dedikasi Copyright@Yuspar Uzer 97 \| Wahana Dedikasi Copyright@Yuspar Uzer 97 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 98 \| Wahana Dedikasi Copyright@Yuspar Uzer Menurut Muthiah (dikutip Hutahaean, 2014), teks anekdot adalah teks yang berisi pengalaman seseorang yang tidak biasa. Pengalaman yang tidak biasa tersebut disampaikan kepada orang lain dengan tujuan untuk menghibur si pembaca. terkenal.Anekdot semata-mata tidak hanya menyajikan hal-hal yang lucu- lucu, guyonan, ataupun humor. Akan tetapi, terdapat pula tujuan lain di balik cerita lucunya itu, yakni berupa pesan yang diharapkan bisa memberikan pelajaran kepeda khalayak (Kosasih, 2014). Anekdot merupakan cerita singkat yang menarik karena lucu dan mengesankan, biasanya mengenai orang penting atau terkenal dan berdasarkan kejadian yang sebenarnya (Kemendikbud, 2013). Ada pengertian lain bahwa anekdot dapat merupakan cerita rekaan yang tidak harus didasarkan pada kenyataan yang terjadi di masyarakat, yang menjadi partisipan atau pelaku di dalamnya pun tidak harus orang penting. Selain itu, teks anekdot juga dapat berisi peristiwa- peristiwa yang membuat jengkel atau konyol bagi partisipan yang mengalaminya. Perasaan jengkel dan konyol seperti itu merupakan krisis yang ditanggapi dengan reaksi dari pertentangan antara nyaman dan tidak nyaman, puas dan frustrasi, serta tercapai dan gagal. Berdasarkan pendapat- pendapat tersebut bahwa teks anekdot merupakan teks yang mengandung unsur lucu (humor) sekaligus mengandung kritik dan pesan yang akan disampaikan kepada pembaca. Teks anekdot tergolong ke dalam teks berbentuk cerita (narasi). Di dalamnya ada tokoh, alur atau rangkaian peristiwa, serta latar. Berdasarkan fungsi umumnya, anekdot sama dengan teks-teks cerita lainnya, seperti cerita pendek ataupun novel. Anekdot berfungsi untuk menyampaikan sebuah cerita baik fiksi maupun nonfiksi, sehingga pembaca seolah-olah menyaksikan peristiwa yang diceritakan itu. Adapun rangkaiannya itu dibentuk oleh bagian-bagian seperti berikut. 98 \| Wahana Dedikasi Copyright@Yuspar Uzer 98 \| Wahana Dedikasi Copyright@Yuspar Uzer Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 99 \| Wahana Dedikasi Copyright@Yuspar Uzer adanya kekonyolan yang menggeliti dan mengundang tawa. adanya kekonyolan yang menggeliti dan mengundang tawa. 1) Abstrak merupakan pendahuluan yang menyatakan latar belakang atau gambaran umum tentang isi suatu teks. 1) Abstrak merupakan pendahuluan yang menyatakan latar belakang atau gambaran umum tentang isi suatu teks. 1) Abstrak merupakan pendahuluan yang menyatakan latar belakang atau gambaran umum tentang isi suatu teks. 4) Reaksi merupakan tanggapan atau respons atas krisis yang ditanyakan sebelumnya, yang dimaksud adalah sikap mencela atau menertawakan. 2) Orientasi merupakan bagian cerita yang mengarah pada terjadinya suatu krisis, konflik, atau peristiwa utama. 5) Koda merupakan penutup atau kesimpulan sebagai pertanda berakhirnya cerita. 3) Krisis atau komplikasi merupakan bagian dari inti peristiwa suatu anekdot. Pada bagian itulah Contoh Teks Anekdot Neil Amstrong Bukan Manusia Pertama ke Bulan Neil Amstrong sering disebut-sebut sebagai manusia pertama yang menginjakkan kaki di bulan. Namun, ternyata predikat itu tidak benar. Buktinya, ketika Mas Amstrong sedang berjalan-jalan dengan bangganya di bulan, dia bertemu dengan orang dari negeri cina dan Indonesia. Keduanya sudah jauh lebih dulu berada di sana. Neil Amstrong, yang terbang ke bulan dengan Apollo 11, kaget dan bertanya kepada si orang Cina bagaimana caranya dia sampai ke bulan. “Kami bekerja sama dengan saling naik pundak seluruh penduduk Cina, akhirnya sampailah saya di sini,” jawab yang ditanya. Wah, piker Amstrong, satu miliar manusia rupanya bias ditumpuk-tumpuk, dan akhirnya bias ke bulan. “Kalau Anda, bagaimana caranya bisa sampai disini?” Tanya Amstrong kepada orang Indonesia. “Saya naik tumpukan kertas-kertas seminar.” Ha ha ha …. dan Indonesia pergi ke bulan. Meskipun demikian, cerita tersebut dimanfaatkan tokoh faktual, yakni dan Indonesia pergi ke bulan. Meskipun demikian, cerita tersebut dimanfaatkan tokoh faktual, yakni dan Indonesia pergi ke bulan. Meskipun demikian, cerita tersebut dimanfaatkan tokoh faktual, yakni Cerita tersebut tentu saja bersifat fiktif karena, tidak ada cerita yang membuktikan ada orang Cina 99 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Neil Amstrong agar ceritanya seolah- olah nyata. Percaya atau tidaknya pembaca terhadap isi cerita tersebut memang tidak penting karena tujuan menulis bukanlah hal tersebut. Maksud dari anekdot tersebut adalah menyindir perilaku masyarakat Indonesia yang lebih senang berteori di ruangan seminar sehingga kertas pun menumpuk sampai-sampai bisa dipakai naik ke bulan. Proses berpikir itu sendiri biasanya dilakukan melalui tanya jawab antara guru dan siswa. Menurut Suryosubroto (2014), model pembelajaran inkuiri adalah discovery yang merupakan bagian dari inkuiri, atau inkuiri perluasan proses discovery yang digunakan lebih dalam. Inkuiri yang dalam bahasa inggris inquiry, berarti pertanyaan, pemeriksaan, atau penyelidikan. Inkuiri sebagai suatu proses umum yang dilakukan manusia untuk mencari atau memahami informasi. 100 \| Wahana Dedikasi Copyright@Yuspar Uzer Model Pembelajaran Inkuiri Model pembelajaran inkuiri merupakan pembelajaran yang berasal dari pemikiran John Dewey.Dia pertama kali memperkenalkannya dengan istilah berfikir reflektif artinya berusaha aktif, teliti, dan melakukan pengujian yang tepat atas dukungan teori yang kuat dan kenyataan (Rosdiani, 2013). Berdasarkan pendapat- pandapat tersebut dapat disimpulkan bahwa model pembelajaran inkuiri lebih menekankan pada proses mencari atau menemukan dengan tujuan untuk memahami informasi. Pembelajaran inkuiri memiliki prinsip-prinsip berikut ini. Pembelajaran inkuiri memiliki prinsip-prinsip berikut ini. inkuiri Menurut Hamdayama (2014), model pembelajaran inkuiri merupakan rangkaian kegiatan pembelajaran yang menekankan pada proses berpikir secara kritis dan analitis untuk mencari dan menemukan sendiri jawaban dari suatu masalah yang dipertanyakan. 1. Berorientasi pada pengembangan intelektual, tujuan utama model inkuiri adalah mengembangkan kemampuan berpikir. Dengan demikian, model pembelajaran inkuiri selain berorientasi kepada 1. Berorientasi pada pengembangan intelektual, tujuan utama model inkuiri adalah mengembangkan kemampuan berpikir. Dengan demikian, model pembelajaran inkuiri selain berorientasi kepada 100 \| Wahana Dedikasi Copyright@Yuspar Uzer ( ) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 101 \| Wahana Dedikasi Copyright@Yuspar Uzer 101 \| Wahana Dedikasi Copyright@Yuspar Uzer Multimedia dan Pengajaran Writing skill dengan menggunakan Multimedia Media visual yaitu media yang hanya dapat dilihat, seperti foto, gambar dan poster. b. Media audio yaitu media yang hanya dapat didengar saja seperti kaset audio, MP3, dan radio. terjadinya proses belajar”. Dengan demikian dapat disimpulkan media adalah segala alat fisik yang dapat menyajikan pesan serta merangsang siswa untuk belajar seperti film, buku, dan kaset. Pengaturan media pembelajaran harus sedemikiaan rupa sehinggga mendukung suasana belajarmengajar. Kegiatan belajar mengajar adalah sebuah system. Aktivitas belajar mengajar memiliki komponen-komponen tertentu. Tanpa adanya salah satukomponen saja, maka kegiatan belajar mengajar tidak dapat berlangsung dengan sempurna. Ibarat sebuah mobil, mobil tidak akan berjalan dengan baik jika bannya jelek atau tidak ada. Fungsi media pembelajaran yaitu: 1. Untuk mewujudkan situasi pembelajaran yang efektif 2. Penggunaan media merupakan bagian internal dalam system pembelajaran. 4. Penggunaan media dalam pembelajaran adalah untuk mempercepat proses pembelajaran dan membantu siswa dalam upaya memahami mater yang disajikan oleh Guru dalam kelas. 5. Penggunaan media dalam pembelajaran dimaksudkan untuk mempertinggi mutu pendidikan. Pengelompokan jenis-jeni media pembelajaran banyak disampaikan oleh para ahli media pembelajaran, d antaranya Asra (2007 mengelompokkan media pembelajaran menjad beberapa jenis, yaitu: a. Media visual yaitu media yang hanya dapat dilihat, seperti foto gambar dan poster. b. Media audio yaitu media yang hanya dapat didengar saja sepert kaset audio, MP3 dan radio 102 \| Wahana Dedikasi Copyright@Yuspar Uzer Multimedia dan Pengajaran Writing skill dengan menggunakan Multimedia hasil belajar juga berorientasi pada proses belajar. 2. Prinsip interaksi, baik proses interaksi antara siswa maupun interaksi antara siswa dengan guru, bahkan interaksi siswa dengan lingkungan sekitar. Menurut Heinich, Molenida, dan Russel (1993) berpendapat bahwa“teknologi atau media pembelajaran sebagai penerapan ilmiah tentang prosesbelajar pada manusia dalam tugas praktis belajar mengajar. Menurut Ali (1992) 3. Prinsip bertanya, yang harus dilakukan guru dalam model pembelajaran inkuri adalah guru sebagai penanya, sebab kemampuan siswa untuk menjawab setia pertanyaan pada dasarnya sudah merupakan bagian dari proses berpikir. berpendapat bahwa “Media adalah berbagai jenis komponen dalam lingkungan siswa yang dapar memberikan rangsangan untuk belajar”. Menurut Gagne (1990) 4. Prinsip belajar untuk berpikir, bukan hanya mengingat sejumlah fakta, tetapi belajar adalah proses berpikir, yakni proses pengembangan seluruh otak. berpendapat bahwa “Kondisi yang berbasis media meliputi jenis penyajian yang disampaikan kepada para pembelajar dengan penjadwalan, pengurutan danpengorganisasian. Menurut Miarso (2004) berpendapat bahwa “Mediapembelajaran adalah segala sesuatu yang digunakan untuk menyalurkan pesan 5. Prinsip keterbukaan, suatu proses mencoba berbagai kemungkinan. Segala sesuatu mungkin saja terjadi. Oleh sebab itu, anak perlu diberikan kebebasan untuk mencoba sesuai dengan kemampuan perkembangan logika dan nalarnya. serta dapat merangsang pikiran, perasaan, perhatian, dan kemauan si belajarsehingga dapat mendorong 101 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) ( ) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Perangkat Multimedia terjadinya proses belajar”. Dengan demikian dapat disimpulkan media adalah segala alat fisik yang dapat menyajikan pesan serta merangsang siswa untuk belajar seperti film, buku, dan kaset. Pengaturan media pembelajaran harus sedemikiaan rupa sehinggga mendukung suasana belajarmengajar. Kegiatan belajar mengajar adalah sebuah system. Aktivitas belajar mengajar memiliki komponen-komponen tertentu. Tanpa adanya salah satukomponen saja, maka kegiatan belajar mengajar tidak dapat berlangsung dengan sempurna. Ibarat sebuah mobil, mobil tidak akan berjalan dengan baik jika bannya jelek atau tidak ada. Fungsi media pembelajaran yaitu: 1. Untuk mewujudkan situasi pembelajaran yang efektif 2. Penggunaan media merupakan bagian internal dalam system pembelajaran. 3 Media pembelajaran penting 4. Penggunaan media dalam pembelajaran adalah untuk mempercepat proses pembelajaran dan membantu siswa dalam upaya memahami materi yang disajikan oleh Guru dalam kelas. 5. Penggunaan media dalam pembelajaran dimaksudkan untuk mempertinggi mutu pendidikan. Pengelompokan jenis-jenis media pembelajaran banyak disampaikan oleh para ahli media pembelajaran, di antaranya Asra (2007) mengelompokkan media pembelajaran menjadi beberapa jenis, yaitu: a. c. Media audio visual yaitu media yang dapat dilihat sekaligus didengar seperti pendengaran saja, contohnya tape recorder, dan radio. c. Media audio visual adalah film, video, program TV, dan lain sebagainya. film suara, video, televise dan sound slide. film suara, video, televise dan sound slide. d. Multimedia adalah media yang dapat menyajikan unsur media secara lengkap 4. Penggunaan media dalam pembelajaran adalah untuk mempercepat proses 4. Penggunaan media dalam pembelajaran adalah untuk mempercepat proses terjadinya proses belajar”. Dengan demikian dapat pembelajaran dan membantu siswa dalam upaya memahami materi yang merangsang siswa untuk belajar seperti film, buku, dan kaset. Pengaturan media merangsang siswa untuk belajar seperti film, buku, dan kaset. Pengaturan media disajikan oleh Guru dalam kelas. 5. Penggunaan media dalam pembelajaran dimaksudkan untuk mempertinggi mutu pendidikan. pembelajaran harus sedemikiaan rupa sehinggga mendukung suasana belajarmengajar. Kegiatan belajar mengajar adalah sebuah system. Aktivitas belajar mengajar memiliki komponen-komponen tertentu. Tanpa adanya salah satukomponen saja, maka kegiatan belajar mengajar tidak dapat berlangsung dengan sempurna. Ibarat sebuah mobil, mobil tidak akan berjalan dengan baik jika bannya Pengelompokan jenis-jenis media pembelajaran banyak disampaikan oleh para ahli media pembelajaran, di antaranya Asra (2007) mengelompokkan media pembelajaran menjadi beberapa jenis, yaitu: a. Media visual yaitu media yang hanya dapat dilihat, seperti foto, gambar dan poster. b. Media audio yaitu media yang hanya dapat didengar saja seperti kaset audio, MP3, dan radio. jelek atau tidak ada. jelek atau tidak ada. Fungsi media pembelajaran yaitu: Fungsi media pembelajaran yaitu: 1. Untuk mewujudkan situasi pembelajaran yang efektif b. Media audio yaitu media yang hanya dapat didengar saja seperti kaset audio, 2. Penggunaan media merupakan bagian internal dalam system pembelajaran. MP3, dan radio. MP3, dan radio. 3. Media pembelajaran penting dalam rangka mencapai tujuan pembelajaran. 102 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 103 \| Wahana Dedikasi Copyright@Yuspar Uzer pendengaran saja, contohnya tape recorder, dan radio. c. Media audio visual adalah film, video, program TV, dan lain sebagainya. c. Media audio visual yaitu media yang dapat dilihat sekaligus didengar seperti Perangkat Multimedia seperti suara, animasi, video, grafis dan film. Secara umum, proses pembelajaran dengan menggunakan model pembelajaran inkuiri dapat mengikuti langkah-langkah sebagai berikut. e. Media realia yaitu semua media nyata yang ada di lingkungan alam, seperti tumbuhan, batuan, air, sawah, dan sebagainya. tumbuhan, batuan, air, sawah, dan sebagainya. 1. Orientasi adalah langkah untuk membina suasana atau iklim pembelajaran yang responsif. Pada langkah ini, guru mengkondisikan agar siswa siap melaksanakan proses pembelajaran. Langkah orientasi merupakan langkah yang sangat penting, keberhasilan model pembelajaran inkuiri sangat tergantung pada kemampuan siswa untuk beraktivitas menggunakan kemampuannya dalam memecahkan masalah. 1. Orientasi adalah langkah untuk membina suasana atau iklim pembelajaran yang responsif. Pada langkah ini, guru mengkondisikan agar siswa siap melaksanakan proses pembelajaran. Langkah orientasi merupakan langkah yang sangat penting, keberhasilan model pembelajaran inkuiri sangat tergantung pada kemampuan siswa untuk beraktivitas menggunakan kemampuannya dalam memecahkan masalah. Pengelompokan jenis-jenis media pembelajaran juga diungkapkan oleh Ashar (2011) yaitu: a. Media visual yaitu jenis media yang digunakan hanya mengandalkan indra 2. Merumuskan masalah merupakan langkah membawa siswa kepada 2. Merumuskan masalah merupakan langkah membawa siswa kepada 103 \| Wahana Dedikasi Copyright@Yuspar Uzer 103 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 104 \| Wahana Dedikasi Copyright@Yuspar Uzer model pembelajaran inkuiri, mengumpulkan data merupakan proses mental yang sangat penting dalam pengembangan intelektual. sesuatu persoalan yang mengandung teka-teki persoalan yang menantang siswa untuk memecahkan teka-teki, dan siswa didorong untuk mencari jawaban yang tepat. Proses mencari jawaban itu yang sangat penting, melalui proses tersebut siswa akan memperoleh pengalaman yang sangat berharga sebagai mengembangkan mental melalui proses berpikir. sesuatu persoalan yang mengandung teka-teki persoalan yang menantang siswa untuk memecahkan teka-teki, dan siswa didorong untuk mencari jawaban yang tepat. Proses mencari jawaban itu yang sangat penting, melalui proses tersebut siswa akan memperoleh pengalaman yang sangat berharga sebagai mengembangkan mental melalui proses berpikir. 5. Menguji hipotesis adalah proses menentukan jawaban yang dianggap diterima sesuai dengan data atau informasi yang diperoleh berdasarkan pengumpulan data. Yang terpenting dalam menguji hipotesis adalah mencari tingkat keyakinan siswa atas jawaban yang diberikannya. Selain itu, menguji hipotesis juga berarti mengembangkan kemampuan berpikir rasional. Artinya, kebenaran jawaban yang diberikan bukan hanya berdasarkan argumentasi, tetapi harus didukung oleh data yang ditemukan dan dapat dipertanggung jawabkan. 3. Mengajukan hipotesis adalah jawaban sementara dari suatu permasalahan yang sedang dikaji, hipotesis perlu diuji kebenarannya. Salah satu cara yang dapat dilakukan guru untuk mengembangkan kemampuan menebak (berhipotesis) pada setiap anak dengan mengajukan berbagai pertanyaan yang dapat mendorong siswa untuk merumuskan jawaban sementara atau dapat merumuskan berbagai perkiraan. 6. Merumuskan kesimpulan adalah proses mendeskripsikan temuan yang diperoleh berdasarkan hasil pengujian hipotesis. 6. Merumuskan kesimpulan adalah proses mendeskripsikan temuan yang diperoleh berdasarkan hasil pengujian hipotesis. 4. Mengumpulkan data adalah aktivitas menjaring informasi yang dibutuhkan untuk mengkaji hipotesis yang diajukan. Dalam BAHAN DAN METODE Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Adapun masalah yang akan diteliti yaitu mengenai Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG. Metode pengumpulan data pada penelitian ini dilakukan dengan observasi proses belajar mengajar dan tes. Hasil penelitian dari setiap siklus dan tindakan dideskripsikan, dianalisis, dan direfleksikan untuk mengetahui kelebihan dan kekurangan dalam kegiatan pembelajaran yang ditujukan untuk perbaikan pelaksanaan tindakan dan pembelajaran selanjutnya. Penelitian dilaksanakan dalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG yaitu metode penelitian tindakan kelas. Model penelitian tindakan kelas (PTK) yang digunakan dalam penelitian ini adalah model penelitian tindakan kelas berdasarkan teori John Elliot. Model ini pada setiap siklusnya terdiri terdiri dari beberapa aksi, yaitu antara tiga sampai lima aksi (tindakan). Tindakan ini diambil berdasarkan pemikiran bahwa dalam suatu mata pelajaran terdapat beberapa pokok bahasan dan setiap pokok bahasan terdiri atas beberapa materi yang tidak dapat diselesaikan dalam satu kali tindakan.”Sementara itu, setiap tindakan terdiri dari beberapa langkah yang terealisasi dalam bentuk kegiatan belajar-mengajar. 105 \| Wahana Dedikasi Copyright@Yuspar Uzer 106 \| Wahana Dedikasi Copyright@Yuspar Uzer Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG dapat menghubungkan pengetahuan yang telah dimiliki sebelumnya dan memiliki tujuan menulis yang jelas. Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG merupakan strategi yang baru. Pembelajaran menulis yang selama ini dilakukan hanya dengan strategi yang konvensional yaitu dengan ceramah dan pemberian tugas pada siswa untuk menulis wacana. Pembelajaran seperti itu ternyata kurang mendapat respon positif dari siswa. Masih banyak siswa yang kurang antusias dengan pembelajaran. Berdasarkan data hasil dari penelitian ini, ternyata Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNGmampu meningkatkan kemampuan membaca pemahaman. Peningkatan tersebut cukup berarti, baik dari segi proses maupun hasil. Dari segi proses, Metode Speed Reading ternyata mampu menarik perhatian siswa. Siswa terlihat lebih antusias dalam pembelajaran. Faktor- Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG dapat menghubungkan pengetahuan yang telah dimiliki sebelumnya dan memiliki tujuan menulis yang jelas. Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG merupakan strategi yang baru. Pembelajaran menulis yang selama ini dilakukan hanya dengan strategi yang konvensional yaitu dengan ceramah dan pemberian tugas pada siswa untuk menulis wacana. Pembelajaran seperti itu ternyata kurang mendapat respon positif dari siswa. Masih banyak siswa yang kurang antusias dengan pembelajaran. Berdasarkan data hasil dari penelitian ini, ternyata Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNGmampu meningkatkan kemampuan membaca pemahaman. Peningkatan tersebut cukup berarti, baik dari segi proses maupun hasil. Dari segi proses, Metode Speed Reading ternyata mampu menarik perhatian siswa. Siswa terlihat lebih antusias dalam pembelajaran. Faktor- faktor yang mempengaruhi kegiatan membaca pemahaman seperti, sulit memahami wacana karena banyak kosakata yang tidak tahu maknanya dan menganggap sulit terlebih dahulu terhadap pembelajaran membaca pemahaman sudah bisa di atasi. Selain itu, secara keseluruhan peran aktif siswa juga lebih meningkat meski terdapat satu atau dua siswa yang kurang aktif. Dilihat dari segi hasil (produk), jelas terlihat bahwa jumlah siswa yang hasil belajarnya telah memenuhi standar KKM meningkat dari pratindakan hingga siklus II. Pada saat pratindakan terdapat 5 (23,81%) siswa yang memenuhi standar KKM, pada akhir siklus I meningkat menjadi 16 (72,73%) siswa yang memenuhi KKM. Pada akhir siklus II terjadi peningkatan lagi yaitu 22 (95,65%) siswa telah memenuhi KKM. PEMBAHASAN Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG. Latar belakang pengetahuan yang dimiliki seseorang sangat memengaruhi proses dan hasil membaca. Hal ini memperkuat pernyataan Rahim (2011: 44) bahwa dengan Penerapan Model Pembelajaran Inkuiri Dalam Alasan penulis menggunakan desain Penelitian Tindakan Kelas yaitu karena adanya kesamaan karakteristik yang terdapat dalam PTK dengan masalah yang ada. 105 \| Wahana Dedikasi Copyright@Yuspar Uzer 105 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 106 \| Wahana Dedikasi Copyright@Yuspar Uzer harus mengalami kesulitan. Selian itu, kemampuan menyimakpun terdapat peningkatan, karena dengan adanya peningkatan dalam menyimak, siswa akan konsentrasi dalam pembelajaran pada saat materi bahasa Indonesia bagian membaca. Keterampilan membaca, menyimak serta membaca cepat merupakan satu „kesatuan yang sulit dipisahkan‟ karena saling „terkait satu dengan yang lain. Didalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG siswa juga harus dapat menyimak dengan baik terkait isi yang ada didalam suatu bacaan dalam menulis cerita pendek. antara lain: untuk mengerti ide pokok, mampu memahami fakta dan detail khusus, meningkatkan kekayaan pengetahuan umum, dan mendapatkan informasi tentang apa yang terjadi di dunia dalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG. Hasil penelitian ini memperkuat pernyataan Johnson dan Pearson (dalam Zuchdi, 2008: 23-24) bahwa faktor yang berasal dari dalam diri dan faktor yang berasal dari luar pembaca dapat mempengaruhi terhadap kemampuan membaca pemahaman. Lebih lanjut, hasil penelitian ini juga selaras dengan pernyataan Paul D. Leedy (dalam Soedarso, 2005: 120-121) yang mengemukakan beberapa tujuan membaca pemahaman. Tujuan tersebut faktor yang mempengaruhi kegiatan membaca pemahaman seperti, sulit memahami wacana karena banyak kosakata yang tidak tahu maknanya dan menganggap sulit terlebih dahulu terhadap pembelajaran membaca pemahaman sudah bisa di atasi. Selain itu, secara keseluruhan peran aktif siswa juga lebih meningkat meski terdapat satu atau dua siswa yang kurang aktif. Dilihat dari segi hasil (produk), jelas terlihat bahwa jumlah siswa yang hasil belajarnya telah memenuhi standar KKM meningkat dari pratindakan hingga siklus II. Pada saat pratindakan terdapat 5 (23,81%) siswa yang memenuhi standar KKM, pada akhir siklus I meningkat menjadi 16 (72,73%) siswa yang memenuhi KKM. Pada akhir siklus II terjadi peningkatan lagi yaitu 22 (95,65%) siswa telah memenuhi KKM. Hasil penelitian ini memperkuat pernyataan Johnson dan Pearson (dalam Zuchdi, 2008: 23-24) bahwa faktor yang berasal dari dalam diri dan faktor yang berasal dari luar pembaca dapat mempengaruhi terhadap kemampuan membaca pemahaman. Lebih lanjut, hasil penelitian ini juga selaras dengan pernyataan Paul D. Leedy (dalam Soedarso, 2005: 120-121) yang mengemukakan beberapa tujuan membaca pemahaman. Tujuan tersebut 106 \| Wahana Dedikasi Copyright@Yuspar Uzer 106 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan k l i di (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 107 \| Wahana Dedikasi Copyright@Yuspar Uzer KESIMPULAN Dapat disimpulkan yakni dalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG siswa mengalami peningkatan dalam membaca tanpa Dapat disimpulkan yakni dalam Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill dengan Menggunakan Perangkat Multimedia di SMAN 2 KAYU AGUNG siswa mengalami peningkatan dalam membaca tanpa DAFTAR PUSTAKA Jeremy Harmer, Cambrige, UK. 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Sussel, I.D. & Smaldiro, S.E.(1996) Instructional Media and Technologies forLearning 107 \| Wahana Dedikasi Copyright@Yuspar Uzer 107 \| Wahana Dedikasi Copyright@Yuspar Uzer 108 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia BudayaStanley J. Baran, Penerbit Erlangga Jakarta. Sadtono, Vugene, Antologi Pengajaran Bahasa Asing khususnya Bahasa Inggris. Jakarta :Deparartemen Pendidikan dan Kebudayaan. khususnya Bahasa Inggris. Jakarta :Deparartemen Pendidikan dan Kebudayaan. 108 \| Wahana Dedikasi Copyright@Yuspar Uzer 109 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 109 \| Wahana Dedikasi Copyright@Yuspar Uzer 109 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 110 \| Wahana Dedikasi Copyright@Yuspar Uzer 110 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 111 \| Wahana Dedikasi Copyright@Yuspar Uzer 112 \| Wahana Dedikasi Copyright@Yuspar Uzer Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia Yuspar Uzer, Muhammad Ali, Sri Wahyu Indrawati, Yus Ver Nandes Uzer, Herlina, Noviati (2021) Penerapan Model Pembelajaran Inkuiri Dalam Peningkatan Writing Skill Dengan Menggunakan Perangkat Multimedia 112 \| Wahana Dedikasi Copyright@Yuspar Uzer
https://openalex.org/W4383532655	https://www.thetherapist.com.pk/index.php/tt/article/download/119/82	English	null	Trend of Snakebite Cases and their Management at Holy Family Hospital Rawalpindi During 2022	The therapist	2,023	cc-by	3,524	Corresponding Author: Rizwana Shahid Rizwana Shahid Department of Community Medicine, Rawalpindi Medical University, Rawalpindi, Pakistan th Received Date: 14 April, 2023 th Received Date: 14 April, 2023 th Acceptance Date: 27 May, 2023 th Acceptance Date: 27 May, 2023 th Published Date: 30 June, 2023 th Published Date: 30 June, 2023 1 2* 1 Shazia Zeb , Rizwana Shahid and Farzana Fatima ¹Holy Family Hospital, Rawalpindi, Pakistan ¹Holy Family Hospital, Rawalpindi, Pakistan ²Department of Community Medicine, Rawalpindi Medical University, Rawalpindi, Pakistan THE THERAPIST JOURNAL OF THERAPIES & REHABILITATION SCIENCES https://thetherapist.com.pk/index.php/tt Volume 4, Issue 2 (April-June 2023) How to Cite: Zeb, S. ., Shahid, R. ., & Fatima, F. . (2023). Trend of Snakebite Cases and their Management at Holy Family Hospital Rawalpindi During 2022: Trend of Snakebite Cases. THE THERAPIST (Journal of Therapies &Amp; Rehabilitation Sciences), 4(02). https://doi.org/10.54393/tt.v4i02.119 Key Words: Snakebite is a neglected public health problem of tropical and subtropical regions globally. Millions of cases are reported annually worldwide and about half of them are bitten by poisonous snakes. Objectives: To determine trend of snakebite cases and their management at Holy Family Hospital during 2022. Methods: A retrospective hospital-record based study was done to identify the trend of snakebite cases reported at Holy Family Hospital Rawalpindi during 2022. The data was gathered from hospital administrators pertaining to age, gender, residential address, types of snakebite and treatment given. Data were analysed by SPSS software version 25.0 and MS Excel 2016. Descriptive statistics were computed. Independent sample t-test was applied to measure statistically signiïcant gender-based difference in mean age of the snake bite victims. P < 0.05 was considered signiïcant. Results: Of the 90 snakebite cases, 64.1% were males. Mean age of the victims was 34.7 ±14.8 years. Difference in mean age of male and female victims was statistically insigniïcant (P > 0.67). Majority (33%) was resident of Rawalpindi, followed by 22% and 12.3% from Attock and Azad Jammu & Kashmir respectively. Peak of the cases was during July and August. As most (91.1%) of them were bitten by vasculotoxic snakes, so out of 1,117 anti-snake venom ampules about 93.1% were administered to those cases. None of the cases succumbed to snakebite. Conclusions: Snakebite has frequently been reported among residents of Rawalpindi and its neighbouring areas during summer season. The victims were promptly treated for their survival. Snakebite, Vasculotoxic, Anti-snake venom ampules, Survived Original Article Trend of Snakebite Cases and their Management at Holy Family Hospital Rawalpindi During 2022 Trend of Snakebite Cases Trend of Snakebite Cases Trend of Snakebite Cases Trend of Snakebite Cases DOI: https://doi.org/10.54393/tt.v4i02.119 THE THERAPIST JOURNAL OF THERAPIES & REHABILITATION SCIENCES https://thetherapist.com.pk/index.php/tt Volume 4, Issue 2 (April-June 2023) JAN - JUN 2020 3 23 Copyright © 2023. THE THERAPIST, Published by Crosslinks International Publishers M E T H O D S A retrospective hospital-record based study was carried out among snakebite cases reported to Holy Family Hospital Rawalpindi during 2022. The month-wise data of snakebite cases was gathered with informed consent of hospital administrators. Data were collected pertaining to age, gender, residential address, type of snakebite and treatment received by the cases. Data were analysed by SPSS software version 25.0 and MS Excel 2016. Descriptive statistics were applied. Gender based difference in mean age of the snake bite victims was statistically determined by independent sample t-test. P < 0.05 was taken as signiïcant. 61-70 years 51-60 years 41-50 years 31-40 years 21-30 years 11-20years 0 50 100 150 200 250 300 350 400 Anti-snake venom ampules adminstered Patients subjected to snake bite I N T R O D U C T I O N The current study was hence planned to envisage the snakebite cases reported at a public sector tertiary care hospital of Rawalpindi during 2022 and their management accomplished by provision of antivenoms with an intention to measure the frequency of this problem in our zone. This will not only aid to perceive the frequency of snakebites cases registering in a tertiary healthcare facility of Rawalpindi from diverse territories but will also highlight the management of envenomation executed for the survival of victims. In addition, this study will also enable our healthcare administrators as well as strategic planners to take necessary initiatives for coping with this problem in future. Figure 1: Residential area of snakebite cases Frequent cases were reported during July and August 2022 as shown below in Figure 2. g 25 20 15 10 5 0 March April May June July August September October 2 8 5 9 22 20 17 7 Figure 2: Trend of snakebite cases during 2022 March April May June July August Figure 2: Trend of snakebite cases during 2022 Total 927 and 190 anti-snake venom ampules were administered to male and female patients respectively. Out of 1,117 ampules, most (93.1%) were administered to those who were subjected to vasculotoxic snake bite. Majority of the cases receiving anti-snake venom facility were 21-40 years of age as illustrated below in Figure 3. I N T R O D U C T I O N Majority of the cases receiving anti-snake venom facility were 21-40 years of age as illustrated below in Figure 3. Figure 2: Trend of snakebite cases during 2022 61-70 years 51-60 years 41-50 years 31-40 years 21-30 years 11-20years 0 50 100 150 200 250 300 350 400 Anti-snake venom ampules adminstered Patients subjected to snake bite Figure 3: Age groups of the snake bitten cases & number of anti- snake venom ampules used Majority (91.1%) of our patients were subjected to vasculotoxic snake bite as revealed below in Figure 4. All cases were swifty treated and recovered. 8.90% vasculotoxic neurotixic Frequent cases were reported during July and August 2022 as shown below in Figure 2. 35% 30% 25% 20% 15% 10% 5% 0% 33% 22% 12.30% 8.20% 6.70% 2.70% 2.70% 2.70% 2.70% 1.40% 1.40% 1.40% 1.40% 1.40% Rawalpindi Attock AJK Jhelum Chakwal Islamabad Murree Huripur Mianwali Khanpur Gujrat Gojar Khan pindigheb Abbotabad Figure 1: Residential area of snakebite cases 25 20 15 10 5 0 March April May June July August September October 2 8 5 9 22 20 17 7 Figure 2: Trend of snakebite cases during 2022 Frequent cases were reported during July and August 2022 as shown below in Figure 2. 35% 30% 25% 20% 15% 10% 5% 0% 33% 22% 12.30% 8.20% 6.70% 2.70% 2.70% 2.70% 2.70% 1.40% 1.40% 1.40% 1.40% 1.40% Rawalpindi Attock AJK Jhelum Chakwal Islamabad Murree Huripur Mianwali Khanpur Gujrat Gojar Khan pindigheb Abbotabad Figure 1: Residential area of snakebite cases 25 20 15 10 5 0 March April May June July August September October 2 8 5 9 22 20 17 7 Figure 2: Trend of snakebite cases during 2022 Frequent cases were reported during July and August 2022 35% 30% 25% 20% 15% 10% 5% 0% 33% 22% 12.30% 8.20% 6.70% 2.70% 2.70% 2.70% 2.70% 1.40% 1.40% 1.40% 1.40% 1.40% Rawalpindi Attock AJK Jhelum Chakwal Islamabad Murree Huripur Mianwali Khanpur Gujrat Gojar Khan pindigheb Abbotabad Figure 1: Residential area of snakebite cases venom and subsequent healthcare outcomes of envenomation, snakebite cases have suíciently been reported for mismanagement globally due to poor knowledge about the composition of venoms and devenomizing approach[10]. Moreover, due to insuícient epidemiology known to us about snakebite and its greatest occurrence in rural areas and hence non-reporting to our healthcare centres [11], there is very meagre information about its propensity in our country. I N T R O D U C T I O N 20%[5]. Although most of the snakes found in Pakistan are non-venomous [6]; however, detection of four venomous snake types predominantly in Sindh and Punjab provinces has imposed World Health Organization (WHO) to categorize these regions of Pakistan as the highest risk due to increased vulnerability of the respective population to snakebite [7]. A study by Shah et al., among Southern Punjab inhabitants revealed a misconception of about 80% of the snakes being poisonous and around 50% acknowledged the recovery of victims on apt management [8]. Venomous snakebite in addition to certain communicable diseases has been recognized as the prime contributor to mortality in third world countries[9]. Despite having adequate information pertaining to types of snake Snakebite is a renowned occupational hazard that is now being perceived as a public health issue across the globe [1]. Approximately 1.8 to 2.7 million people worldwide are subjected to snake bite annually with resultant 80,000- 138,000 deaths [2]. Human beings when bitten by a venomous snakes are injected with mixture of toxins [3]; henceforth, critical emergencies might be attributed to this mishap [4]. The greatest burden of snake bite associated mortality and morbidity has been acknowledged in African and Asian regions of the world[2]. Mortality among Russell's viper bite cases of Myanmar has been documented as high as 10% due to subjection of the cases to severe neurotoxicity. Victims below 12 years of age were notiïed with the highest Case Fatality Ratio (CFR) of THE THERAPIST VOL. 4 Issue 2 April-June 2023 THE THERAPIST VOL. 4 Issue 2 April-June 2023 Copyright © 2023. THE THERAPIST, Published by Crosslinks International Publishers 05 Trend of Snakebite Cases Zeb S et al., DOI: https://doi.org/10.54393/tt.v4i02.119 DOI: https://doi.org/10.54393/tt.v4i02.119 Frequent cases were reported during July and August 2022 as shown below in Figure 2. 35% 30% 25% 20% 15% 10% 5% 0% 33% 22% 12.30% 8.20% 6.70% 2.70% 2.70% 2.70% 2.70% 1.40% 1.40% 1.40% 1.40% 1.40% Rawalpindi Attock AJK Jhelum Chakwal Islamabad Murree Huripur Mianwali Khanpur Gujrat Gojar Khan pindigheb Abbotabad Figure 1: Residential area of snakebite cases 25 20 15 10 5 0 March April May June July August September October 2 8 5 9 22 20 17 7 Total 927 and 190 anti-snake venom ampules were administered to male and female patients respectively. Out of 1,117 ampules, most (93.1%) were administered to those who were subjected to vasculotoxic snake bite. D I S C U S S I O N [21]. Sindh province across pakistan particularly its Rajanpur district is of paramount signiïcane with respect to snakebite cases. A descriptive study analyzing 2000- 2007 hospital record of cases highlighted maximum propensity (57%) of cobra bitten cases that indicated the envenomization of majority by neurotoxi snakes. However, likewise our study, antisnake venom services were also available there [22]. World Health Organization has also released the guidelines for managing snakebite cases particularly in South East Asian Region alongwith treatment of complications and measures to prevent snakebite [23]. Likwise Sustainable Development Goals (SDGs) to be attained by 2030, WHO Regional Director for SEARO (South East Asian Region Organization) has also speciïed a goal to reduce snakebite assocaited deaths and diasbilities by 50% by the end of 2030. Global strategy for prevention and control of snakebite envenoming has also been launched for this purpose [24]. These aspects not only highlight the signiïcance of reporting the snakebite casses to healthcare centres immediately after onset but also emphasize training of the staff for prompt provision of managerial facilities. Snakebite cases are maximally reported from highly populated provinces of pakistan like those of Punjab and Sindh that is chieðy attributed to preponderance of agricultural activities [12]. The greatest number of snakebite cases in current study have been reported during July and August when humidity in the climate prevails due to monsoon. According to a study carried out in Brazil, ecology of venomous snakes is remarkably associated with the climate of any region [13]. On the other hand, a colombian study concluded that snakebite envoming is attributed to rainfall only in extremely dried regions; however, temperature was not proven to moderate the occurrence of snakebite cases [14]. A study done among snakebite cases reporting to a tertiary healthcare centre of South Indian region elucidated the crowning of snakebite cases from September to December [15]. World Health Organization (WHO) has included snakebite envenoming in the list of category A Neglected Tropical Diseases (NTDs) during 2009 due to devastating healthcare consequences [16]. Ecological characteristics of any region that increase the likelihood of snakebite should thoroughly be investigated so that predictive modelling could facilitate the concerned strategic planners substantially in mitigating the cases. The male to female ratio of snakebite cases in our study was 4:1 with mean age of 34.7 ± 14.8 years. D I S C U S S I O N likewise, a cross-sectional study done in Emergency Medicine department of a tertiary care faciliity elucidated the age of cases from 15-45 years with male to female ration of 3:1 [17]. Similarly another retrospective study carried out on 2014-2021 data of asian snakebite cases explored that 70% of the victims were males with majority (45%) of them being 18-30 years old[18]. On the other hand, an observational study by Tchoffo et al., among inhabitants of Cameroon revealed about 62% snake envenomization among females which was predominantl attributed to non- reporting of substantial cases to healthcare centres for treatment [19]. According to a study done among Sri Lankan populates, about 51.3% of snake-bitten cases were those of males [20]. In additon to gender and age based diversities in snakebite incidnece, occupation of the cases should also be scrutinized with an intention to provide all necessary protective measures as data pertainng to this variable was not avaiable from hospital record. Of the total 90 snakebite victims, about 33% were residents of rawalpindi city while 22% and 12.3% belonged to Attock and Azad Jammu & Kashmir (AJK) respectively. Most (91.1%) were bitten by vasculotoxic snakes. Being a public sector tertiary healthcare facilitiy, Holy Family Hospital of rawalpindi located in midcity makes it an ideal healthcare centre for the dwellers of nearby towns like Attock, Murree, Jhelum Chakwal Haripur etc for accessing healthcare R E S U L T S Of the total 90 snakebite cases reporting to Holy Family Hospital Rawalpindi during 2022, 72 (64.1%) and 18 (33.7%) were males and females respectively. Mean age of the cases was 34.7 ± 14.8 years. Gender-wise disparity in mean age of the snakebite cases was statistically insigniïcant on applying independent sample t-test as depicted below in Table 1. Anti-snake venom ampules adminstered Figure 3: Age groups of the snake bitten cases & number of anti- snake venom ampules used Majority (91.1%) of our patients were subjected to vasculotoxic snake bite as revealed below in Figure 4. All cases were swifty treated and recovered. Majority (91.1%) of our patients were subjected to vasculotoxic snake bite as revealed below in Figure 4. All cases were swifty treated and recovered. y Figure 4: Category of snakebite cases 8.90% 91.10% vasculotoxic neurotixic Table 1: Gender-based difference in mean age of snakebite victims Mean age of snakebite cases (mean ± SD) P-value Males (n = 72) Females (n = 18) 34.11± 15.9 years 35.9 ±14.7 years 0.67 Most (33%) of our cases belonged to Rawalpindi city as displayed below in Figure 1. Figure 4: Category of snakebite cases THE THERAPIST VOL. 4 Issue 2 April-June 2023 06 Trend of Snakebite Cases DOI: https://doi.org/10.54393/tt.v4i02.119 Trend of Snakebite Cases Zeb S et al., DOI: https://doi.org/10.54393/tt.v4i02.119 C O N C L U S I O N S Snakebite is very common in rawalpindi district and its nearby regions mainly during july and August. Reporting of signiïcant number of snakebite cases depicts non- availability of anti-snake venom in other regional hospitals. 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https://openalex.org/W2946611572	https://aspects.org.ua/index.php/journal/article/download/446/439	Ukrainian	null	Efficiency improvement of the authorities personnel potential concerning measures implementing in the sphere of the national and patriotic education of youth	Aspekti publìčnogo upravlìnnâ	2,018	cc-by	7,502	Корнієвський С. В., Управління молоді і спорту Дніпропетровської облдержадміністрації У статті визначено, що в сучасних умовах стає актуальною необхідність вироблення методо логічного забезпечення практичної діяльності органів влади в сфері національно-патріотичного виховання молоді. Проводиться аналіз проблемного поля формування та реалізації політики націо нально-патріотичного виховання та надається методика підвищення кваліфікації працівників ор ганів влади з метою ефективної організації та здійснення заходів політики. Зазначається що, норма тивно-правова база, яка забезпечує впровадження політики національно-патріотичного виховання, потребує певного удосконалення на рівні цільового, суб’єктно-об’єктного, змістовного компонентів виховного процесу. Надається перелік конкретизованих вимог організації національно-патріотично го виховання: проведення виховання на системній основі; забезпечення його планового характеру; координація діяльності органів влади та громадських організацій; постійний пошук нових напрям ків, форм і методів; підвищення кваліфікації посадових осіб. Автор вважає, що в сучасних умовах готовність і здатність держави проводити політику націо нально-патріотичного виховання молоді залежать, в першу чергу, від її позиції і відповідей щодо низки актуальних питань. По-перше, це визначення смислових концептів національно-патріотич ного виховання як першочергового завдання органів державної влади, втілюючи прагнення мільй онів українців щодо збереження незалежності та територіальної цілісності держави. По-друге, це усвідомлення того, що процеси національно-патріотичного виховання сьогодні потребують більш універсальних підходів. По-третє, це визначення змістовного поля і характеру взаємодії державних органів та інститутів громадянського суспільства в постановці і вирішенні життєво важливих для країни проблем. емо розглядаються науково-методичні проблеми національно-патріотичного виховання. Ключові слова: національно-патріотичне виховання; цілі національно-патріотичного виховання; суб’єк ти національно-патріотичного виховання; об’єкти національно-патріотичного виховання; проблематика національно-патріотичного виховання; методика національно-патріотичного виховання Підвищення ефективності кадрового потенціалу органів влади щодо реалізації заходів у сфері національно-патріотичного виховання молоді УДК: 331.108:325.452 DOI: 10.15421/151854 УДК: 331.108:325.452 УДК: 331.108:325.452 Аспекти публічного правління Том 6 № 9 2018 Аспекти публічного правління Том 6 № 9 2018 www.aspects.org.ua DOI: 10.15421/151854 Підвищення ефективності кадрового потенціалу органів влади щодо реалізації заходів у сфері національно-патріотичного виховання молоді Підвищення ефективності кадрового потенціалу органів влади щодо реалізації заходів у сфері національно-патріотичного виховання молоді Корнієвський С. В., Управління молоді і спорту Дніпропетровської облдержадміністрації Efficiency improvement of the authorities personnel potential concerning measures implementing in the sphere of the national and patriotic education of youth Efficiency improvement of the authorities personnel potential concerning measures implementing in the sphere of the national and patriotic education of youth It is determined in the article that under the modern conditions the necessity of developing methodological practice support of state authorities in the sphere of national and patriotic education of young people becomes urgent. An analysis of the problem field of the formation and implementation of the policy of national and patriotic education is conducted and a methodology for raising the skill level of the employees of state authorities, local governments for the purpose of effective organization and implementation of measures for national and patriotic education at the local level is provided. The basic specified requirements of the organization of the national and patriotic education should be attributed: conducting of the national and patriotic education of young people on a systematic basis; ensuring the planned nature of the national and patriotic education; coordinating of the state authorities activity, local government bodies and public organizations in the field of national and patriotic education; permanent searching for new directions, forms and methods of the national and patriotic education of youth; skill developing of persons involved in the processes of the national and patriotic education. The author believes that in present-day conditions, the readiness and ability of the state to pursue the policy of the national and patriotic education of young people depend primarily on its position and responses to a number of topical issues. Firstly, it is the definition of semantic concepts of national and patriotic education as a priority task of the state authorities with embodying the aspirations of millions of Ukrainians to preserve the independence and territorial integrity of the state. Secondly, it is awareness that 52 Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua today the processes of the national and patriotic education require more universal approaches. Thirdly, it is a definition of the semantic space and the interaction nature of the state bodies and civil society institutions in the statement and solution of vital problems for the country. Another current issue is the problem differentiation of the national and patriotic education into its constituent elements on different grounds and a more in-depth study of them. Efficiency improvement of the authorities personnel potential concerning measures implementing in the sphere of the national and patriotic education of youth Keywords: national and patriotic education, goals of national and patriotic education; subjects of national and patriotic education; objects of national and patriotic education; problems of national and patriotic education; methods of national and patriotic education Повышение эффективности кадрового потенциала органов власти по реализации мероприятий в сфере национально-патриотического воспитания молодежи Корниевский С. В., Управление молодежи и спорта Днепропетровской облгосадминистрации Корниевский С. В., Управление молодежи и спорта Днепропетровской облгосадминистрации В статье определено, что в современных условиях становится актуальной необходимость вы работки методологического обеспечения практической деятельности органов власти в сфере на ционально-патриотического воспитания молодежи. Проводится анализ проблемного поля фор мирования и реализации политики национально-патриотического воспитания и предоставляется методика повышения квалификации работников органов власти с целью эффективной организации и осуществления мероприятий политики. Отмечается что, нормативно-правовая база, которая обе спечивает внедрение политики национально-патриотического воспитания, требует определенного совершенствования на уровне целевого, субъектно-объектного, содержательного компонентов вос питательного процесса. Предоставляется перечень конкретизированных требований организации национально-патриотического воспитания: проведение воспитания на системной основе; обеспече ния его планового характера; координация деятельности органов власти и общественных организа ций; постоянный поиск новых направлений, форм и методов; повышение квалификации должност ных лиц. Автор считает, что в современных условиях готовность и способность государства проводить политику национально-патриотического воспитания молодежи зависит, в первую очередь, от ее по зиции и ответов по ряду актуальных вопросов. Во-первых, это определение смысловых концептов национально-патриотического воспитания как первоочередной задачи органов государственной власти, воплощая стремление миллионов украинцев по сохранению независимости и территориаль ной целостности государства. Во-вторых, это осознание того, что процессы национально-патриоти ческого воспитания сегодня требуют более универсальных подходов. В-третьих, это определение содержательного поля и характера взаимодействия государственных органов и институтов граждан ского общества в постановке и решении жизненно важных для страны проблем. б Отдельно рассматриваются научно-методические проблемы национально-патриотического вос питания. Ключевые слова: национально-патриотическое воспитание; цели национально-патриотического вос питания; субъекты национально-патриотического воспитания; объекты национально-патриотического воспитания; проблематика национально-патриотического воспитания; методика национально-патриоти ческого воспитания Аналіз досліджень і публікацій. Аналіз досліджень і публікацій. суспільства в постановці і вирішенні життєво важливих для країни проблем. Значний внесок у закладення теоретич них основ формування політики національ но-патріотичного виховання молоді внесли такі науковці як І. Бех, Є. Бородін, О. Боя ринцев, М. Вавринчук, О. Діденко, А. Заго родня, В. Кротюк, Н. Майборода, С. Ориш ко, О. Рак, К. Чорна, М. Яруш. Однією з ключових проблем національ но-патріотичного виховання молоді є про блема його вдосконалення як виховного про цесу на рівні всіх компонентів – цільового, суб’єктно-об’єктного, змістовного, опера ційно-діяльнісного, аналітико-результатив ного [3, с. 26]. Проблеми національно-патріотичного виховання підростаючого покоління в освіт ній сфері досліджували В. Івашковський, О. Боярчук, О. Глазова, О. Докукіна, К. Жур ба, О. Каплуновська, В. Ложкіна, О. Матвій чук, В. Назаров, А. Нетудихатка, С. Пасацька, В Сорока, Т. Черкащина, В. Яхонтова та ін. Зупинимося на цільовій спрямованості національно-патріотичного виховання. В Концепції національно-патріотичного вихо вання дітей і молоді його метою визначено формування у молодого покоління високої патріотичної свідомості, почуття вірності, любові до Батьківщини, турботи про благо свого народу, готовності до виконання гро мадянського і конституційного обов’язку із захисту національних інтересів, цілісності, незалежності України, сприяння становлен ню її як правової, демократичної, соціальної держави [7]. Зважаючи на таку увагу науковців, можна обґрунтовано зазначити, що національно-па тріотичне виховання сьогодні є найбільш до сліджуваним з усіх інших видів виховання, а одержувані в ході досліджень результати мають значний вплив на якісний розвиток знань в теорії виховання в цілому, але на рівні методологічного забезпечення діяль ності органів державної влади ці питання потребують більш глибокого вивчення. З наведеного переліку цілей видно, що інтегровані завдання громадянського, духов но-морального та соціального виховання в цілому віднесені до цільової області націо нально-патріотичного виховання, тобто па тріотичне виховання вживається в значенні громадського виховання в цілому, включає складовими елементами всі інші види (на прямки) виховання. З наведеного переліку цілей видно, що інтегровані завдання громадянського, духов но-морального та соціального виховання в цілому віднесені до цільової області націо нально-патріотичного виховання, тобто па тріотичне виховання вживається в значенні громадського виховання в цілому, включає складовими елементами всі інші види (на прямки) виховання. Метою статті є аналіз проблемного поля формування та реалізації політики націо нально-патріотичного виховання та надання методики підвищення кваліфікації праців ників органів державної влади, органів міс цевого самоврядування з метою ефективної організації та здійснення заходів із націо нально-патріотичного виховання на місце вому рівні. Постановка проблеми. го наукового обґрунтування. Прийняті Стра тегія національно-патріотичного виховання дітей та молоді на 2016 – 2020 роки та Кон цепція національно-патріотичного виховання дітей і молоді містять в собі теоретичні по зиції за основними аспектами патріотичного виховання. Ефективне виконання зазначених актів актуалізує необхідність вироблення методологічного забезпечення практичної діяльності органів влади у сфері національ но-патріотичного виховання молоді. Е ф п в і Е фективна реалізація основних на прямів національно-патріотичного виховання молоді неможлива без консолідації діяльності органів державної влади всіх рівнів, органів місцевого самовря дування, інститутів громадянського суспіль ства, а також наукових і освітніх установ. Практика реалізації політики національ но-патріотичного виховання не може бути цілеспрямованою і системною без серйозно 53 Аспекти публічного правління Том 6 № 9 2018 www.aspects.org.ua Аналіз досліджень і публікацій. З комплексних позицій гуманітарних і со ціально-економічних наук, формування осо бистості людини відбувається в трьох його найбільш важливих сферах життя – свідо мості, відносинах і діяльності. Якщо резуль тати національно-патріотичного виховання в сфері свідомості і відносин відображені чітко (патріотична свідомість та почуття любові і вірності Батьківщині), то про необхідність патріотичної поведінки і діяльності громадя нина в Концепції йдеться невиразно, не про водиться найважливіша думка – патріотизм, в першу чергу, повинен проявлятися в патріо тичних вчинках, соціальній активності. З комплексних позицій гуманітарних і со ціально-економічних наук, формування осо бистості людини відбувається в трьох його найбільш важливих сферах життя – свідо мості, відносинах і діяльності. Якщо резуль тати національно-патріотичного виховання в сфері свідомості і відносин відображені чітко (патріотична свідомість та почуття любові і вірності Батьківщині), то про необхідність патріотичної поведінки і діяльності громадя нина в Концепції йдеться невиразно, не про водиться найважливіша думка – патріотизм, в першу чергу, повинен проявлятися в патріо тичних вчинках, соціальній активності. у р Виклад основного матеріалу. р у В сучасних умовах готовність і здатність держави проводити політику національно-па тріотичного виховання молоді залежать, в першу чергу, від її позиції і відповідей щодо низки актуальних питань. По-перше, це визна чення смислових концептів національно-па тріотичного виховання як першочергового завдання органів державної влади, втілюючи прагнення мільйонів українців щодо збере ження незалежності та територіальної ціліс ності держави. По-друге, це усвідомлення того, що процеси національно-патріотичного виховання сьогодні потребують більш універ сальних підходів. По-третє, це визначення змістовного поля і характеру взаємодії дер жавних органів та інститутів громадянського Не можна забувати і про те, що на цільо вому рівні національно-патріотичне вихо вання дітей та молоді, крім спрямованості на формування соціально значущих якостей, фокусується на досягненні інших цілей ви ховного процесу – успішної соціалізації 54 Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua підростаючих поколінь в сучасних умовах і саморозвитку людини як суб’єкта діяль ності, як особистості і як індивідуальності. підростаючих поколінь в сучасних умовах і саморозвитку людини як суб’єкта діяль ності, як особистості і як індивідуальності. ховання дітей і молоді ці об’єкти не наве дені. Стратегія національно-патріотичного виховання дітей та молоді на 2016 – 2020 роки визначає цільові групи, до яких відно сяться молоді громадяни, котрі визначають свої життєві перспективи та потребують, насамперед, світоглядної опори, що не су перечить їхньому практичному досвіду, але розкриває нові можливості, а також діти з огляду на актуальність для них прикладів для наслідування, способів поведінки, що дають їм визнання в колективі однолітків і серед дорослих [9]. Аналіз досліджень і публікацій. Важливе питання патріотично орієнто ваного виховного процесу – його суб’єктна база. Якщо знову звернутися до Концепції національно-патріотичного виховання дітей і молоді, то коло соціальних інститутів ви ховання визначається органами державної влади, громадськими організаціями, сім’єю, освітніми закладами, іншими соціальними інститутами. Актуальним завданням сучасного проце су національно-патріотичного виховання є надання йому суб’єктно-суб’єктного характе ру, забезпечення активної ролі дітей і молоді в своєму морально-патріотичному розвитку. Без відображення їх активної суб’єктної по зиції в умовах життя в демократичному су спільстві дезавуюються демократичний ха рактер виховання підростаючого покоління в нашій країні, такі найважливіші принципи виховання, як гуманізація виховного проце су, ціннісно-демократична орієнтація управ ління розвитком молоді, пріоритетний вплив у виховному процесі на реальну соціальну ситуацію, в якій здійснюється життєдіяль ність молодої людини та її сім’ї. На нашу думку, необхідно надати більш чітке визначення об’єктів національно-па тріотичного виховання. Також актуальним є диференціація про блематики національно-патріотичного ви ховання на її складові елементи за різними підставами та більш глибоке вивчення їх – то бто питання патріотичних ідеалів, ціннісних орієнтацій і позицій, установок, світогляду, формування патріотичних почуттів, сто сунків, поведінки тощо, особливості змісту та організації національно-патріотичного вихо вання з різними категоріями дітей та молоді (соціально дезадаптованими дітьми, дітьми з внутрішньо переміщених родин та ін.). Розглядаючи міждисциплінарні пробле ми національно-патріотичного виховання, тобто такі, що входять в предмет дослід ження інших гуманітарно-соціальних наук (філософії, соціології, політології, історії, психології тощо), пріоритетним видається питання: «Що ж таке патріотизм в сучасних умовах, що він в себе включає на рівні кон кретної особистості людини?». Важливість правильної відповіді на це питання вкрай велика, тому що виведені з нього якості є ме тою національно-патріотичного виховання дітей та молоді, тісно корелюють з цінностя ми демократичної держави, що має європей ський вектор свого розвитку. Ця теза ґрунтується на визначенні мо лодіжної роботи, яке наведене у Портфоліо молодіжної роботи Ради Європи. За цим визначенням молодіжна робота є «ключовим словом» для всіх видів діяльності соціально го, культурного, освітнього або політичного характеру, що робиться разом із молоддю, для молоді та силами самої молоді [6]. Також аналіз європейського досвіду ро звитку політичних інститутів показує, що в умовах поширення інструментів колектив ної участі, органи влади можуть система тично отримувати різноманітну допомогу і залучати до вирішення проблем все більше представників молодіжної громадськості. Молоді люди хочуть і готові спільно шукати відповіді на питання, що мають відношення до їх життя, що, з одного боку, призводить до більшої легітимності, а з іншого – до підви щення ефективності прийняття рішень [5]. В. А. Аналіз досліджень і публікацій. Кротюк про понує наступне визначення патріотизму: «Па тріотизм – одна з найбільш значущих цінно стей, притаманних усім сферам суспільства і держави, є найважливішим духовним надбан ням особистості, характеризує вищий рівень її розвитку і виявляється в активно-діяльнісній самореалізації на благо Вітчизни. Патріотизм уособлює любов до своєї Вітчизни, причет ність до її історії, культури, досягнень, стано вить духовно-моральну основу особистості, що формує її громадянську позицію і потре бу в гідному, самовідданому, жертовному служінні Батьківщині. Патріотизм є своєрід ним фундаментом суспільного і державного будівництва, ідеологічним підґрунтям його життєздатності, однією з першоосновних умов ефективного функціонування всієї си стеми соціальних і державних інститутів» [2]. Аналіз поглядів науковців дає підста ви стверджувати, що перелік якостей гро мадянина-патріота корелює з основними структурними компонентами патріотизму – патріотичними свідомістю, відносинами і діяльністю. Патріотизм виступає в єдності духовності, громадянськості та соціальної активності особистості; в цьому одночас но виявляються цілісність і нерозривність процесу виховання як соціального явища, неможливість розмежувати його в реальній практиці на самостійні напрямки. Наступна група актуальних проблем на ціонально-патріотичного виховання – ор ганізаційні. До них можна віднести наступ ні: структура та організаційне забезпечення реалізації державної політики національ но-патріотичного виховання дітей та молоді; організаційні механізми координації діяль ності органів державної влади всіх рівнів, органів місцевого самоврядування, наукових і освітніх установ, інститутів громадянсь кого суспільства; організація міжсекторної взаємодії у сфері національно-патріотично го виховання; підвищення кваліфікації і про фесійна перепідготовка працівників органів влади, на яких покладені питання націо нально-патріотичного виховання, активістів громадських організацій національно-па тріотичного спрямування та інших суб’єктів національно-патріотичного виховання тощо. р у [ ] Аналіз поглядів науковців дає підста ви стверджувати, що перелік якостей гро мадянина-патріота корелює з основними структурними компонентами патріотизму – патріотичними свідомістю, відносинами і діяльністю. Патріотизм виступає в єдності духовності, громадянськості та соціальної активності особистості; в цьому одночас но виявляються цілісність і нерозривність процесу виховання як соціального явища, неможливість розмежувати його в реальній практиці на самостійні напрямки. р З метою активізації роботи щодо вирі шення зазначених проблем на урядовому рівні створено Міжвідомчу комісію з питань національно-патріотичного виховання [10], а на рівні місцевих державних адміністрацій – координаційні ради з питань національ но-патріотичного виховання [8]. Аналіз поглядів науковців дає підста ви стверджувати, що перелік якостей гро мадянина-патріота корелює з основними структурними компонентами патріотизму – патріотичними свідомістю, відносинами і діяльністю. Патріотизм виступає в єдності духовності, громадянськості та соціальної активності особистості; в цьому одночас но виявляються цілісність і нерозривність процесу виховання як соціального явища, неможливість розмежувати його в реальній практиці на самостійні напрямки. Аналіз досліджень і публікацій. Кротюк, проводячи аналіз дослід жень проблеми патріотизму, вказує на різно манітність і неоднозначність тлумачення тер міна «патріотизм». Автор вважає, що багато в чому це пояснюється складною природою даного явища, неординарністю його структу ри, багатоманітністю форм вияву тощо. Крім того, проблема патріотизму розглядається в Стосовно об’єктів національно-патріо тичного виховання, потрібно зазначити, що в Концепції національно-патріотичного ви 55 Аспекти публічного правління Том 6 № 9 2018 www.aspects.org.ua різних історичних, соціально-політичних і економічних умовах, залежно від сформова них поглядів і виробленої під впливом різних чинників особистої громадянської позиції, в тому числі стосовно своєї Вітчизни. Науковець умовно диференціював різ номаніття підходів до розуміння патріо тизму, наявних у науково-дослідній літера турі, за низкою напрямків, які наведені в таблиці 1. Табл. 1. Підходи до розуміння патріотизму Напрям Сутність піднесено-емоційний фіксація позитивного ставлення до Батьківщини в абстрактній формі активно-діяльнісний почуття, втілене в дієву спонукальну силу явище суспільної свідомості потенціал відродження суспільства на переломних етапах його розвитку державний виявлення суб'єктами патріотизму почуття національної гордості за державу особистісний першочергова роль надається суб'єкту патріотизму, тобто особистості духовно-релігійний патріотизм - вершина духовно-релігійного самовиявлення особистості Табл. 1. Підходи до розуміння патріотизму За результатами аналізу В. А. Кротюк про понує наступне визначення патріотизму: «Па тріотизм – одна з найбільш значущих цінно стей, притаманних усім сферам суспільства і держави, є найважливішим духовним надбан ням особистості, характеризує вищий рівень її розвитку і виявляється в активно-діяльнісній самореалізації на благо Вітчизни. Патріотизм уособлює любов до своєї Вітчизни, причет ність до її історії, культури, досягнень, стано вить духовно-моральну основу особистості, що формує її громадянську позицію і потре бу в гідному, самовідданому, жертовному служінні Батьківщині. Патріотизм є своєрід ним фундаментом суспільного і державного будівництва, ідеологічним підґрунтям його життєздатності, однією з першоосновних умов ефективного функціонування всієї си стеми соціальних і державних інститутів» [2]. Наступна група актуальних проблем на ціонально-патріотичного виховання – ор ганізаційні. До них можна віднести наступ ні: структура та організаційне забезпечення реалізації державної політики національ но-патріотичного виховання дітей та молоді; організаційні механізми координації діяль ності органів державної влади всіх рівнів, органів місцевого самоврядування, наукових і освітніх установ, інститутів громадянсь кого суспільства; організація міжсекторної взаємодії у сфері національно-патріотично го виховання; підвищення кваліфікації і про фесійна перепідготовка працівників органів влади, на яких покладені питання націо нально-патріотичного виховання, активістів громадських організацій національно-па тріотичного спрямування та інших суб’єктів національно-патріотичного виховання тощо. За результатами аналізу В. А. Аналіз досліджень і публікацій. Окремо необхідно розглянути науко во-методичні проблеми національно-па тріотичного виховання. Це методика організації національно-патріотичного ви ховання в різних типах освітніх установ; 56 Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua методика організації виховання у громад ських об’єднаннях, військово-патріотичних і спортивних молодіжних клубах; відбір змісту освіти за навчальними предметами і актуалізація в ньому національно-патріо тичних ідей; міждисциплінарна інтеграція патріотичних знань; використання актив них форм, методів і прийомів навчання у процесах національно-патріотичного ви ховання дітей і молоді; узагальнення, си стематизація та поширення накопиченого передового досвіду у сфері національно-па тріотичного виховання. • специфіку основних сфер національ но-патріотичного виховання; • специфіку основних сфер національ но-патріотичного виховання; • особливості використання виховного потенціалу обраної території у національ но-патріотичному вихованні дітей та молоді; р у ; • особливості управлінських дій щодо визначення змісту, етапів, методів, важелів та інституцій політики національно-патріо тичного виховання. Вміти: Вміти: Вміти: Вміти: • формулювати мету і завдання політи ки національно-патріотичного виховання; • виявляти загальні тенденції щодо змін у суспільній свідомості місцевого співтовариства; З метою підвищення кваліфікації пра цівників райдержадміністрацій, виконавчих комітетів міських рад та об’єднаних тери торіальних громад, на яких покладені пи тання національно-патріотичного виховання дітей і молоді, в управлінні молоді і спорту Дніпропетровської облдержадміністрації розроблена методика проведення навчаль них семінарів для зазначених осіб за темою: «Формування заходів політики національ но-патріотичного виховання дітей та молоді на основі використання власного виховного потенціалу територій». З метою підвищення кваліфікації пра цівників райдержадміністрацій, виконавчих комітетів міських рад та об’єднаних тери торіальних громад, на яких покладені пи тання національно-патріотичного виховання дітей і молоді, в управлінні молоді і спорту Дніпропетровської облдержадміністрації розроблена методика проведення навчаль них семінарів для зазначених осіб за темою: «Формування заходів політики національ но-патріотичного виховання дітей та молоді на основі використання власного виховного потенціалу територій». • мати навички щодо використання ін дикаторів ефективності реалізації заходів із національно-патріотичного виховання дітей та молоді; • розробляти рекомендації щодо впро вадження заходів із національно-патріотич ного виховання; • опанувати практичні навички щодо розрахунку показників ефективності заходів із національно-патріотичного виховання. На першому етапі семінару з учасниками проводиться обговорення проблемної ситуа ції, яка склалась у сфері національно-патріо тичного виховання, а саме: Навчальна ціль семінару – узагальнен ня теоретичних знань щодо процесу фор мування та реалізації політики національ но-патріотичного виховання молоді на регіональному та місцевому рівні і набуття практичних навичок щодо аналізу ефектив ності її заходів. Аналіз досліджень і публікацій. - відсутність ефективного механізму формування та реалізації державної політи ки у сфері національно-патріотичного ви ховання; брак комунікацій з громадянським суспільством з питань національно-патріо тичного виховання; В ході семінару його учасники на підставі вивчення і аналізу теоретичного матеріалу щодо проблем національно-патріотично го виховання дітей та молоді мають визна чити рівень впливу політики у цій сфері на суспільну свідомість місцевого співтова риства; сформулювати висновки стосовно впровадження заходів політики національ но-патріотичного виховання на місцевому рівні; розробити рекомендації щодо доціль ності впровадження заходів політики; визна чити основні можливі пріоритетні напрями діяльності на майбутнє. Аналіз досліджень і публікацій. - недостатні зусилля держави у сфері політики консолідації суспільства, форму вання активної громадянської позиції та на ціонально-патріотичної свідомості громадян України, особливо дітей і молоді; р , ; - брак духовності і моральності у су спільстві; - наявність істотних відмінностей у си стемах цінностей, світоглядних орієнтирах груп суспільства, населення певних тери торій держави, окремих громадян; у Очікувані навчальні результати семінару: - наявність імперсько-тоталітарних ру диментів у суспільній свідомості та зумов лених нищенням української духовно-куль турної спадщини та історичної пам’яті розбіжностей в уявленнях про історичне ми Очікувані навчальні результати семінару: Знати: • засади формування та реалізації політики національно-патріотичного вихо вання; Аспекти публічного правління Том 6 № 9 2018 www.aspects.org.ua ховних цінностей та громадянських ідеалів культом «золотого тільця»; ховних цінностей та громадянських ідеалів культом «золотого тільця»; нуле, зокрема про тоталітарну добу, голодо мори і політичні репресії; - незавершеність процесу формування національного мовно-культурного простору, стійкості його ціннісної основи до зовніш нього втручання; - незавершеність процесу формування національного мовно-культурного простору, стійкості його ціннісної основи до зовніш нього втручання; - на зміну концепції виховання колек тивізму почала впроваджуватись ідея форму вання індивідуалізму, егоцентризму, замість виховання патріотизму почало пропагува тись зневажливе ставлення до вітчизняної історії [1]; - відсутність єдиної державної інфор маційно-просвітницької політики щодо пи тань організації та висвітлення заходів із на ціонально-патріотичного виховання; - з поширенням інтертнету посилився негативний вплив кіберпростору на молодь, у тому числі щодо поняття «патріотизм»; - перетворення інформаційного про стору на поле маніпуляцій суспільною сві домістю, продукування ціннісної дезорієн тації; - відсутність наукових досліджень, присвячених військово-патріотичному вихо ванню молоді та її професійній орієнтації на військову службу; - недостатня нормативно-правова уре гульованість сфери національно-патріотич ного виховання; - завдання, які були визначені низкою нормативно-правових актів з національ но-патріотичного виховання, не були досяг нуті з причин недостатнього фінансування та неефективного конторлю; - завдання, які були визначені низкою нормативно-правових актів з національ но-патріотичного виховання, не були досяг нуті з причин недостатнього фінансування та неефективного конторлю; - недостатній рівень гармонізації зако нодавства й управлінських практик у сфері формування громадянської позиції з зако нодавством та кращими практиками держав Європейського Союзу та інших розвинутих держав; - незадовільний рівень знань та на вичок спеціалістів у центральних, регіо нальних та місцевих органах влади, які відповідають за реалізацію політики націо нально-патротичного виховання, відсутність можливостей для професійного розвитку та підвищення кваліфікації (внаслідок відсут ності коштів і плинності кадрів); - незадовільний рівень знань та на вичок спеціалістів у центральних, регіо нальних та місцевих органах влади, які відповідають за реалізацію політики націо нально-патротичного виховання, відсутність можливостей для професійного розвитку та підвищення кваліфікації (внаслідок відсут ності коштів і плинності кадрів); - відсутність єдиних методичного та термінологічного підходів до процесу націо нально-патріотичного виховання; - відсутність єдиних методичного та термінологічного підходів до процесу націо нально-патріотичного виховання; - відсутність єдиних стандартів щодо процесів, суб’єктів, їх компетенції та повно важень, якості діяльності у сфері національ но-патріотичного виховання; - відсутність повної статистичної ін формації на обласному рівні та рівні міст і районів, яка дозволяла б моніторинг ефек тивності заходів політики національно-па тротичного виховання, відповідно до сучас них пріоритетів та завдань політики, та була б порівнювальна з європейськими стати стичними стандартами. Аналіз досліджень і публікацій. - відсутність повної статистичної ін формації на обласному рівні та рівні міст і районів, яка дозволяла б моніторинг ефек тивності заходів політики національно-па тротичного виховання, відповідно до сучас них пріоритетів та завдань політики, та була б порівнювальна з європейськими стати стичними стандартами. - недостатність кваліфікованого кадро вого потенціалу в органах державної влади, органах місцевого самоврядування, у на вчальних закладах для організації та здійс нення заходів із національно-патріотичного виховання, нерозвиненість низової ланки в системі координації виховних процесів у цьому напрямі; Тож проблема, винесена для обговорення і вирішення в ході семінару, полягає в тому, що території через неефективну політику національно-патріотичного виховання не спроможні використати на належному рівні власний виховний потенціал у зазначеній сфері. у р - низький рівень матеріально-техніч ного забезпечення та розвитку інфраструк тури у сфері національно-патріотичного ви ховання [9]. На наступному етапі визначаються при чини, як привели до виникнення вищенаве дених проблем: На підставі аналізу особливостей реалі зації політики національно-патріотичного виховання на певній території необхідно: - відсутність у системі виховання ком плексної програми впливу на особистість з метою розвитку патріотичних почуттів; - здійснити поглиблене вивчення те оретичної сутності політики національ но-патріотичного виховання, особливостей її реалізації в умовах зовнішніх загроз та викликів; - в умовах економічної кризи відбу вається процес соціальної та моральної де градації значної частини молоді, заміна ду 58 р у Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua на основі дослідження оцінити тенденції змін суспільної свідомості в громаді; - сформулювати дієві рекомендації щодо оперативного впровадження заходів політики для забезпечення позитивної ди наміки розвитку місцевої спільноти. на основі дослідження оцінити тенденції змін суспільної свідомості в громаді; - провести узагальнення особливостей реалізації заходів політики на обраній тери торії (місто, район, ОТГ); - сформулювати дієві рекомендації щодо оперативного впровадження заходів політики для забезпечення позитивної ди наміки розвитку місцевої спільноти. р ( р ) - дослідити основні індикатори ефек тивності реалізації заходів із національ но-патріотичного виховання дітей та молоді, Табл. 2. Форма звітності для виконання завдань семінару Табл. 2. Форма звітності для виконання завдань семінар Етапи роботи Форма звітності Узагальнення теоретичної сутності політики національно-патріотичного виховання, особливостей її реалізації в умовах зовнішніх загроз та викликів, виявлення переваг і недоліків чинного законодавства в цій сфері та надання практичних рекомендацій щодо формування заходів політики. Обґрунтувати переваги і недоліки чинного законодавства стосовно національно- патріотичного виховання та надати результати у вигляді компаративного аналізу в розрізі: зарубіжний досвід / чинне національне законодавство. Аналіз досліджень і публікацій. Характеристики діяльності органів державної влади та місцевого самоврядування, громадських організацій у сфері національно- патріотичного виховання на місцевому рівні. Використовуючи індикатори та показники, визначається характеристика діяльності органів державної влади та місцевого самоврядування, громадських організацій у сфері національно-патріотичного виховання на місцевому рівні Експертизи доцільності впровадження заходів політики, їх відповідності Стратегії національно- патріотичного виховання дітей та молоді на 2016-2020 роки. На підставі даних щодо виконання Плану заходів з національно-патріотичного виховання, відповідності їх заходів цілям та пріоритетам Стратегії національно- патріотичного виховання дітей та молоді на 2016-2020 роки оцінюється доцільність та ефективність впровадження заходів Плану. Оцінки власного виховного потенціалу обраної території та можливостей його використання у національно-патріотичному вихованні. Використовуючи приклади, визначається рівень власного виховного потенціалу обраної території. Узагальнення отриманих результатів дослідження, формулювання висновків та визначення можливих пріоритетних напрямів роботи на майбутнє. Проводиться узагальнення отриманих результатів дослідження (етапи 1 – 4), формулюються висновки та визначаються можливі пріоритетні напрями роботи на майбутнє. В таблиці 2 наведені зазначені етапи ро боти та відповідна форма звітності. 3. Оцінки рівня впливу політики націо нально-патріотичного виховання на на су спільну свідомість громади та визначення загальної тенденції щодо її зміни. Практичні рекомендації для території, обраної для дослідження (місто, район, ОТГ), мають зводитись до наступного: 4. Експертизи доцільності впроваджен ня заходів політики, їх відповідності цілям Стратегії національно-патріотичного ви ховання дітей та молоді на 2016–2020 роки та визначенні можливих пріоритетних на прямів роботи на майбутнє. 1. Узагальнення теоретичної сутності політики національно-патріотичного вихо вання, особливостей її реалізації в умовах зовнішніх загроз та викликів, виявлення пе реваг і недоліків чинного законодавства в цій сфері та надання практичних рекоменда цій щодо формування заходів політики. р р у 5. Виявлення факторів, що впливають на формування у молоді національно-патріо тичної свідомості. 2. Характеристики діяльності органів дер жавної влади та місцевого самоврядування, гро мадських організацій у сфері національно-па тріотичного виховання на місцевому рівні. Для характеристики діяльності органів державної влади та місцевого самовряду вання, громадських організацій у сфері на Для характеристики діяльності органів державної влади та місцевого самовряду вання, громадських організацій у сфері на 59 Аспекти публічного правління Том 6 № 9 2018 www.aspects.org.ua прямів діяльності на майбутнє використову ються дані щодо виконання Плану заходів із національно-патріотичного виховання (за твердженого на території, що досліджуєть ся), відповідності його заходів цілям та пріо ритетам зазначеної Стратегії. Аналіз досліджень і публікацій. ціонально-патріотичного виховання на міс цевому рівні обирається для дослідження територія (місто, район, ОТГ) та визнача ються наступні індикатори: - участь влади у проведенні заходів, спря мованих на реалізацію патріотичного вихо вання в закладах системи освіти, культури, спорту; Для оцінки власного виховного потенціа лу обраної території та можливостей його використання у національно-патріотично му вихованні необхідно проаналізувати на явність наступних прикладів: - участь влади у підтримці молодіжних волонтерських проектів; - участь влади у підтримці молодіжних волонтерських проектів; - участь влади у заходах молодіжної про фесійної й самодіяльної творчості; - героїчної боротьби за самовизначення і творення власної держави, ідеалів свободи, соборності та державності у різні історичні періоди, а саме княжої доби, українських ко заків, Українських Січових Стрільців, армій Української Народної Республіки, учасників антибільшовицьких селянських повстань, Української повстанської армії, україн ців-повстанців у сталінських концтаборах, учасників дисидентського руху в Україні; - героїчної боротьби за самовизначення і творення власної держави, ідеалів свободи, соборності та державності у різні історичні періоди, а саме княжої доби, українських ко заків, Українських Січових Стрільців, армій Української Народної Республіки, учасників антибільшовицьких селянських повстань, Української повстанської армії, україн ців-повстанців у сталінських концтаборах, учасників дисидентського руху в Україні; - участь влади у залученні до патріотич ного виховання дітей та молоді учасників бойових дій на Сході України, членів сімей Героїв Небесної Сотні, бійців АТО та їхніх сімей, діячів сучасної культури, мистецтва, науки, спорту, які виявляють активну грома дянську і патріотичну позицію; д д р , Героїв Небесної Сотні, бійців АТО та їхніх сімей, діячів сучасної культури, мистецтва, науки, спорту, які виявляють активну грома дянську і патріотичну позицію; - участь влади у проведенні заходів, спря мованих на утвердження здорового спосо бу життя молодих громадян з урахуванням принципів національно-патріотичного вихо вання; - мужності та героїзму учасників револю ційних подій в Україні у 2004, 2013–2014 ро ках, учасників антитерористичної операції в Донецькій та Луганській областях; - участь влади у налагодженні співпраці з військовими формуваннями України з ме тою мотивації готовності молоді до вибору військових професій; - героїзму українців, які боролися в ар міях держав - учасників Антигітлерівської коаліції та в рухах опору нацистській Німеч чині та її союзникам під час Другої світової війни 1939–1945 років, учасників міжнарод них операцій з підтримки миру і безпеки; - сприяння органами влади роботі клубів за місцем проживання, центрів патріотично го виховання, які здійснюють патріотичне виховання молоді; - сприяння органами влади роботі клубів за місцем проживання, центрів патріотично го виховання, які здійснюють патріотичне виховання молоді; - внесків у скарбницю світової цивіліза ції, зокрема у сферах освіти, науки, культу ри, мистецтва, спорту. БІБЛІОГРАФІЧНІ ПОСИЛАННЯ 1. Бояринцев О. А. Громадські молодіжні організації як чинник патріотичного виховання молоді / О. А. Бояринцев // Віс ник Глухівського національного педагогічного університету імені Олександра Довженка. Сер. : Педагогічні науки. – 2012. – Вип. 20. – С. 322-327. 1. Бояринцев О. А. Громадські молодіжні організації як чинник патріотичного виховання молоді / О. А. Бояринцев // Віс ник Глухівського національного педагогічного університету імені Олександра Довженка. Сер. : Педагогічні науки. – 2012. – Вип. 20. – С. 322-327. 2. Кротюк В. А. Сучасні концепції дослідження патріотизму / В. А. Кротюк // Вісник Національної юридичної академії України імені Ярослава Мудрого. Сер. : Філософія, філософія права, політологія, соціологія. – 2013. – № 5. – С. 52-58. 3. Лозова В. І Теоретичні основи виховання і навчання: навч. посіб. / В. І. Лозова, Г. В. Тройко. – 2-ге вид., випр. і доп. – Харків: ОВС, 2002. – 400 с . 2. Кротюк В. А. Сучасні концепції дослідження патріотизму / В. А. Кротюк // Вісник Національної юридичної академії України імені Ярослава Мудрого. Сер. : Філософія, філософія права, політологія, соціологія. – 2013. – № 5. – С. 52-58. 3. Лозова В. І Теоретичні основи виховання і навчання: навч. посіб. / В. І. Лозова, Г. В. Тройко. – 2-ге вид., випр. і доп. – Харків: ОВС, 2002. – 400 с . р , 4. Корнієвський С. В. Методологія аналізу діяльності органів державної влади та місцевого самоврядування в сфері на ціонально-патріотичного виховання молоді / С. В. Корнієвський // Молодіжна політика та молодіжна робота : матеріали міжрегіональної наук.-практ. конф., 23 грудня 2016 р., м. Дніпро. – Д.: ДРІДУ НАДУ, 2016. – С. 38-41. р 4. Корнієвський С. В. Методологія аналізу діяльності органів державної влади та місцевого самоврядування в сфері на ціонально-патріотичного виховання молоді / С. В. Корнієвський // Молодіжна політика та молодіжна робота : матеріали міжрегіональної наук.-практ. конф., 23 грудня 2016 р., м. Дніпро. – Д.: ДРІДУ НАДУ, 2016. – С. 38-41. 5. Корнієвський С. В. Розширення доступу молоді до прийняття політичних рішень / С. В. Корнієвський // Молодіжна політика та молодіжна робота : матеріали ІІ міжрегіональної наук.-практ. конф., 14 грудня 2017 р., м. Дніпро. – Д.: ДРІДУ НАДУ, 2017. – С. 65-67. оліо молодіжної роботи Ради Європи. – Режим доступу : http://www. dsmsu.gov.ua/media/2017/03/20/1/PDF_Uk olio.pdf. _ 7. Про затвердження Концепції національно-патріотичного виховання дітей і молоді, Заходів щодо реалізації Концепції національно-патріотичного виховання дітей і молоді та Методичних рекомендацій щодо національно-патріотичного вихо вання у загальноосвітніх навчальних закладах: наказ МОН України від 16 черв. Аналіз досліджень і публікацій. - участь влади у проведенні всеукраїн ської дитячої військово-патріотичної гри «Сокіл» («Джура»); Також одним із важливих чинників ви ховного потенціалу території має бути поширення інформації про земляків, які стали жертвами комуністичного та інших тоталітарних режимів в Україні, Голодомору, політичних репресій і депортацій. - рівень співпраці органів влади з дитя чими і молодіжними громадськими організа ціями (об’єднанями), які здійснюють патріо тичне виховання молоді; - організаційна та фінансова підтримка органами влади програм, проектів громадсь ких організацій, спрямованих на національ но-патріотичне виховання дітей та молоді. На заключному етапі семінару прово диться узагальнення отриманих результатів дослідження (етапи 1-4), формулюються висновки та визначаються можливі пріори тетні напрями роботи на майбутнє. Показниками зазначених індикаторів ма ють бути кількість проведених заходів та кількість охоплених осіб. Наведена методика обговорювалась в ході проведення міжрегіональної науко во-практичної конференції «Молодіжна політика та молодіжна робота» [4]. Для експертизи доцільності впроваджен ня заходів політики, їх відповідності цілям Стратегії національно-патріотичного ви ховання дітей та молоді на 2016–2020 роки та визначенні можливих пріоритетних на Ефективність запропонованої методи ки була доведена під час проведення низ 60 Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua ки семінарів із працівниками структурних підрозділів із питань молоді райдержад міністрацій, виконавчих комітетів міських рад та об’єднаних територіальних громад Дніпропетровської області. ціонально-патріотичного виховання, що пе редбачає затвердження відповідної держав ної цільової програми; - координація діяльності органів держав ної влади, органів місцевого самоврядуван ня та громадських організацій у сфері націо нально-патріотичного виховання; Д р р Висновки. Висновки. 1. Нормативно-правова база, яка забез печує впровадження політики національ но-патріотичного виховання, в цілому сфор мована і є надійною теоретичною основою для реалізації заходів політики, але потребує певного удосконалення на рівні цільового, суб’єктно-об’єктного, змістовного компо нентів виховного процесу. 1. Нормативно-правова база, яка забез печує впровадження політики національ но-патріотичного виховання, в цілому сфор мована і є надійною теоретичною основою для реалізації заходів політики, але потребує певного удосконалення на рівні цільового, суб’єктно-об’єктного, змістовного компо нентів виховного процесу. - постійний пошук нових напрямків, форм і методів національно-патріотичного виховання молоді; - підвищення кваліфікації осіб, які задіяні в процесах національно-патріотичного вихо вання. 3. Поширення запропонованої автором методики формування заходів політики на ціонально-патріотичного виховання дітей та молоді на основі використання власного виховного потенціалу територій сприятиме підвищенню кадрового потенціалу органів влади у зазначеній сфері. у 2. До основних конкретизованих вимог організації національно-патріотичного ви ховання слід віднести: - проведення національно-патріотичного виховання молоді на системній основі; - забезпечення планового характеру на БІБЛІОГРАФІЧНІ ПОСИЛАННЯ 2015 р. № 641. – Режим доступу: http://old. mon.gov.ua/ru/ аЬои^тіпіє^у/погта^уе/4068. g у у 8. Про затвердження Типового положення про координаційну раду з питань національно-патріотичного виховання при місцевій державній адміністрації: Постанова Кабінету Міністрів України від 07 груд. 2016 р. № 999. – Режим доступу: https://www.kmu.gov.ua/ua/npas/249625271. p g p 9. Про Стратегію національно-патріотичного виховання дітей та молоді на 2016 – 2020 роки: Указ Президента України від 13 жовт. 2015 р. № 580/2015. – Режим доступу: zakon.rada.gov.ua/laws/show/580/2015 . p g p 9. Про Стратегію національно-патріотичного виховання дітей та молоді на 2016 – 2020 роки: Указ Президента України від 13 жовт. 2015 р. № 580/2015. – Режим доступу: zakon.rada.gov.ua/laws/show/580/2015 . 10. Про утворення Міжвідомчої комісії з питань національно-патріотичного виховання: Постанова Кабінету Міністрів р у у g . Про утворення Міжвідомчої комісії з питань національно-патріотичного виховання: Постанова Кабінету раїни від 22 липн. 2015 р. № 524. – Режим доступу: http://zakon2.rada.gov.ua/laws/show/en/524-2015-п. REFERENCES 1. Boiaryntsev, O.A. (2012). Hromadski molodizhni orhanizatsii yak chynnyk patriotychnoho vykhovannia molodi [Public youth organizations as a factor in the patriotic upbringing of young people]. 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Metodolohiia analizu diialnosti orhaniv derzhavnoi vlady ta mistsevoho samovriaduvannia v sferi natsionalno-patriotychnoho vykhovannia molodi [Methodology for analyzing the activities of state authorities and local self-government bodies in the field of national-patriotic education of youth]. Youth policy and youth work. Dnipro: DRIPA NAPA [in Ukrainian]. g _ _ _ p [ ] 7. Pro zatverdzhennia Kontseptsii natsionalno-patriotychnoho vykhovannia ditei i molodi, Zakhodiv shchodo realizatsii Kontseptsii natsionalno-patriotychnoho vykhovannia ditei i molodi ta Metodychnykh rekomendatsii shchodo natsionalno-patriotychnoho vykhovannia u zahalnoosvitnikh navchalnykh zakladakh [On Approval of the Concept of National-Patriotic Education of Children and Youth, Measures to Implement the Concept of National-Patriotic Education of Children and Youth and Methodical Recommendations on National-Patriotism Education in General Educational Institutions]. Order of the Ministry of Education and Science of Ukraine. June 16, 2015. № 641. Retrieved from: http://old.mon.gov.ua/ru/ aoy^tipiie^u/pohta^ue/4068 [in Ukrainian]. 8. Pro zatverdzhennia Typovoho polozhennia pro koordynatsiinu radu z pytan natsionalno-patriotychnoho vykhovannia pry mistsevii derzhavnii administratsii [On Approval of the Model Regulations on the Coordination Council on National Patriotism Education at the Local State Administration]. Resolution of the Cabinet of Ministers of Ukraine. Deceber 07, 2016. № 999. – Retrieved from: https://www.kmu.gov.ua/ua/npas/249625271 [in Ukrainian]. p g p [ ] 9. Pro Stratehiiu natsionalno-patriotychnoho vykhovannia ditei ta molodi na 2016 – 2020 roky [About the Strategy of National- Patriotic Education of Children and Youth for 2016-2020]. Decree of the President of Ukraine. October 13, 2015. № 580/2015. Retrieved from: zakon.rada.gov.ua/laws/show/580/2015 [in Ukrainian]. g [ ] 10. Pro utvorennia Mizhvidomchoi komisii z pytan natsionalno-patriotychnoho vykhovannia [On the formation of the Interdepartmental Commission on national-patriotic education]. Resolution of the Cabinet of Ministers of Ukraine. July 22, 2015. № 524. – Retrieved from: http://zakon2.rada.gov.ua/laws/show/en/524-2015-p [in Ukrainian]. Корнієвський Сергій Володимирович р р р Кандидат наук з державного управління і і і і Управління молоді і спорту Дніпропетровської облдержадміністрації 49006, Дніпро, вул. В. Антоновича, 70 Управління молоді і спорту Дніпропетровської облдержадміністрації 49006, Дніпро, вул. В. Антоновича, 70 Korniievskyi Sergii PhD in public administration y g PhD in public administration p Department of Youth and Sports of Dnipropetrovsk Regional State Administration 70, V. Antonovicha St., Dnipro. 49006, Ukraine Email: unexp@ukr.net ійшла / Article arrived: 06.09.2018 Схвалено до друку / Accepted: 18.09.2018 Концептуалізація управління процесами розвитку територіальних громад Бобровська О. Ю., Дніпропетровський регіональний інститут державного управління Національної академії державного управління при Президентові України Розглянуто першооснову формування явища розвитку суспільства на рівні територіальних гро мад. Наведено результати дослідження розширення можливостей розвитку територіальних громад як первісного рівня існування суспільства в умовах модернізації місцевого самоврядування шляхом запровадження процесного підходу. Наведено докази пріоритетної ролі процесів розвитку територій і необхідність створення систем управління процесом розвитку як фрактальної системи місцевого самоврядування. Зазначено, що управління процесами розвитку на місцевому рівні ще не доведено до теоре тико-методичної чіткості; не надано теоретичні характеристики, параметри і показники процесів розвитку; не змодельовано технології побудови і плину; не визначено підходи до оцінювання резуль татів процесів відповідно до векторів розвитку громад, що засвідчує відсутність дієвого управління явищем розвитку, починаючи з первісного рівня його виникнення. Для вирішення проблеми інтенсифікації і підвищення темпів розвитку територіальних громад розроблено концептуальне бачення процесного управління, створення системи управління проце сами, яка розглядається як фрактальна складова частина системи місцевого самоврядування. Вона представлена понятійним апаратом управління процесами, запропонованим визначенням понять «процес», «процес розвитку», контуром управління процесами, структурою механізму управління процесу розвитку в контурі управління. Визначено критерії і показники керованості розвитком процесів шляхом дотримання допусти мих меж їх коливань. Надані пропозиції поглиблюють теоретико-методичну основу процесного управління. Запропонований алгоритм проектування процесів розвитку на першому етапі їх жит тєвого циклу дозволяє упорядковувати діяльність з управління процесами розвитку, скоротити шлях і час на їх реалізацію. Ключові слова: алгоритм побудови процесу; критерії і показники оцінювання; управління; органи управ ління; механізм управління DOI: 10.15421/151855 DOI: 10.15421/151855 УДК: 352-04 Концептуалізація управління процесами розвитку територіальних громад Email: unexp@ukr.net Цитування: Корнієвський С. В. Підвищення ефективності кадрового потенціалу органів влади щодо реалізації заходів у сфері національно-патріотичного виховання молоді / С. В. Корнієвський // Аспекти публічного управління. – 2018. – Т. 6. – № 9. – С. 52-62. Citation: Korniievskyi, S.V. (2018). Pidvyshchennia efektyvnosti kadrovoho potentsialu orhaniv vlady shchodo realizatsii zakhodiv u sferi natsionalno-patriotychnoho vykhovannia molodi [Efficiency improvement of the authorities personnel potential concerning measures implementing in the sphere of the national and patriotic education of youth]. Public administration aspects, 6 (9), 52-62. Схвалено до друку / Accepted: 18.09.2018 62 Public Administartion Aspects 6 (9) 2018 www.aspects.org.ua Conceptualization of the management of the processes of development of territorial communities Bobrovska O. Y., Dnipropetrovsk Regional Institute of Public Administration of the National Academy of Public Administration under the President of Ukraine Considered the first principle of the formation of the phenomenon of development of society at the level of territorial communities. The results of the study of the expansion of the possibilities of development of territorial communities as the initial level of society existence in the conditions of modernization of local self-government through the introduction of a process approach are given. The proofs of the priority role of the processes of development of territories and the necessity of creating control systems for the development process as a fractal system of local self-government are given. It is noted that the management of development processes at the local level has not yet been brought to theoretical and methodological clarity; not provided theoretical characteristics, parameters and indicators of development processes; the technology of construction and flow is not simulated; There are no defined approaches to the evaluation of the results of processes in accordance with the vectors of community development, which testifies to the lack of effective management of the phenomenon of development, starting with the initial level of its occurrence. To solve the problem of intensification and increase of the development of territorial communities, a conceptual vision of process management, creation of a system of process management, which is considered as a fractal component of the system of local self-government is developed. It is represented by a conceptual apparatus for controlling processes proposed by the definition of the concepts of «process», «development process», the path of process management, the structure of the mechanism for managing the development process in the control circuit. Criteria and indicators of controllability of development of processes are determined by adhering to the allowable limits of their fluctuations. The given proposals deepen the theoretical and methodological basis of process management. The proposed algorithm for designing development processes at the first stage of their 63
https://openalex.org/W2779306242	http://revistas.udc.es/index.php/reipe/article/download/reipe.2017.0.12.3044/pdf	Portuguese	null	Mercado de capitais em currículos de cursos de engenharia: proposta de análise	Revista de estudios e investigación en psicología y educación	2,017	cc-by-sa	3,895	Introdução “O currículo do profissional de nível superior é apresentado sob a forma de itens de conteúdo, não sendo capaz de esclarecer o que o aprendiz deve estar apto a fazer após ter sido submetido a esses conteúdos ou classes de informações”,ou seja, o ensino é concebido como adesão a informações e adoção de práticas e procedimentos conhecidos e difundidos, e não como desenvolvimento de uma atuação transformadora da realidade (Rebelatto, 1999 apud Nose; Rebelatto, 2001, p.30). Abstract In Brazil, the Capital Markets area has been increasingly occupied by professionals trained in several Engineering courses. Currently, there is a tendency to consider Engineering as the most appropriate course for the area. In this sense, the objective of this work is to present a research proposal to investigate whether the curriculum of the Engineering (Civil, Electrical, Mechanical and Production) courses taught in three Brazilian Universities, together with the strategies, resources And the evaluation methods, the development of skills also in the area of Capital Markets. A formação de qualquer profissional de nível superior está invariavelmente fundamentada na implementação de um currículo. Pode-se dizer que o currículo é o caminho que será trilhado pelo ingressante no ensino superior, para tornar se um profissional preparado para enfrentar os desafios do mercado de trabalho e para cumprir seu juramento feito na ocasião de sua colação de grau (Rebelatto, 1999 apud Nose; Rebelatto, 2001, p.30). Keywords: Curriculum, engineering courses, capital market, skills. p ) Todavia, o mesmo autor, sustenta que: p Todavia, o mesmo autor, sustenta que: REVISTA DE ESTUDIOS E INVESTIGACIÓN EN PSICOLOGÍA Y EDUCACIÓN eISSN: 2386-7418, 2017, Vol. Extr., No. 12 DOI: https://doi.org/10.17979/reipe.2017.0.12.3044 REVISTA DE ESTUDIOS E INVESTIGACIÓN EN PSICOLOGÍA Y EDUCACIÓN eISSN: 2386-7418, 2017, Vol. Extr., No. 12 DOI: https://doi.org/10.17979/reipe.2017.0.12.3044 REVISTA DE ESTUDIOS E INVESTIGACIÓN EN PSICOLOGÍA Y EDUCACIÓN eISSN: 2386-7418, 2017, Vol. Extr., No. 12 DOI: https://doi.org/10.17979/reipe.2017.0.12.3044 Correspondencia : João de Azevedo, jdazev@hotmail.com; Selección y peer-review bajo responsabilidad del Comité Organizador del XIV Congreso Internacional Galego-Portugués de Psicopedagogía Mercado de capitais em currículos de cursos de engenharia: proposta de análise Capital market in curricula of engineering courses: proposal of analysis João de Azevedo , Maria Palmira Carlos Alves , Elisa Henning** Udesc/Uminho, Uminho, *Udesc João de Azevedo , Maria Palmira Carlos Alves , Elisa Henning* Udesc/Uminho, Uminho, **Udesc Superior ofereçam uma maior e melhor qualificação aos seus formandos. Resumo No Brasil a área de Mercado de Capitais vem sendo ocupada cada vez mais por profissionais formados nos diversos cursos de Engenharia. Atualmente, existe uma tendência em se considerar a Engenharia como o curso mais adequado para a área. Neste sentido, o objetivo deste trabalho consiste em apresentar uma proposta de pesquisa para investigar se a grade curricular dos cursos de Engenharia (Civil, Elétrica, Mecânica e de Produção), lecionados em três Universidades brasileiras, permite em conjunto com as estratégias, os recursos e os métodos de avaliação, o desenvolvimento de competências também na área do Mercado de Capitais. Em face dessas exigências, cada vez mais pessoas ingressam no Ensino Superior, com o objetivo de desenvolverem competências para uma determinada área específica. Porém, muitas vezes, ao fazerem um estágio durante, ou no final de seu curso de Graduação, sentem-se um pouco desapontadas para enfrentar os desafios impostos no dia-a-dia pela atividade profissional. Se for verdade que os contextos profissionais estão em constante mudança e que nunca o graduado estará totalmente apto para enfrentar as exigências que ser lhe colocam, não é menos verdade que os currículos acadêmicos estão pouco atualizados, baseiam-se na transmissão de conteúdos, na reprodução dos mesmos e em processos de avaliação final, (Alves, 2004), sendo que a preparação para a prática, para a transformação da realidade, vem sendo descurada, nos currículos, apesar da importância que estes assumem: Palavras-chave: currículo, cursos de engenharia, mercado de capitais, competências. Apresentação do tema e contextualização do problema Pode-se entender a Globalização, como um processo de “intensificação de relações sociais à escala mundial, estabelecendo ligações entre localidades de maneira a fazer com que acontecimentos locais sejam moldados por eventos ocorrendo a muitas milhas de distância” (Giddens, 1990). As mudanças que vêm ocorrendo no mundo, como consequência da Globalização, se refletem em muitas dimensões: cultural, econômica, educativa. Na dimensão econômica as mudanças geram maior competitividade exigindo que a educação seja confrontada com uma maior exigência dos mercados de trabalho, fazendo com que as instituições de Ensino A formação profissional de um ingressante no ensino superior começa a ser construída em sala de aula e prolonga-se durante toda a sua Graduação, que se fundamenta num conjunto de disciplinas que são ministradas no cumprimento de uma grade curricular. Um curso de Graduação estruturado numa grade curricular que permita aos graduandos desenvolverem AZEVEDO, ALVES, HENNING paga além de salários altos, e também bônus de acordo com o seu desempenho. competências quer numa determinada área específica e direcionadas às exigências do mercado de trabalho, quer competências transversais, ajudá-los-á a tornarem-se profissionais preparados para enfrentar os desafios decorrentes das exigências da atividade profissional, possibilitando-lhes uma boa probabilidade de “Empregabilidade”. A constatação de que existem Engenheiros trabalhando em outras áreas, diferentes da de sua formação, está de acordo com os dados do Conselho Federal de Engenharia e Agronomia – CONFEA, que segundo o qual, no Brasil existem 800 mil engenheiros registrados, mas apenas um terço atua na parte técnica. As outras partes se dividem entre os que criaram a própria empresa, os que oferecem consultorias e os que trabalham em diversos departamentos nas empresas: de Compras, de Finanças e de Administração. Existindo também, como já foi dito, os que atuam na área do Mercado de Capitais, junto a Instituições Financeiras, Corretoras e entre outras. A Empregabilidade também pode ser compreendida como um conjunto de competências e habilidades necessárias para que os graduandos possam se inserir no mercado de trabalho e nele permanecer; de também lhes poder favorecer a busca por uma nova oportunidade de trabalho fora da organização em que estejam atuando profissionalmente. Pode-se dizer também que é a capacidade de os profissionais encontrarem, com uma probabilidade alta, um novo emprego quando demitidos (Morosini, 2001). Também de acordo com um levantamento realizado pela Confederação Nacional da Indústria (CNI), atualmente, no Brasil, a maioria dos engenheiros brasileiros não exerce função nas áreas em que se formam. Apresentação do tema e contextualização do problema Esse é o resultado de análise feita pela CNI, com base em estatísticas do Ministério da Educação (MEC) e do Instituto Brasileiro de Geografia e Estatística (IBGE). O levantamento aponta que de 680.526 engenheiros empregados, apenas 286.302 (42%) trabalham na área de formação. (UOL economia Empregos e carreiras, 2014). Diante desse contexto, a probabilidade de Empregabilidade dos graduandos depende da maneira como as universidades os preparam para o mercado de trabalho, podendo ser potencializada pelo desenvolvimento de competências, não só disciplinares, mas também transdisciplinares, alinhadas com as exigências sociais e econômicas. O perfil exigido das pessoas para exercerem suas atividades profissionais, sofre pressões e inovações às quais a Universidade não pode ficar indiferente, pois a formação acadêmica não pode ficar longe da realidade, colocando em risco a Empregabilidade presente ou futura dos graduandos. (Rebelatto, 1999). Em minha opinião, um dos motivos pelos quais se acentuou a imigração de engenheiros para o Mercado de Capitais, se deve ao fato de que nas décadas de 80 e 90, com as crises econômicas pelas qual o Brasil passou, tenha havido uma desaceleração no desenvolvimento das Infraestruturas do País. Com isso, a demanda por engenheiros diminuiu. Pois, nem o ramo da construção civil conseguia empregar todos os engenheiros formados. Para que os graduados sejam capazes de atuar no mundo em constantes mudanças, é importante que as instituições de Ensino Superior se preocupem com os conteúdos das grades curriculares dos seus cursos de Graduação, nomeadamente, que eles resultem de uma articulação entre as forças vivas da sociedade, nas quais se inclui o mercado de trabalho. O que pode ter facilitado essa imigração de engenheiros para a área de Mercado de Capitais, é que os cursos de Engenharia têm, em suas grades curriculares, várias disciplinas na área de Matemática, que proporcionam uma forte formação nessa área para os seus graduandos. Além de um bom embasamento matemático, essas disciplinas também proporcionam também aos graduandos em Engenharia, o desenvolvimento de suas capacidades de abstração, de raciocínio lógico, que são qualidades fundamentais em qualquer área de atuação do mercado de trabalho. Para tal, torna-se necessária uma reformulação, tanto nos currículos dos cursos, quanto na maneira de ensinar, devendo proporcionar-se aos estudantes, durante a sua graduação, a oportunidade de desenvolver competências diversas, que contribuirão para uma boa probabilidade de Empregabilidade presente ou futura. MERCADO DE CAPITAIS NOS CURSOS DE ENGENHARIA abre-se a possibilidade da implantação de experiências inovadoras de organização curricular. aos engenheiros mais facilidades de se adaptarem ao mercado de trabalho em outras áreas, além daquela de sua formação. O que me leva a pensar que as grades curriculares dos cursos de Engenharia continuam distanciadas em relação às necessidades do mercado de trabalho na área do Mercado de capitais são os cursos de Especialização MBA’s. Esses vieram, de certo modo, para corrigir certas deficiências, que os engenheiros recém-formados possuem na área de Administração e Finanças. Os cursos de graduação que mais cedem profissionais para o Mercado Financeiro brasileiro, são os de Administração, Economia e Engenharia. Podendo se dizer, que é um setor que pode abrigar pessoas com várias áreas de formações, além das tradicionais em Administração e Economia. Isso nos leva a refletir sobre a questão da educação continuada (Quintas, 2008), pois o profissional vive em um ambiente de intensas mudanças e são necessários novos conhecimentos a cada momento da vida profissional. O mercado de renda fixa, por ser o maior, absorve grande parte dos engenheiros que atuam na área de finanças. Contudo, com o crescimento do mercado de renda variável a partir de 2004, cada vez mais engenheiros passaram a trabalhar com o Mercado Acionário, com análise e gestão de ações, sendo que, atualmente, existe uma tendência em se considerar a Engenharia como o curso mais adequado para a área. (Rocha, 2012). Diante desse contexto, tem que se pensar nos princípios para um design de currículo que atenda a essas novas demandas sociais, humanas e para os profissionais que carecem de novas escolhas (Leite, 2007). As grades curriculares dos cursos de Graduação, muitas vezes, refletem também em parte a questão cultural das pessoas envolvidas na sua concepção, deixando um pouco de lado o contexto real de necessidades da sociedade. Contudo, dependendo da Instituição de Ensino Superior, que tenham cursos de Engenharia Civil, Elétrica, Mecânica e de Produção, esses ou alguns desses, possuem em sua grade curricular, alguma(s) disciplina(s) que contemple algum conteúdo voltado para á área de Mercado de Capitais. Será que os cursos que têm em suas grades curriculares, disciplinas com algum conteúdo voltado para essa área, esses estão suficientemente articulados com as necessidades do mercado de trabalho,no que diz respeito ao conhecimento do funcionamento do Mercado de Capitais? Apresentação do tema e contextualização do problema Dependendo da área de formação de um profissional, às vezes, algumas das competências desenvolvidas por esse, durante a sua fase de graduação, pode beneficiá-lo a atuar em outras áreas do mercado de trabalho, diferentes da área de sua formação. Isso acontece com profissionais formados em várias áreas no mercado de trabalho, entre as quais, também na de Engenharia. De Acordo com (Araujo, 2016), têm-se engenheiros trabalhando profissionalmente fora da área de Engenharia, tal como na área de gestão em Engenharia, e na de gestão Financeira. Apesar de essas áreas serem diferentes, (Araujo, 2016), ainda diz que: No Brasil, existem muitos profissionais formados na área de Engenharia Civil, Elétrica, Mecânica e de Produção, ocupando a função de gestores em empresas e trabalhando também na área do Mercado Financeiro, que no Brasil, esse divide-se em quatro mercados: Mercado de Crédito; Mercado Monetário; Mercado de Câmbio; e Mercado de Capitais. (Czelusniak, 2011; Universia Brasil, 2005). “Se, por um lado, o engenheiro é um tipo de profissional fundamental para a elevação da produtividade e para a inovação, por outro, ele tem bastante flexibilidade com respeito às opções ocupacionais, devido à formação versátil em ciências e matemática.” Com todas essas capacidades desenvolvidas, os graduandos de Engenharia, acabam tendo mais facilidade para compartimentar problemas, ter uma visão sistêmica, relacionar e isolar variáveis e desenvolver soluções para situações complexas. Isso dá Pois, os engenheiros que abandonam a carreira e passam a trabalhar em bancos, consultorias e outras entidades do mercado financeiro, encontra um setor que R Est Inv Psico y Educ, 2017, Extr.(12), A12-76 MERCADO DE CAPITAIS NOS CURSOS DE ENGENHARIA Uma grade curricular de um curso de Graduação, é um plano pedagógico e institucional que orienta a aprendizagem dos alunos de forma sistemática. No sentido tradicional, o currículo é compreendido como “currículo”, “conteúdos de ensino” ou “conjunto de disciplinas” (Pacheco, 2001), que conforme forem ministradas potencializem e valorizem as habilidades e também desenvolvam competências individuais e coletivas. Assim, a nossa problemática consiste em investigar se a grade curricular dos cursos de Engenharia (Civil, Elétrica, Mecânica e de Produção), lecionados em três Universidades Brasileiras, assim como as estratégias, os recursos e os métodos de avaliação para o desenvolvimento de competências, nomeadamente, na área do Mercado de Capitais, desenvolve competências quanto ao conhecimento do funcionamento do Mercado de Capitais. A grade curricular de qualquer curso de Graduação não deve ser entendida como um plano totalmente acabado e definido, mas como um ponto de partida das metas a serem alcançadas dependentes das condições da sua aplicação. Assim, para acompanhar as transformações estruturais significativas com o advento das novas tecnologias por meio dos avanços da ciência, a Educação deve ser repensada começando pelas políticas curriculares, especialmente no Ensino Superior, pois o currículo não é estático, pelo contrário, ele foi e continua sendo construído (Goodson, 2001). Problemática e Objetivos da Investigação Nessas últimas duas décadas, devido ao contexto da Globalização, além das habilidades específicas da profissão, um profissional tem que entender claramente o mercado de trabalho em que esteja inserido. Pois, só assim esse terá condições de responder às mudanças das necessidades do mercado de trabalho (Giddens, 1990). Surge, assim, a preocupação com a questão do currículo: como deve ser o ensino tendo em vista a formação do engenheiro em suas diversas áreas de atuação? E como garantir que ele aprenda aquilo que é realmente necessário para uma atuação efetiva, responsável, ética, levando em conta as particularidades do contexto social onde se insere? Que processo de avaliação deve ser contemplado? (Alves, 2004) Mas será que as grades curriculares dos cursos de Engenharia, estão em sintonia com as necessidades dos empregadores do mercado de trabalho, devido ao efeito da Globalização? A Globalização afeta a Educação por incidir sobre os sujeitos com ela envolvidos, os conteúdos dos currículos e as formas de aprender. Ou seja, vão-se tornando indispensáveis os conhecimentos aquém de sua área de atuação. Dessa forma, se faz necessário entender o currículo como uma oportunidade de refletir sobre as mudanças que serão necessárias para que os graduandos possam enfrentar este novo cenário globalizado e com perspectivas para mudanças permanentes (Pinar, 2007). Prevalecendo o ideário da flexibilidade curricular, de forma que esse esteja em sintonia com as necessidades do mercado de trabalho, os graduandos terão pela frente uma boa probabilidade de Empregabilidade presente ou futura. A Resolução CNE/CES, de 11 de março de 2002, institui Diretrizes Curriculares para os cursos de graduação em Engenharia ditando as tendências para estes cursos. Nesta proposta de Diretrizes Curriculares,tem-se a possibilidade de novas formas de estruturação dos cursos. Ao lado da tradicional estrutura de disciplinas organizadas através da grade curricular, R Est Inv Psico y Educ, 2017, Extr.(12), A12-77 AZEVEDO, ALVES, HENNING Assim, para a pergunta de Moraes (2007, p.133): Também Costa (2001) afirma que se pode, na mesma pesquisa, reunir os aspectos qualitativos e quantitativos, evidenciando a abordagem mista. Dessa forma, a pesquisa pode ser ‘qualiquantitativa’ ou ‘quantiqualitativa’, dependendo da ênfase nas informações qualitativas ou quantitativas, respectivamente. “De que forma as sociedades atuais poderão garantir que seus membros sejam suficientemente dotados de condições intelectuais e instrumentais que garantam sua permanência na própria vida social sem se transformar em sobrecarga para o Sistema”? Instrumentos de coletas de dados Ou ainda, as propostas curriculares deverão ser de forma a garantir que os membros de uma sociedade possam nela permanecer de forma que, ao invés de se tornarem umas sobrecargas, possam sim com ela colaborar. Esse objetivo será alcançado mediante o contínuo desenvolvimento das grades curriculares dos cursos de Graduação, de forma que essa esteja sempre em alinhamento com as necessidades do mercado de trabalho. Assim, teremos pela frente uma Educação de nível Superior, de maneira que os que dela usufruírem tenham condições de se inserirem no mercado de trabalho com uma alta probabilidade de Empregabilidade presente ou futura. A pesquisa terá uma coleta de dados dividida em três etapas. A primeira etapa da pesquisa será apresentar o referencial teórico, proveniente da literatura disponível e dos referenciais curriculares dos cursos de Engenharia que preparam profissionais para atuarem na área de Mercado de Capitais. Procurar-se-á, também, buscar similaridades que se apresentam nas instituições de ensino superior no Brasil que possuem cursos de Engenharia. Na segunda etapa, far-se-á uma pesquisa de campo, utilizando um inquérito por entrevista com o uso de questionário com perguntas fechadas junto a: professores de disciplinas de Mercado de Capitais e/ou de disciplinas que contenham algum conteúdo relacionado com a área; engenheiros já atuando profissionalmente na área de Mercado de Capitais; empregadores do Mercado de Capitais; e por um questionário com 92 perguntas utilizando a escala Likert, aplicado a graduandos em Engenharia, de modo a formar uma amostra representativa da população. Metodologias abordadas Há dois paradigmas que orientam as pesquisas científicas do ponto de vista da metodologia: a qualitativa associada à subjetividade; e a quantitativa que, conforme o nome já diz, está associada a números. Segundo Bento (2012): Na terceira etapa, será feito um tratamento dos dados primários coletados via entrevistas e questionário, analisados com recurso à estatística descritiva e inferencial. A investigação educacional tem sido descrita como quantitativa ou qualitativa. Estes termos referem as duas tradições diferentes de investigação, cada uma com a sua terminologia, métodos e técnicas. Por muitas décadas, a maioria da investigação educacional era baseada na tradição quantitativa, que é o que é conhecido como a filosofia positivista de como geramos o conhecimento. Como refletido nas ciências exatas e na psicologia, o positivista acredita que há fatos com realidade objetiva que podem ser expressos numericamente. Consequentemente,há uma pesada dependência em números, medidas, experiências, relações e descrições numéricas (Bento, 2012, p.1). Os dados resultantes das entrevistas serão tratados com recurso à análise de conteúdo (Bardin, 1996). Problemática e Objetivos da Investigação Como uma das respostas à pergunta anterior, será na construção ou na elaboração dos modelos e das propostas curriculares que se define que tipo de sociedade e de cidadão se deseja construir, o que a escola faz, para quem faz ou deixa de fazer. Ou seja, é na construção ou definição das propostas que são selecionados conteúdos, que ajudarão as pessoas a entender melhor a sua história e a compreender o mundo que as cerca. Atendendo ao problema, aos objetivos e às questões relativas à pesquisa aqui proposta e na perspectiva ora colocada, a metodologia a ser utilizada nesta pesquisa terá uma abordagem mesclada: “qualiquantitativa” ou “quantiqualitativa”, por acreditar-se que assim se alcançará uma visão mais ampla das questões discutidas e dos resultados advindos da pesquisa de campo. Considerações finais Com o trabalho proposto, desejamos verificar o que os graduandos em Engenharia (Civil, Mecânica, Elétrica e de Produção), de três Universidades Brasileiras, conhecem a respeito do funcionamento do Mercado de Capitais, e também o que os empregadores do mercado de trabalho nessa área, esperam dos engenheiros que desejam trabalhar na mesma área. Na discussão sobre a abordagem que uma pesquisa científica, se qualitativa ou quantitativa, não devemos aceitar que haja uma dicotomia entre essas duas abordagens, se aceitarmos, essas se tornam com paradigmas distintos e incompatíveis (Moraes e Neves, 2007). Esperamos também contribuir, com sugestões para alguma alteração do conteúdo de disciplinas que fazem parte das grades curriculares dos cursos de Engenharia, e que abordam o funcionamento do Mercado de Capitais e/ou de disciplinas que contenham algum conteúdo relacionado com a área, de forma que os graduandos de Engenharia, que desejarem trabalhar nessa área, estejam mais capacitados. Ainda conforme Demo (2000) o ideal seria reunir no método as duas possibilidades de estudar o mensurável e o imensurável, a quantidade e a qualidade, o linear e o não linear, ou seja, mesclá-las reunindo na mesma pesquisa os aspectos qualitativos e quantitativos, evidenciando assim uma abordagem mista. R Est Inv Psico y Educ, 2017, Extr.(12), A12-78 MERCADO DE CAPITAIS NOS CURSOS DE ENGENHARIA MERCADO DE CAPITAIS NOS CURSOS DE ENGENHARIA Tese (Doutorado) - Escola de Engenharia de São Carlos, Universidade de São Paulo. Referencias Alves, M. P. (2004). Currículo e Avaliação. Uma perspectiva integrada. Porto: Porto Editora. Rocha, André. (2012, Junho 11). ValorInveste. Disponível em: http://www.valor.com.br/valor- investe/o-estrategista/2697982/qual-melhor-formacao- para-trabalhar-no-mercado-acionario. ARAÚJO, B. C. P. O. Carreiras de Jovens Engenheiros no Brasil, São Paulo. Tese (Doutorado) – Departamento de Engenharia de Produção, Universidade de São Paulo. 2016. (2005, Janeiro 03). Universia Brasil. Disponível em: http://noticias.universia.com.br/destaque/noticia/2005/ 01/03/491939/cresce-migrao-engenheiros-mercado- financeiro.html Bento, A. Investigação quantitativa e qualitativa: Dicotomia ou complementaridade?. (2012).Revista JA (Associação Académica da Universidade da Madeira), nº 64, ano VII (pp. 40-43). UOL economia Empregos e carreiras. (2014, Abril 09). Disponível em: http://economia.uol.com.br/empregos-e- carreiras/noticias/redacao/2014/04/09/maioria-dos- formados-em-engenharia-nao-trabalha-como- engenheiro-diz-cni.htm. Bardin, L. Análise de conteúdo. 3 ed. Lisboa: Edições 70, 2004. BRASIL. (2002, março 11).Ministério da Educação – Conselho Nacional de Educação - Câmara de Ensino Superior. Resolução CNE/CES, que institui Diretrizes Curriculares para os cursos de graduação em Engenharia. Brasília: Ministério da Educação, 2002a. Disponível em http://www.mec.gov.br. Acesso em 13/05/2015. Agradecimientos Este trabalho foi financiado por Fundos Nacionais através da FCT (Fundação para a Ciência e a Tecnologia) e cofinanciado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) no âmbito do CIEC (Centro de Investigação em Estudos da Criança da Universidade do Minho) com a referência POCI-01-0145-FEDER-007562. Czelusniak. A. (2011, Julho 11). Gazeta do Povo. Disponível em: http://www.gazetadopovo.com.br/educacao/vida-na- universidade/vestibular/engenheiros-cada-vez-mais- gestores-dgnyzgg9n3oub49wl1a3bttu6. Demo, P. (2000). Metodologia do Conhecimento Científico. São Paulo: Atlas. Giddens, A. The Consequences of Modernity. Stanford: Stanford University Press, 1990. Goodson, I. (2001). O Currículo em Mudança. Estudos na Construção social do currículo. Porto: Porto Editora. Leite, C. (2007). A avaliação e o ensino-aprendizagem em função de competências: Porque? Como?. Qualiforma, 1, 5-9. Morais, A. M.; Neves, I. P. (2007). Fazer investigação usando uma abordagem metodológica mista. Revista Portuguesa de Educação, vol. 20, n. 2, (55-104), Braga, Universidade do Minho. MOROSINI, M. C. The quality of higher education: isomorphism, diversity and fairness, Interface _ Comunic, Saúde, Educ, v.5, n.9, p.89-102, 2001. Disponível em: <http://www.scielo.br/scielo.php?pid=S1414- 32832001000200006 &script =sci abstract>. Acesso em: 09 mai. 2015. Nose, M. M.; Rebelatto, D. A. do N. (2001). O Perfil do Engenheiro Segundo as Empresas. Cobenge. Disponível em: http://www.abenge.org.br/Cobenge Anteriores/2001/trabalhos/DTC008.pdf. Acesso em 06/05/2015. Pacheco, J. A. (2001). Currículo: Teoria e Práxis. Porto: Porto Editora. Pinar, W. (2007). O que é a teoria do Currículo?. Porto: Porto Editora. Quintas, H. L. M. (2008).Educação de adultos: vida no currículo, currículo na vida – perspectivas e reflexões. Lisboa: Agência Nacional para a qualificação. IP. Rebelatto, D. A. N. (1999). O campo de atuação profissional do engenheiro de produção: inter-relações com as áreas de economia e finanças, São Carlos. R Est Inv Psico y Educ, 2017, Extr.(12), A12-79
https://openalex.org/W4213153639	https://discovery.ucl.ac.uk/id/eprint/10122900/13/Powner_anie.202101376.pdf	English	null	Selective Prebiotic Synthesis of α‐Threofuranosyl Cytidine by Photochemical Anomerization	Angewandte Chemie	2,021	cc-by	5,321	Selective Prebiotic Synthesis of a-Threofuranosyl Cytidine by Photochemical Anomerization B W F C l ill d M tth W P * Selective Prebiotic Synthesis of a-Threofuranosyl Cytidine by Photochemical Anomerization Figure 1. Nucleic acid backbones of TNA, RNA (R=OH, R1=H), ANA (R=H, R1=OH), and DNA (R=H, R1=H). Truncated 3’-2’-phospho- diester of TNA (blue) compared to natural 5’-3’-phosphodiesters (red). In TNA the a-anomer of l-threose nucleic acid is observed to Watson– Crick base pair with RNA/DNA. B=nucleobase. Abstract: The structure of lifes first genetic polymer is a question of intense ongoing debate. The “RNA world theory” suggests RNA was lifes first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl- selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2’,3’-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life. Figure 1. Nucleic acid backbones of TNA, RNA (R=OH, R1=H), ANA (R=H, R1=OH), and DNA (R=H, R1=H). Truncated 3’-2’-phospho- diester of TNA (blue) compared to natural 5’-3’-phosphodiesters (red). In TNA the a-anomer of l-threose nucleic acid is observed to Watson– Crick base pair with RNA/DNA. B=nucleobase. Nu Nucleic acids are essential to modern biology, and several theories for the origin of life rely on the chemical (non- enzymatic) emergence of nucleic acids.[1,2] The “RNA world theory”,[3] for example, which postulates that RNA emerged as lifes first genetic polymer, is supported by the universal dual genetic and phenotypic functions of RNA in biology.[4,5] It has been suggested that selective chemical synthesis of RNAs monomers may have foreshadowed its selective inclusion into life. However, alternative scenarios have been proposed,[6] and it remains unclear whether RNA was the first genetic polymer to emerge during the evolution of life,[7] therefore alternatives must be evaluated. The structure and composition of a nucleic acids backbone is a key property that dictates its ability to form a Watson–Crick duplex, and is therefore crucial to heritable information transfer. Selective Prebiotic Synthesis of a-Threofuranosyl Cytidine by Photochemical Anomerization B W F C l ill d M tth W P * Threose nucleic acid (TNA), built from a 4-carbon tetrose sugar, was identified as a potential progenitor to RNA (Figure 1).[8] TNA can form stable duplexes with not only itself but also with RNA and DNA.[8–10] Although TNAs backbone con- nectivity results from a truncated 3’-2’-phosphodiester linkage (whereas RNA and DNA are linked by 5’-3’-phosphodiest- ers), TNAs trans-vicinal phosphodiesters can stretch into a conformation that can accommodate Watson–Crick base pairing (Figure 1). Homo-duplexes of TNA are more hydro- lytically stable than RNA, instead comparable to DNA.[11–14] Mixed pools of TNA and RNA monomers could form chimeric oligomers, which have been shown to promote the formation of homogeneous oligomers during template- directed ligation.[15] TNAs can also fold into tertiary struc- tures that bind targets with high affinity and specificity.[16] These properties of TNA have enabled the in vitro evolution of TNAs, which can be synthesized by DNA poly- merases,[10,17–19] RNA-dependent RNA polymerases,[20] and even TNA polymerases.[21] (whereas RNA and DNA are linked by 5’-3’-phosphodiest- ers), TNAs trans-vicinal phosphodiesters can stretch into a conformation that can accommodate Watson–Crick base pairing (Figure 1). Homo-duplexes of TNA are more hydro- lytically stable than RNA, instead comparable to DNA.[11–14] Mixed pools of TNA and RNA monomers could form chimeric oligomers, which have been shown to promote the formation of homogeneous oligomers during template- directed ligation.[15] TNAs can also fold into tertiary struc- tures that bind targets with high affinity and specificity.[16] These properties of TNA have enabled the in vitro evolution of TNAs, which can be synthesized by DNA poly- merases,[10,17–19] RNA-dependent RNA polymerases,[20] and even TNA polymerases.[21] Despite TNAs remarkable pairing properties and recent progress toward the chemical synthesis of RNA mono- mers,[2,22–30] the prebiotic synthesis of TNAs monomers has yet to be thoroughly evaluated. TNA is a simpler structure than RNA, with one fewer chiral carbon atom in its sugar moiety, and conceptually, TNAs sugar moiety can be assembled from two achiral C2 building blocks (whereas RNAs sugar moiety requires one C2 and one chiral C3 building block).[8] TNA nucleosides (adenosine, inosine, uridine) have been observed in glycosylation reactions,[25,26] but these glycosylation reactions require preformed nucleo- bases and invoke formose-type reactions to deliver separately formed tetrose sugars. [] B. W. F. Colville, Prof. Dr. M. W. Powner Department of Chemistry, UCL 20 Gordon Street, London, WC1H 0AJ (UK) E-mail: matthew.powner@ucl.ac.uk Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.202101376. 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH. 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. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angewandte Chemie Angewandte Chemie Communications Communications How to cite: International Edition: doi.org/10.1 German Edition: doi.org/10.1 How to cite: International Edition: doi.org/10.1002/anie.202101376 German Edition: doi.org/10.1002/ange.202101376 How to cite: International Edition: doi.org/10.1002/anie.202101376 German Edition: doi.org/10.1002/ange.202101376 Prebiotic Chemistry Hot Paper Prebiotic Chemistry Hot Paper Communications these (RNA, ANA, DNA) syntheses has been common aminooxazoline intermediates, and it is of note that tetrose aminooxazolines (i.e., threose aminooxazoline, TAO and erythrose aminooxazoline, EAO) can also be efficiently synthesized by the same reactions that yield pentose amino- oxazolines (e.g., ribose aminooxazoline, RAO) (Figure 2).[31] Moreover, we have previously demonstrated that equimolar reaction of 2AO with C2 and C3 sugars (glycolaldehyde 5, glyceraldehyde, and dihydroxyacetone) significantly favours synthesis of TAO over RAO (7:1, see ref. [27] and Supple- mentary Figure 19 & 20 therein). Therefore, we envisaged a prebiotic synthesis that yields TAO would in turn provide access to TNA.[22,32] competing cyclization to oxazolidinones, via attack of the C2’- hydroxy on the C2-position of the base, was problematic for pyrimidine ribonucleoside anomerization.[33] Sutherland and co-workers have recently demonstrated that irradiation (254 nm) of a-ribo-thiocytidine in neutral water with hydro- gen sulfide gave a much higher photoanomerization yield of thiocytidine 2 than previously observed for anomerizations of cytidines,[24] even without blocking the 2’-hydroxy moiety to prevent oxazolidinone formation.[41,42] Proposed TNA pre- cursor aminooxazolines (e.g., TAO) have been shown to form alongside those aminooxazolines that can yield RNA,[31] but the transformation of TAO to TNA has not been reported. Accordingly, we set out to investigate the protecting-group- free, prebiotic synthesis of threo-cytidine 3 by UV-light driven photoanomerization (Figure 2). Irradiation with UV-light provides a powerful mechanism to bring about molecular change in prebiotic chemis- try,[2,22,24,28,30,33,34] and UV-light would have been an important energy source on the prebiotic Earth.[35] The prebiotic Earths atmosphere is predicted to have been significantly more transparent to UV-light, due to lower O2/O3 concentration than the modern atmosphere, leading to more UV-light (l > 204 nm) irradiating the surface of Earth.[36–39] Pioneering work by Orgel and co-workers demonstrated that irradiation of the unnatural a-anomer of cytidine gave a 4% yield of the natural b-anomer 1.[40] While low yielding, this demonstrated the plausibility of photochemical anomerization for prebiotic cytidine syntheses. Communications Further studies have revealed that the Previous work has established that cyanamide 4 and glycolaldehyde 5 react efficiently to form 2-aminooxazole 2AO (> 80%)[22] and that tetrose aminooxazolines, TAO and EAO, can be formed in high yields (> 90%) from the reaction of 2AO with glycolaldehyde 5 (Figure 2).[27,31,32] Here, we also observed the reaction of TAO and EAO, with cyanoacetylene (HC3N),[43,44] to give near-quantitative conversion to their respective anhydronucleosides, 6 and 7 (Figure 3 and Supple- mentary Figure 1). We next turned our attention to the synthesis of tetrose nucleoside 3. We envisaged H2S addition Figure 2. Divergent synthesis of RNA and TNA cytidines through the reaction of cyanamide with prebiotically plausible aldehydes. The five- carbon RNA backbone requires sequential reaction of glycolaldehyde 5 (blue) and glyceraldehyde (red) generating four furanosyl pentose aminooxazolines (RAO, AAO, XAO, and LAO) and one pyranosyl pentose aminooxazolines (p-LAO). The four-carbon TNA backbone requires only glycolaldehyde 5 (blue) and only generates two furanosyl isomers (EAO and TAO). Figure 3. Synthesis of tetrose nucleosides by photochemical anomeri- zation. Tetrose aminooxazolines (EAO and TAO) react with cyanoace- tylene (HC3N) to yield tetrose 2,2’-anhydrocytidines 6 and 7. Reversible thiolysis of 6 and 7, followed by selective photoanomerization of the 1’,2’-cis-nucleosides 9 and 10 yields 1’,2’-trans-nucleosides 8 and 11. Oxidative hydrolysis of trans-1’,2’-thiocytidine 8 and 11 furnishes cytidines 3 and 15, whereas oxidation of cis-1’,2’-thiocytidines 9 and 10 furnishes anhydrocytidines 6 and 7. Figure 2. Divergent synthesis of RNA and TNA cytidines through the reaction of cyanamide with prebiotically plausible aldehydes. The five- carbon RNA backbone requires sequential reaction of glycolaldehyde 5 (blue) and glyceraldehyde (red) generating four furanosyl pentose aminooxazolines (RAO, AAO, XAO, and LAO) and one pyranosyl pentose aminooxazolines (p-LAO). The four-carbon TNA backbone requires only glycolaldehyde 5 (blue) and only generates two furanosyl isomers (EAO and TAO). Figure 2. Divergent synthesis of RNA and TNA cytidines through the reaction of cyanamide with prebiotically plausible aldehydes. The five- carbon RNA backbone requires sequential reaction of glycolaldehyde 5 (blue) and glyceraldehyde (red) generating four furanosyl pentose aminooxazolines (RAO, AAO, XAO, and LAO) and one pyranosyl pentose aminooxazolines (p-LAO). The four-carbon TNA backbone requires only glycolaldehyde 5 (blue) and only generates two furanosyl isomers (EAO and TAO). Selective Prebiotic Synthesis of a-Threofuranosyl Cytidine by Photochemical Anomerization B W F C l ill d M tth W P We, and others, have recently eluci- dated prebiotically plausible syntheses of RNA, ANA, and DNA nucleosides which bypass these challenging glycosyla- tion reactions,[22,27–30] and now we extend this work to evaluate this mode of prebiotic synthesis for TNA. A key element of 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH 1 1 These are not the final page numbers! Angewandte Chemi Communications 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021, 60, 1 – 6 Communications Communications Figure 4. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 6.1– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (18 mm) and 10 (16 mm) at pH 7 with H2S (113 mm): a) before the irradiation; and after b) 1 day; c) 2 days; d) 3 days irradiation. Spectrum e) following addition of authentic 8. Spectrum f) following addition of authentic 11. (C6)H and (C1’)H resonances are labelled: 9 (!), 10 (~), 8 (), and 11 (J). to tetrose anhydronucleosides 6 and 7 would regioselectively position a sulfur atom at C2 on the nucleobase and enable photochemical anomerization to yield the TNA monomer 3 (via thiocytidine 8). As anticipated, we observed smooth thiolysis of 6 and 7 to their corresponding b-thiocytidines 9 and 10 in formamide (78%, Figure 3 and Supplementary Figure 2). Therefore, we sought to investigate the stereochemical inversion of the anomeric position for the tetrose series to understand if this would enable delivery of TNA monomers. We then envisaged the C2-sulfur would be removed by a facile oxidation,[28] or by hydrolysis,[24] to yield the canonical nucleobase. After 1 day of irradiation with UV-light (254 nm) in water at pH 7 with H2S (26 mm) we observed conversion of b-threo- thiocytidine 9 (25 mm) to a-threo-thiocytidine 8 (46%). After 1.5 days irradiation a-threo-thiocytidine 8 (51%) was observed to be the major product (Supplementary Figure 6). Spiking with an authentic standard of 8 (See Supplementary Information for synthesis of authentic 8) confirmed the effective photochemical anomerization. Concomitant photoanomerization of threo- and erythro- thiocytidine 9 and 10 was observed to occur at a similar rate (Figure 4), to furnish 8 (40%) and 11 (42%) after 1 day. Maximum conversion was observed after 2 days (8 52%, 11 51%). Conversely, when 8 and 11 were irradiated (254 nm) in neutral water for 3 days with hydrogen sulfide we observed significant formation of 2-thiocytosine 12 from both a- isomers, but interestingly little reversion to the b-anomers (Supplementary Figure 14). As anticipated the sulfur atom at C2 can be facilely removed, to generate the canonical nucleobase. For example, reaction with hydrogen peroxide[28] rapidly converted 8 to the TNA nucleoside, a-threo-cytidine 3, in quantitative yield (Supplementary Figure 4). www.angewandte.org Angew. Chem. Int. Ed. 2021, 60, 1 – 6 These are not the final page numbers! These are not the final page numbers! Angewandte Chemie Angewandte Chemi Communications Interestingly, however, cis-1’,2’- isomers 9 and 10 were observed to rapidly cyclize to reform their corresponding anhydronucleoside 6 and 7 (Figure 3 & Supplementary Figure 3) upon exposure to hydrogen perox- ide, rather than undergo conversion to their respective cytidine hydrolysis products. Encouraged by the anomerization of tetrose thiocytidines, we next considered the role and timing of phosphorylation in the sequence towards TNA. Phosphorylation of prebiotic nucleosides has been demonstrated in the dry state with urea[45] and we sought to utilize this simple phosphorylation in our work. In the ribo-series, photoanomerization and con- version of thiocytidine 2 to cytidine 1 must occur prior to phosphorylation.[24] These constraints are imposed by the formation of 2’,3’-cyclic phosphates which, when in a trans- configuration with respect to the 2-thiocytidine nucleobase, undergo (destructive) cyclization to 2,2’-thioanhydronucleo- tides.[29] We envisage that this process, in the tetrose cytidines, could furnish the selectivity required to synthesize the threo- isomer (i.e. TNA cytidine) by selective destruction of the erythro-isomer of thiocytidine. Figure 4. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 6.1– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (18 mm) and 10 (16 mm) at pH 7 with H2S (113 mm): a) before the irradiation; and after b) 1 day; c) 2 days; d) 3 days irradiation. Spectrum e) following addition of authentic 8. Spectrum f) following addition of authentic 11. (C6)H and (C1’)H resonances are labelled: 9 (!), 10 (~), 8 (), and 11 (J). after undergoing photoanomerization from b-anomer to a- anomer, attack the C2’-position of the sugar moiety, leading to nucleotide degradation. Pleasingly, upon irradiation we observed rapid destruction of cyclic phosphate 13; 2-thiocy- tosine 12 was the sole observed cytosine product after irradiating 13 for 2 days (Supplementary Figure 18). We next irradiated a mixture of erythro-cyclic phosphate 13 and threo-9; after 3 days 8 was the major nucleoside (47%), whilst 13 was observed to degrade in only 1 day (Figure 6 and Supplementary Figures 16 and 17), demonstrating that irra- b-erythro-thiocytidine 10 underwent highly selective urea- mediated phosphorylation,[23,45] with only one equivalent of ammonium dihydrogen phosphate, to yield 2’,3’-cyclic phos- phate 13 as the major product (Figure 5A and Supplementary Figure 15). We anticipated that the C2-sulfur of 13 would, 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021, 60, 1 – 6 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org Irradiation of this mixture of acyclic phosphates leads to anomerization of b- threo to a-threo nucleotides, which was unambiguously confirmed (following treatment with bovine intestinal phos- phatase) by sample spiking with a-threo-thiocytidine 8 (Supplementary Figure 20). However, the high temperature required for urea-mediated phosphorylation resulted in some conversion of 9 back to anhydrocytidine 6 and concomitant phosphorylation and thermal rearrangement to yield photo- stable a-erythro-cytidine-2’,3’-cyclic phosphate 14 (Supple- mentary Figure 20). Figure 6. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 5.85– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (12 mm) and 13 (12 mm) at pH 7 with H2S (109 mm): a) before the irradiation; and after b) after 1 day; c) 2 days; d) 3 days; e) 4 days irradiation. Spec- trum f) following addition of authentic 8. Spectrum g) following addition of authentic 12. (C6)H and (C1’)H resonances are labelled for 9 (!), 13 (~), and 8 (). (C6)H and (C5)H resonances are labelled for 12 (J). Figure 6. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 5.85– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (12 mm) and 13 (12 mm) at pH 7 with H2S (109 mm): a) before the irradiation; and after b) after 1 day; c) 2 days; d) 3 days; e) 4 days irradiation. Spec- trum f) following addition of authentic 8. Spectrum g) following addition of authentic 12. (C6)H and (C1’)H resonances are labelled for 9 (!), 13 (~), and 8 (). (C6)H and (C5)H resonances are labelled for 12 (J). are photostable. A milder phosphorylation strategy would be required to further enhance the observed selection in the intermediate thiocytidines and avoid conversion of 9 to 14.[46] However, the trans-disposition of hydroxy groups in cytidine 3 means that phosphorylation cannot yield a 2’,3’-phosphate, whereas the cis-disposition of hydroxy groups in 11 (and its cytidine congener 15) mean that 2’,3’-phosphate formation is inevitable in erythro-nucleotides. Therefore, the sluggish chemical reactivity of 2’,3’-cyclic phosphates[47–51] may provide the final selection required, during polymer synthesis, to selectively incorporate TNA into nucleic acids. Whilst 3 can be chemically activated to polymerisation (for example, as an imidazolide[52,53]), activation in the erythro-isomer 15 will be Our results demonstrate the TNA monomers can be accessed by the same photochemical strategy as RNA monomers. We also observed that, following phosphorylation and irradiation, threose thiocytidine 8 can be photochemically selected. www.angewandte.org 3 3 3 3 These are not the final page numbers! diation, when coupled with erythro-specific 2’,3’-cyclic phos- phate synthesis allows the photochemical selection of threo Figure 5. Photochemical selection of threo-thiocytidine induced by erythro-specific 2’,3’-cyclic phosphate synthesis. A) Urea-mediated phosphorylation of erythro-thiocytidine 10 yields cyclic phosphate 13 in near quantitative yield. 13 is observed to undergo rapid and complete photodegradation to free base 12. B) Urea-mediated phosphorylation of b-threo-thiocytidine 9 yields a mixture of b-threo-thiocytidine phos- phates that photoanomerize to their a-anomers. Annulation of 9 is also observed during urea-mediated phosphorylation to yield anhydro- nucleoside 6, which phosphorylates and rearranges to yield photo- stable erythro-cytidine 14. R=H or (PO3 )n. Angewandte Chemie Communications coupled with erythro-specific 2’,3’-cyclic phos- is, allows the photochemical selection of threo- from a mixture of erythro- and threo-thiocyti- ,3’-trans-configuration of threose means that ides cannot form 2’,3’-cyclic phosphates, and A thiocytidine is inherently protected from ive reactions. Phosphorylation of b-threo-thio- nnot furnish b-threo-2’,3’-cyclic phosphates, but lds a mixture of nucleoside phosphates (Fig- Supplementary Figure 20). Irradiation of this yclic phosphates leads to anomerization of b- hreo nucleotides, which was unambiguously ll i t t t ith b i i t ti l h hemical selection of threo-thiocytidine induced by 2’,3’-cyclic phosphate synthesis. A) Urea-mediated of erythro-thiocytidine 10 yields cyclic phosphate 13 in e yield. 13 is observed to undergo rapid and complete n to free base 12. B) Urea-mediated phosphorylation tidine 9 yields a mixture of b-threo-thiocytidine phos- toanomerize to their a-anomers. Annulation of 9 is uring urea-mediated phosphorylation to yield anhydro- hich phosphorylates and rearranges to yield photo- tidine 14. R=H or (PO3 )n. Figure 6. 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 5.85– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (12 mm) and 13 (12 mm) at pH 7 with H2S (109 mm): a) before the irradiation; and after b) after 1 day; c) 2 days; d) 3 days; e) 4 days irradiation. Spec- trum f) following addition of authentic 8. Spectrum g) following addition of authentic 12. (C6)H and (C1’)H resonances are labelled for 9 (!), 13 (~), and 8 (). (C6)H and (C5)H resonances are labelled for 12 (J). Angewandte Chemie Communications Communications Communications Communications Figure 6. www.angewandte.org 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH These are not the final page numbers! www.angewandte.org 1H NMR (700 MHz, noesygppr1d, H2O/D2O (9:1), 5.85– 8.3 ppm) spectra showing the irradiation (254 nm) of 9 (12 mm) and 13 (12 mm) at pH 7 with H2S (109 mm): a) before the irradiation; and after b) after 1 day; c) 2 days; d) 3 days; e) 4 days irradiation. Spec- trum f) following addition of authentic 8. Spectrum g) following addition of authentic 12. (C6)H and (C1’)H resonances are labelled for 9 (!), 13 (~), and 8 (). (C6)H and (C5)H resonances are labelled for 12 (J). Angewandte Chemie nications Figure 5. Photochemical selection of threo-thiocytidine induced by erythro-specific 2’,3’-cyclic phosphate synthesis. A) Urea-mediated phosphorylation of erythro-thiocytidine 10 yields cyclic phosphate 13 in near quantitative yield. 13 is observed to undergo rapid and complete photodegradation to free base 12. B) Urea-mediated phosphorylation of b-threo-thiocytidine 9 yields a mixture of b-threo-thiocytidine phos- phates that photoanomerize to their a-anomers. Annulation of 9 is also observed during urea-mediated phosphorylation to yield anhydro- nucleoside 6, which phosphorylates and rearranges to yield photo- stable erythro-cytidine 14. R=H or (PO3 )n. diation, when coupled with erythro-specific 2’,3’-cyclic phos- phate synthesis, allows the photochemical selection of threo- thiocytidine 8 from a mixture of erythro- and threo-thiocyti- dines. The 2’,3’-trans-configuration of threose means that TNA nucleosides cannot form 2’,3’-cyclic phosphates, and therefore TNA thiocytidine is inherently protected from these destructive reactions. Phosphorylation of b-threo-thio- cytidine 9 cannot furnish b-threo-2’,3’-cyclic phosphates, but reversibly yields a mixture of nucleoside phosphates (Fig- ure 5B and Supplementary Figure 20). Irradiation of this mixture of acyclic phosphates leads to anomerization of b- threo to a-threo nucleotides, which was unambiguously confirmed (following treatment with bovine intestinal phos- phatase) by sample spiking with a-threo-thiocytidine 8 (Supplementary Figure 20). However, the high temperature required for urea-mediated phosphorylation resulted in some conversion of 9 back to anhydrocytidine 6 and concomitant phosphorylation and thermal rearrangement to yield photo- stable a-erythro-cytidine-2’,3’-cyclic phosphate 14 (Supple- mentary Figure 20). diation, when coupled with erythro-specific 2’,3’-cyclic phos- phate synthesis, allows the photochemical selection of threo- thiocytidine 8 from a mixture of erythro- and threo-thiocyti- dines. The 2’,3’-trans-configuration of threose means that TNA nucleosides cannot form 2’,3’-cyclic phosphates, and therefore TNA thiocytidine is inherently protected from these destructive reactions. Phosphorylation of b-threo-thio- cytidine 9 cannot furnish b-threo-2’,3’-cyclic phosphates, but reversibly yields a mixture of nucleoside phosphates (Fig- ure 5B and Supplementary Figure 20). g 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org However, this selection is only feasible in the thiocytidine intermediates, as b-erythro-cytidines, such as 14, Angew. Chem. Int. Ed. 2021, 60, 1 – 6 www.angewandte.org 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Keywords: nucleotides · origin of life · photochemistry · prebiotic chemistry · TNA Keywords: nucleotides · origin of life · photochemistry · prebiotic chemistry · TNA [33] M. W. Powner, C. Anastasi, M. A. Crowe, A. L. Parkes, J. Raftery, J. D. Sutherland, ChemBioChem 2007, 8, 1170 – 1179. [34] N. Miller, P. Cerutti, Proc. Natl. Acad. Sci. USA 1968, 59, 34 – 38. [35] P. B. Rimmer, J. Xu, S. J. Thompson, E. Gillen, J. D. Sutherland, D. 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W. Powner, Nat. Chem. 2017, 9, 584 – 589. We thank Volkswagen Foundation (94743), Simons Founda- tion (318881FY19, 493895), Leverhulme Trust (RPG-2019- 214), and EPSRC (EP/K004980/1, EP/P020410/1) for finan- cial support, K. Karu, M. Puchnarewicz (UCL Mass Spec- trometry Facility), A. E. Aliev (NMR spectroscopy), S. Islam, and D. Whitaker for helpful discussions and feedback. [28] S. J. Roberts, R. Szabla, Z. R. Todd, S. Stairs, D.-K. Bucˇar, J. Sˇponer, D. D. Sasselov, M. W. Powner, Nat. Commun. 2018, 9, 4073. [29] J. Xu, N. J. Green, C. Gibard, R. Krishnamurthy, J. D. Suther- land, Nat. Chem. 2019, 11, 457 – 462. [30] J. Xu, V. Chmela, N. J. Green, D. A. Russell, M. J. Janicki, R. W. Gra, R. Szabla, A. D. Bond, J. D. Sutherland, Nature 2020, 582, 60 – 66. www.angewandte.org Angew. Chem. Int. Ed. 2021, 60, 1 – 6 4 T These are not the final page numbers! Angewandte Chem Communications [22] M. W. Powner, B. Gerland, J. D. Sutherland, Nature 2009, 459, 239 – 242. quenched by formation of 2’,3’-cyclic phosphate 14. We have demonstrated that prebiotic photoanomerization can selec- tively furnish TNA cytidine, and our results warrant further investigation of selective TNA synthesis in the context of the origins of life. [23] S. Stairs, A. Nikmal, D.-K. Bucˇar, S.-L. Zheng, J. W. Szostak, M. W. Powner, Nat. Commun. 2017, 8, 15270. [24] J. Xu, M. Tsanakopoulou, C. J. Magnani, R. Szabla, J. Sˇponer, R. W. Gra, J. D. Sutherland, Nat. Chem. 2017, 9, 303 – 309. R. W. Gra, J. D. Sutherland, Nat. Chem. 2017, 9, 303 – 3 [25] S. Becker, I. Thoma, A. Deutsch, T. Gehrke, P. Mayer, H. Zipse, T. Carell, Science 2016, 352, 833 – 836. The authors declare no conflict of interest. The authors declare no conflict of interest. The authors declare no conflict of interest. [32] S. Islam, J. A. Aguilar, M. W. Powner, M. Nilsson, G. A. Morris, J. D. Sutherland, Chem. Eur. J. 2013, 19, 4586 – 4595. Keywords: nucleotides · origin of life · photochemistry · prebiotic chemistry · TNA Conflict of interest [31] C. Anastasi, M. A. Crowe, M. W. Powner, J. D. Sutherland, Angew. Chem. Int. Ed. 2006, 45, 6176 – 6179; Angew. Chem. 2006, 118, 6322 – 6325. Angew. Chem. Int. Ed. 2021, 60, 1 – 6 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH 6 www.angewandte.org 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021, 60, 1 – 6 These are not the final page numbers! Angew. Chem. Int. Ed. 2021, 60, 1 – 6 Keywords: nucleotides · origin of life · photochemistry · prebiotic chemistry · TNA Am. Chem. Soc. 2005, 127, 7427 – 7434. [20] D. P. Horning, S. Bala, J. C. Chaput, G. F. Joyce, ACS Synth. Biol. 2019, 8, 955 – 961. Manuscript received: January 28, 2021 Accepted manuscript online: February 28, 2021 Version of record online: && &&, &&&& [21] N. Chim, C. Shi, S. P. Sau, A. Nikoomanzar, J. C. Chaput, Nat. Commun. 2017, 8, 1810. Angew. Chem. Int. Ed. 2021, 60, 1 – 6 Angew. Chem. Int. Ed. 2021, 60, 1 – 6 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH 5 5 Communications Prebiotic Chemistry B. W. F. Colville, M. W. Powner* &&&&—&&&& Selective Prebiotic Synthesis of a- Threofuranosyl Cytidine by Photochemical Anomerization TNA cytidine, a candidate precursor to RNA cytidine at the origin of life, is syn- thesized from prebiotic precursors via a high-yielding, regio- and stereo-selec- tive, protecting-group free synthesis uti- lizing an efficient photochemical anome- rization. Phosphorylation also enables selective photodestruction of the con- taminating erythro-diastereomer enrich- ing any potential pool of TNA monomers. Angewandte Chemie Communications Angewandte Chemi Communications Communications TNA cytidine, a candidate precursor to RNA cytidine at the origin of life, is syn- thesized from prebiotic precursors via a high-yielding, regio- and stereo-selec- tive, protecting-group free synthesis uti- lizing an efficient photochemical anome- rization. Phosphorylation also enables selective photodestruction of the con- taminating erythro-diastereomer enrich- ing any potential pool of TNA monomers. lizing an efficient photochemical anome- rization. Phosphorylation also enables selective photodestruction of the con- taminating erythro-diastereomer enrich- ing any potential pool of TNA monomers.
https://openalex.org/W3129709824	https://eprints.whiterose.ac.uk/173884/1/rme-2020-0164.pdf	English	null	Spatiotemporal readiness is key to preparing regenerative medicine for the clinic	Regenerative medicine	2,021	cc-by	4,918	Article: Article: Stephens, N., Morrison, M., Martin, P. et al. (1 more author) (2021) Spatiotemporal readiness is key to preparing regenerative medicine for the clinic. Regenerative Medicine, 16 (3). pp. 229-235. ISSN 1746-0751 https://doi.org/10.2217/rme-2020-0164 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ This is a repository copy of Spatiotemporal readiness is key to preparing regenerative medicine for the clinic. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/173884/ Version: Published Version Version: Published Version Commentary For reprint orders, please contact: reprints@futuremedicine.com Spatiotemporal readiness is key to preparing regenerative medicine for the clinic Neil Stephens,1 , Michael Morrison2,5 , Paul Martin3,6 & Linda Hogle4 1Social & Political Sciences, Brunel University London, Kingston Lane, Uxbridge, UB8 3PH, UK 2Centre for Health, Law & Emerging Technologies, The Faculty of Law, University of Oxford, St Cross Building, St Cross Road, Oxford, OX1 3UL, UK 3Department of Sociological Studies, University of Sheffield, Elmfield, Northumberland Road, Sheffield, S10 2TU, UK 4University of Wisconsin School of Medicine and Public Health, Health Sciences Learning Center, 750 Highland Ave., M 53705, 608-263-4900, USA 3Department of Sociological Studies, University of Sheffield, Elmfield, Northumberland Road, Sheffield, S10 2TU, UK 4University of Wisconsin School of Medicine and Public Health, Health Sciences Learning Center, 750 Highland Ave., Madison, WI 53705, 608-263-4900, USA 5Institute for Science, Innovation and Society, University of Oxford, 64 Banbury Road, Oxford, OX2 6PN, UK 6iHuman, Interdisciplinary Centre of the Social Sciences, University of Sheffield, 219 Portobello, Sheffield, S1 4DP, UK Author for correspondence: neil.stephens@brunel.ac.uk “Spatiotemporal readiness is key to the deployment of emergent regenerative medicine therapies in clinical settings and requires ongoing attention.” Tweetable abstract: Successful translation of regenerative medicine projects to the clinic requires atten- tion to the complex interaction of spatial and timing issues from manufacturing to clinical use. First draft submitted: 14 October 2020; Accepted for publication: 3 February 2021; Published online: 24 February 2021 Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ 10.2217/rme-2020-0164 C⃝2021 Neil Stephens ywords: cell manufacturing • clinical integration • infrastructure • institutional readiness • space • timing Keywords: cell manufacturing • clinical integration • infrastructure • institutional readiness • space • timing As regenerative medicine products scale up, researchers turn their attention beyond the ongoing technical challenges of collecting, preserving and delivering gene- and cell-based products to manufacturing and business operations, including process control, automation, supply chain and logistics, and the viability of business model alternatives. Assessing readiness for clinical use, however, requires an integrated assessment across all of these crucial components, including how well they ﬁt together (or not), how synergies might enable platform opportunities and, critically, what kinds of infrastructures should be created to support a range of cell- and tissue-based products and services. Infrastructures reveal the way that futures are anticipated while shaping things in the present [1]. Yet large investments, including major changes to physical facilities, organizational structures and processes, can make it difﬁcult to change things later [2]. There is much at stake, then, for institutional readiness in terms of having robust, yet adaptable, infrastructures. We suggest that analysis of the spatial and temporal aspects of infrastructure is a useful way to take a more integrated view across all manufacturing phases, participants and components of regenerative medicine when assessing institutional readiness. We use the concept of institutional readiness [3] as a way to frame the interrelation of material technologies, knowledge bases and social and economic factors affecting translation. We focus on the UK and use the term ‘advanced therapy medicinal products’ to encompass the variety of cell-, tissue- and gene-based therapies that involve altering biological characteristics. Although it is self-evident that there are speciﬁc spatial and temporal needs for different cell types and delivery models, we discuss major planning issues that all share. Our comments derive from an ongoing Wellcome Trust-funded workshop series organized by the authors. The workshops bring together social scientists and biomedical professionals actively developing cell- and tissue- based products with the aim of learning how intended applications and commercialization strategies interact with knowledge production systems and the broader socioeconomic environment. Ongoing workshops center on emerging technological, ﬁnancial and organizational infrastructures and complex production and scale-up techniques and how these interact with regulatory and policy arenas as well as the development of novel business models and valuation strategies. A core group of seven social scientists, all with expertise in science and technology ISSN 1746-0751 229 10.2217/rme-2020-0164 C⃝2021 Neil Stephens Regen. Med. (2021) 16(3), 229–235 Commentary Stephens, Morrison, Martin & Hogle studies, attend all workshops in the series. Each core member has an active portfolio of empirical research regarding cell- and tissue-based products, through which they contribute to the continuing dialogue. Each workshop entails presentations from two invited experts who are actively developing or supporting therapeutic interventions, with in-depth, interactive discussions of speciﬁc challenges they face regarding scale-up. We aim to illuminate challenges that may be less discussed, yet are common across various applications, as well as unique issues as new research and production alternatives appear. In this article, the speciﬁc contributions and details of individual cases remain anonymous. Our analysis represents the comments of the authors only. This commentary also draws upon related research conducted separately by the individual authors that involves interviews with professionals working on engineered skin, blood and muscle and on clinical translation of gene editing, induced pluripotent stem cells and 3D printing in the UK [4–7]. Signed informed consent was received from all interviewees in these studies prior to interview. Spatiotemporal readiness It is important to consider spatiotemporal readiness as a key dimension of overall organizational readiness. Successful translation of regenerative medicine in clinical contexts requires attention to the interrelatedness of the ‘where’ and ‘when’ questions with costs, capacities and effective outcomes. A recent collaborative report identifying engineering processes and logistics challenges for cost-effective and reproducible manufacturing highlights how space, speed, cost and quality issues cannot be viewed separately, as they mutually shape and inform each other [8]. This view was echoed in our workshops through examples in which apparently mundane logistical where and when issues, such as the design and layout of laboratory and production spaces and how people, materials and information pass through them, proved non-trivial in the effective delivery of cell- and tissue-based products. Effectively achieving spatiotemporal readiness is important for the economic and technical success of these endeavors but also has important bioethical implications. Efﬁcient delivery could make therapies available sooner and more cost-effective for end users and, by incorporating a fairness framework for manufacturing capacity allocation, increases the likelihood that the beneﬁts of regenerative medicine will be available for all who need it most [9]. Spatiotemporal readiness enhances product integrity, beneﬁting manufacturers and human subjects who volunteer for clinical trials. However, appropriate spatiotemporal procedures are highly context-speciﬁc and can vary by technology, patient population and economic environment. Importantly, spatiotemporal readiness encompasses both actual and anticipated where and when issues. Visions of future spatiotemporal infrastructures that later prove inappropriate can impede effective delivery. Multiple potential business models are arising [10,11], each including an anticipated spatiotemporal model. Any business model or manufacturing process that does not accurately account for the spatiotemporal organization of the clinics in which therapeutic products would be applied could face difﬁculties, especially if the process design has locked-in elements that are poorly aligned with clinical practice. This is further complicated by the diversity of product and patient contexts. To date, discussion of aligning commercial, regulatory and clinical factors too often implies that commer- cialization is a linear process, and that readiness will allow an acceleration through various steps [6]. Examining spatiotemporal aspects of translation is one way to show how this linear model can be misleading, as time lines take longer than anticipated and, importantly, become iterative through movements within and between preclinical trials and market authorization. Successful acceleration here requires something other than simply speeding forward toward a chosen goal. Spatiotemporal readiness before the clinic International initiatives for manufacturing scale-up are considering multiple levels of scale, from the organization of laboratory space to the geographies of national and international production and use. Although many institutes are offering current GMP facilities for storage, testing and processing, some national-level and government-supported initiatives, such as Canada’s Centre for Commercialization of Regenerative Medicine and the UK Cell and Gene Therapy Catapult, offer centralized facilities. Regulatory-compliant modular spaces and consulting services are established to bridge nascent innovations from academic inventors and commercial entities with industrialization expertise. The UK catapult, for example, has both a 1200 m2 development center with a conﬁgurable pod system to replicate users’ processes and a 7200 m2 manufacturing center with GMP-compliant but segregated modules designed for ﬂexible use. In such contexts, companies must decide whether locating their manufacturing work in such sites and utilizing these facilities is preferable to developing and using their own operations. Larger companies 230 future science group Regen. Med. (2021) 16(3) Spatiotemporal readiness is key to preparing regenerative medicine for the clinic Commentary Spatiotemporal readiness is key to preparing regenerative medicine for the clinic Commentary are more likely to use in-house facilitates and acquire technology from start-ups. Product sponsors may be operating out of academic centers or facilities acquired in company purchases and must decide whether and how to reorganize space to meet requirements for the products they intend to make. Such decisions may be framed by the legacies of previous company acquisitions, as the many takeovers and mergers seen in the sector lead to companies holding a portfolio of spaces and facilities that may or may not map onto their priorities at any particular time. Emerging infrastructures may be built upon existing infrastructure, which possibly brings forward the risk that old constraints may still be in place and can be insufﬁcient to deal with emergent issues and market conditions. Other cases may see the creation of de novo infrastructure or hybrid infrastructures that seek to combine the new with the existing. Such novelty can be costly and more time-consuming. It also retains the risk that the ﬁrst to create a new mode of operating may or may not be the one who reaps the beneﬁts, as novel radical approaches become reproduced by others once initial problems have been worked through. Alternatively, ﬁrst movers may not end up developing what becomes the industry standard and be left with outdated legacy infrastructures. Spatiotemporal readiness before the clinic As with many therapeutics, ﬁrst in class is rarely best in class. The geographical location of these facilities is also important, as locations near major transport links and areas with readily available ancillary services create an advantage. International transportation can also prove burdensome, as logistical boundaries and regulatory jurisdictions provide additional barriers. One eye-catching example occurred when the team producing the ﬁrst tissue-engineered trachea for transplant was turned away from Bristol Airport in 2008, as easyJet representatives refused to allow the biological material onto the ﬂight, despite assurances in advance it would be allowed, necessitating that £14,000 be spent on a private plane to make the 16 h window for use. Such examples highlight the necessity for regenerative medicine infrastructures to ﬁt within existing infrastructures for transport and other needs. In the UK, future border issues could be exacerbated by the assembly of new administrative borders with EU countries and import and export of biological materials. Even within a single country, deciding how and when to transport material is key. The timing of delivery of a therapeutic product to a patient can be critical, but the appropriate timing of a delivery may be contingent on a patient’s complex and variable condition [12]. For example, autologous keratinocyte-based engineered skin should be transplanted within 24 h of leaving the manufacturing facility. But if a patient’s condition deteriorates during the transit period such that surgery is not possible within that window, the patient’s skin, the company’s resources and critical time for healing are all wasted. Within production facilities, the size and modularity of manufacturing spaces affect workﬂow, labor, materials and sequencing. For smaller facilities, having fewer higher-grade clean rooms creates problems with scheduling and sequence of events. One workshop attendee described the challenges of deriving GMP induced pluripotent stem cell lines, which necessarily require open cell culture and thus can monopolize a grade B clean room of any size for an extended period. Other issues include mundane but necessary tasks like cleaning and maintenance. Such challenges demand a careful choreography of laboratory space usage in planning sequential and side-by-side activities, which can have signiﬁcant ﬁnancial implications when these where and when issues are misaligned. Choice of culture system types and automation also must be considered [13,14]. Closed systems can allow manufacturing to move from grade B to C/D clean rooms, with a likely reduction in cost. Spatiotemporal readiness before the clinic Some manufacturing steps can be automated, but there is currently little speed advantage over manual processes. Automated processing still requires skilled operators. As a result, manufacturing remains expensive in terms of high-cost skilled labor time. Many steps still cannot be automated, nor can they be undertaken in fully closed systems, because of the need to feed and sample cells. There is also a relation between closed systems and the cost of labor and reagents. Spatiotemporal readiness for integrating manufacturing with the clinic The way existing facilities map onto the anticipated spatiotemporal dimensions of a business model is also important. Such models have clear spatial economics at the core of their vision that frame where and when priorities. Many manufacturers, including large pharmaceutical companies, favor a production model based on conventional pharmaceuticals, with a limited number of manufacturing sites, each supplying a standardized advanced therapy product to a wide territorial range. This keeps the cost of expensive manufacturing equipment and sites (e.g., GMP- compliant) down and allows centralized quality management, batch control and sign-off by qualiﬁed persons. This model suits standardized products, whether allogeneic therapies or some autologous therapies where each sample is processed in a comparable way, such as chimeric antigen-receptor T-cell therapies. For many products, localized on-site manufacturing is impractical. The volume and conﬁguration of space would be difﬁcult for many existing facilities; the skills needed for processing, maintenance and even data analytics may not be available; and quality f future science group 231 www.futuremedicine.com Commentary Stephens, Morrison, Martin & Hogle control capacity in healthcare facilities is not oriented toward this work. Some clinics may have the capacity for tasks like drug compounding or certain forms of chemistry, but fewer could effectively deliver processing and distribution of live biomaterials. Those hospitals with existing capacity in precedent procedures, such as bone marrow transplants, may face less disruption in introducing cell-based transplants. However, in many cases there may not be enough patients at clinical sites to warrant investing in personnel, equipment and meeting GMP requirements to make investment in distributed models viable. Techniques of additive manufacturing, however, have given a renewed impetus to redistributed manufacturing in health care [15]. One option is highly localized, ‘surgeon-led’ 3D printing of cellular constructs. This would allow very near-patient manufacturing, which can be important for products with a very short shelf life or those that are not amenable to the freezing and thawing cycles needed for more centralized manufacturing strategies. However, the feasibility depends a great deal on the regulatory classiﬁcation of such products. If bioprinting can be considered minimal manipulation of the cells, then a bio-printed construct might be regarded as a transplant and its products as the practice of medicine, but it is more probable that regulators will regard the construct as a product under the advanced therapy medicinal product regulations (most likely as either ‘tissue-engineered medicines’ or combination products) [16]. Spatiotemporal readiness for integrating manufacturing with the clinic In this case, the same prohibitive cost implications of localized manufacture discussed earlier are likely to apply, with each clinic counting as a discrete manufacturing site. As a result, this pathway may be restricted to one-off patient-speciﬁc implants delivered as ‘hospital exemptions’ or ‘hospital specials.’ These bypass the requirement to secure market approval as a medicinal product, but not the requirement for adequate quality management during manufacture, while also limiting the scalability of the technique. l l ﬁ h h b d k h h l d d An alternative spatial conﬁguration is the ‘hub and spokes’ approach where implants are designed, using computer- aided design software, in a centralized facility with centralized quality control and then transmitted to local sites for bioprinting. This looks more like conventional manufacturing in terms of scalability and manufacturing site costs but raises issues regarding the security of transmitted computer-aided design information, which is unavoidably conﬁdential medical data about a patient, and the distribution of liability among the various computer engineers, bioprinter operators, qualiﬁed persons, surgeons and manufacturers of components such as bio-inks and scaffolds that may be utilized to create the ﬁnal implant [16]. In these distrusted manufacturing models, it is not the bio- printed implant that is commercialized but the equipment, protocols and capacity to produce the implant, which is a rather different business model. Readiness here transcends individual institutions and involves a higher-level issue of the capacity of national legal systems (regulation but also issues of manufacturing liability) to support or constrain additive manufacturing. Another model that could apply to both bioprinting and more conventional regenerative medicines involves the creation of specialist centers of excellence (mainly university hospitals). This approach has (some of) the beneﬁts of centralization but is more distributed. Here it would be important to ensure the physical separation of the hospital from the manufacturing site. This centers-of-excellence model requires less upskilling of healthcare staff than a general distribution model and can be used as a test bed to identify key drivers of institutional readiness and work out how best to align academic, industry and hospital systems, with a view to rolling this knowledge out to other sites in the future. This is the approach currently being pursued by the three advanced therapy treatment centers in the UK that operate within the framework of the National Health Service but span academia, small biotechnology companies and pharmaceutical multinationals [17]. Spatiotemporal readiness for integrating manufacturing with the clinic Whatever model is adopted will have profound implications for the development pipeline adopted by companies and their collaborators. As infrastructures become stabilized in particular places, this will dictate the temporal ﬂow of products through these manufacturing systems and place constraints on life cycle management and the creation of next-generation products. Spatiotemporal readiness and the economics of clinical practice It is also vital that spatiotemporal aspects of the operations and business models of hospitals are assessed together with those of the product sponsors. Key issues include where procedures will be performed, how much investment will go into adopting new technologies and the burdens of modifying facilities to support them. The high costs associated with one-off complex therapies do not align well with the periodicity of healthcare budgets, which typically have ﬁxed annual limits. In many countries, hospital cost and revenue models are rapidly changing with the uptake of value-based models. This shift from cost-per-unit thinking to one contingent on clinical outcomes aims to balance healthcare costs with quality, with risk-sharing contracts negotiated between payers and product suppliers. Value-based contracts are being used for high-cost therapies with highly variable outcomes, such as gene therapies [18,19]. Yet this poses 232 Regen. Med. (2021) 16(3) future science group Table 1. Challenges associated with spatiotemporal readiness. Key planning themes Core potential issues Laboratory design and use Efficient use of space; availability of space at the right time; appropriate sizing for application at hand; maintenance scheduling. In-house or external manufacturing facilities Cost; flexibility; speed to start; control and longevity. Movement between sites Speed and shelf life; security; border crossings; trade and regulatory hurdles; route reliability. Adoption of automated systems Deciding when a process is ready to be locked into automation may sacrifice flexibility; securing regulatory approval; potential for portability of closed systems and required expertise. Clinic integration and proximity What level of integration is appropriate for each specific product and each specific clinic; investments and expertise requirements; when to enter the clinic. Regulatory alignment Regulatory category fit; timing of submission; proactive actions for undefined future pathways; unique production complexities (e.g., distributed additive manufacturing). Payment Structuring reimbursement systems with the rhythms and needs of both companies and clinics; (mis)alignment of therapeutic impact and payment model (e.g., with value-based systems). Table 1. Challenges associated with spatiotemporal readiness. particular challenges for regenerative medicine. Outcomes may take considerable time to become apparent for some advanced therapy medicinal product treatments and are more difﬁcult to measure than non-cell-based interventions. This stretches the time line to assess success far beyond the time of treatment, with implications for healthcare institutions and payers as well as product sponsors [7]. Spatiotemporal readiness and the economics of clinical practice Furthermore, recent adoption of alternative ‘real-world evidence’ (used in review of fast-tracked regenerative medicine advanced therapies in the US and elsewhere) calls for monitoring data through patient registries for years post-treatment. How government and commercial payers will build and utilize such evidentiary alternatives to conventional proofs of efﬁcacy and safety will be key to value-based contract negotiations. g Time lines for fund ﬂows are also affected, with differing implications for various stakeholders. Alternative payment schemes have been proposed for high-cost treatments with potentially highly variable outcomes. Annuity or staggered payments involve an instalment time line that can be preferable from a payer’s perspective to spread risk over time [20]. However, such payments may be less amenable to cash–poor small- and mid-size enterprises needing rapid returns to satisfy investors. Value-based payments based on clinical performance also introduce uncertainty for any product sponsor, as the time line and level of payment may be harder to predict and may not meet initial expectations, depending upon outcome. Such possibilities highlight the relationship between business model choices and anticipated payment time lines, as assumptions of future market conditions shape decisions made in the present. Alternative payment schedules may also cause problems for both payer and healthcare facility ﬁnancial reporting using existing cost accounting infrastructures [18]. Novel infrastructures for tracking patients, monitoring outcomes and distributing ﬁnancial reward or liability will thus require further analysis, development and investment. At present, it is unclear who should do this and how such infrastructures should be governed. This may require novel forms of public–private partnership. To address these infrastructure misalignments, both healthcare institutions and innovators need to be resilient, agile and willing to change. However, this may conﬂict with the need to standardize and build viable platforms and requires attentiveness to what processes or ways of thinking become locked in, for how long and what impact this may have. As such, questions of where and when to standardize or be adaptive arise. Acknowledgments The authors thank the participants in our workshops and interviews for their involvement. Financial & competing interests disclosure The authors acknowledge the funding of the Wellcome Trust (WT218807/Z/19/Z, WT208198/Z/17/Z and WT219875/Z/19/Z), the UK Economic and Social Research Council (ES/P002943/1) and the Leverhulme Trust (RPG-2017-330) for the financial support that made this work possible. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. No writing assistance was utilized in the production of this manuscript. Conclusion Our discussions with researchers, both in workshops and individual projects, made clear the ways both actual and anticipated spatiotemporal issues shape, enable and constrain actions. We urgently need to take where and when questions seriously and to recognize their complexities as nonlinear processes. In Table 1, we present some of the key themes institutions planning commercialization should consider in relation to spatiotemporal readiness and the issues these provoke. These and other issues are likely to arise, challenging both actual and anticipated spatiotemporal activities. Focusing on spatiotemporal aspects implies recognizing infrastructure development as a ﬂuid process that requires constant planning, negotiation and maintenance. This attunes us to the interactivity of economic, infrastructural, technical and regulatory aspects, as they inﬂuence and shape each other in complex ways across geographies and time lines. It is vital that considerations of spatiotemporal issues are broad and multifaceted, as planning that focuses on phases or processes in isolation misses broader socio-structural issues that affect the infrastructures of regenerative future science group 233 www.futuremedicine.com Commentary Stephens, Morrison, Martin & Hogle medicine. Spatiotemporal readiness is key to the deployment of emergent regenerative medicine therapies in clinical settings and requires ongoing attention. medicine. Spatiotemporal readiness is key to the deployment of emergent regenerative medicine therapies in clinical settings and requires ongoing attention. Open access This work is licensed under the Creative Commons Attribution 4.0 License. To view a copy of this license, visit http://creativecomm ons.org/licenses/by/4.0/ Papers of special note have been highlighted as: • of interest; •• of considerable interest Papers of special note have been highlighted as: • of interest; •• of considerable interest Papers of special note have been highlighted as: • of interest; •• of considerable interest 1. Gupta A. The future in ruins: thoughts on the temporality of infrastructure. In: The Promise of Infrastructure. Anand N, Gup H (Eds). Duke University Press, NC, USA, 62–79 (2018). 2. Morrison M. Targeted policy support for emerging biomedical innovations (2020). www.openaccessgovernment.org/targeted-policy-support-for-emerging-biomedical-innovations/88436/ 2. Morrison M. Targeted policy support for emerging biomedical innovations (2020). www.openaccessgovernment.org/targeted-policy-support-for-emerging-biomedical-innovations/88436/ 3. Webster A, Gardner J. 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https://openalex.org/W2435118883	https://europepmc.org/articles/pmc5190078?pdf=render	English	null	Increased expression of SKP2 is an independent predictor of locoregional recurrence in cervical cancer <i>via</i> promoting DNA-damage response after irradiation	Oncotarget	2,016	cc-by	9,994	Abstract Although radiation therapy was known to be effective to cervical cancer, loco-regional recurrences are frequently found in patients. We aimed to identify a molecular marker predicting the response of cervical cancer to radiotherapy. We included the patients (n = 149) with cervical cancer who had undergone radiotherapy from 2004 to 2006. Tumor samples were collected to examine the association between the expression of S-phase kinase-associated protein 2 (SKP2) and prognosis in cervical cancer. We found higher expression of SKP2 associated with recurrence (HRs: 2.52, p < 0.001), death (HRs: 2.01, p < 0.001) and higher locoregional recurrence rate (HRs: 3.76, p < 0.001). Cervical cancer cell lines with higher expression of SKP2 showed higher colony formation, cell survival rate and fewer DNA damages after irradiation. SKP2-C25, an inhibitor for SKP2 activity, dose-dependently decreased cell viability after irradiation and knockdown of SKP2 impaired DNA-damage response and sensitized the cervical cancer cells to irradiation. Our data showed the SKP2 represents a promising tool to identify patients with cervical cancer who have a higher risk of locoregional recurrence after radiotherapy. Targeting SKP2 may serve as a potential radiosensitizer for developing effective therapeutic strategies against cervical cancer. Hung-Chun Fu1,2,3, Yi-Chien Yang1,2,4, Yun-Ju Chen1,2, Hao Lin3, Yu-Che Ou3, Chan-Chao Chang Chien3, Eng-Yen Huang5, Hsuan-Ying Huang6, Jui Lan6, Hsi-Ping Chi7, Ko-En Huang2,3, Hong-Yo Kang1,2,3 g , g g 1Graduate Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine, Kaohsiung, Taiwan 2 2Center for Menopause and Reproductive Medicine Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan 3Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 2Center for Menopause and Reproductive Medicine Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan 3Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 3Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 4Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 4Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 5Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 5Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 6Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 6Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 7Medical Sciences Division, University of Oxford, Oxford, England, UK Correspondence to: Hong-Yo Kang, email: hkang3@mail.cgu.edu.tw Keywords: SKP2, local recurrence, radioresistance, cervical cancer, DNA damage Received: March 11, 2016 Accepted: May 16, 2016 Published: June 15, 2016 Keywords: SKP2, local recurrence, radioresistance, cervical cancer, DNA damage Received: March 11, 2016 Accepted: May 16, 2016 Published: June 15, 2016 www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget Increased expression of SKP2 is an independent predictor of locoregional recurrence in cervical cancer via promoting DNA-damage response after irradiation Hung-Chun Fu1,2,3, Yi-Chien Yang1,2,4, Yun-Ju Chen1,2, Hao Lin3, Yu-Che Ou3, Chan-Chao Chang Chien3, Eng-Yen Huang5, Hsuan-Ying Huang6, Jui Lan6, Hsi-Ping Chi7, Ko-En Huang2,3, Hong-Yo Kang1,2,3 Introduction (44%) of the patients experience a recurrence and 35% of recurrent tumors occur loco-regionally [2]. Loco- regional recurrence (LRR) following radiotherapy is still a challenge because successful salvage treatment is difficult [3]. For patients with a high risk of LRR, surgical treatment after radiotherapy is considered for better local control and survival. However, the place and modality of surgery after radiotherapy continue to be debated [4–6]. Cervical carcinoma is the third common malignancy in women worldwide [1]. Radiation therapy with or without cisplatin-containing chemotherapy is the most commonly used treatment modality in patients with advanced cervical cancer. The treatment can achieve a 5-year overall survival rate of 67% [2]. Almost a half www.impactjournals.com/oncotarget Oncotarget 44047 expression level before treatment showed the median percentage of positive cells was 9.0% (range, 0.4–60.3%; mean, 11.6%, Table 1). The mean level of SKP2 in recurrence group (17.2 ± 12.1%) was significantly higher than disease free group (6.8 ± 6.2%) (P < 0.001, Table 1). No correlation was observed with either age at diagnosis, histologic grades, tumor size, chemotherapy, radiotherapy or surgery pattern (Table 1). There were 35 patients (23.5%) received chemotherapy. Chemotherapy was not a significant factor of recurrence (p = 0.746, Table 1). Next, we performed a multivariate Cox regression analysis to assess associations of clinicopathological characteristics with recurrence of disease. As shown in Table 1, staining level of SKP2, histologic types, SCC level, CEA level, and FIGO stage were confirmed to be independent prognostic factors of recurrence in cervical cancer. Molecular profiling of tumor and biomarkers studies have attempted to predict the patients who are more likely to experience a recurrence after standard therapy and develop individualized treatments that improve survival rate. While the expression of EGFR, VEGF, COX-2, galectin-1, Sphingosine kinase 1 and tumor hypoxia may have a major impact on the survival of patients treated with definitive radiotherapy [3, 7–10]. The studies addressing on the molecular biomarkers predicting the locoregional recurrence in cervical cancer after radiotherapy need to be pursued to improve the clinical outcome. S-phase kinase-associated protein 2 (SKP2) protein is an E3 ubiquitin ligase and a part of SKP1-Cul1-F-box (SCF) complexes. SKP2 is overexpressed and associated with poor prognosis in various human cancers [11]. Using genome-wide array comparative genomic hybridization, we previously profiled deoxyribonucleic acid (DNA) copy number alterations and identified SKP2 gene amplification confers tumor aggressiveness in myxofibrosarcoma [12]. Introduction Moreover, integrative genomics analysis [13, 14] and morphoproteomic evidence [15] also identified target over- expressed genes, including SKP2 gene of chromosome 5p gain in cervical cancer may contribute to cervical dysplastic lesion progression to invasive tumor. However, whether SKP2 play important roles on radiotherapy for cervical cancers remains obscure. Thus, our study aimed to (i) determine the immunohistochemical expression of SKP2 as a prognostic marker of patients with cervical cancer receiving radiotherapy, (ii) evaluate expression of SKP2 as a predictor of response to radiotherapy in a cohort of patients and cell lines, and (iii) investigate the role of SKP2 in response of DNA damage of cervical cancer cell lines. Expression of SKP2 presents as a novel biomarker in cervical cancer recurrence after radiation therapy To investigate the role of SKP2 in human cervical cancer, the expression of SKP2 protein was evaluated by immunostaining in a series of 149 primary human cervical cancer specimens before treatment. SKP2 protein was shown in adenocarcinoma and squamous cell carcinoma of cervical cancer tissues. Only cells with a clear nuclear staining were scored as positive. SKP2 expression levels were higher in recurrence group (Figure 1A). Next, we investigated whether the expression level of SKP2 in cervical cancer tissue before treatment could be a biomarker of recurrence after radiotherapy. We performed the receiver operating characteristic (ROC) curve of SKP2 expression in cancer cells for recurrence of cancer analyzed in our study (Figure 1B), the area under the curve was 0.821, indicating very good discrimination. The threshold value of 10.1% was selected as the best cut-off score. The expression of tumor SKP2 could be categorized into lower expression (< 10.1 %) group and higher expression (≥ 10.1%) group. The Kaplan-Meier curves of DFS, which displayed a significant separation between the lower and higher expression of SKP2 groups of patients (P < 0.001 by log-rank test; Figure 1C). Expression of SKP2 of tumor specimen before treatment is a good prediction marker of recurrence of uteri cervical cancer after radiotherapy, as FIGO stage (P < 0.001; Figure 1D) Patient characteristics with SKP2 as an independent prognostic factor in cervical cancer Between January 2004 and December 2006, there were 334 patients showed pathologic proof of cervical cancer. We enrolled 149 patients meeting the criteria of inclusion. The median follow-up duration was 73.1 months (range, 2–113 months). During the follow-up period, recurrence of disease and death were observed in 68 and 74 patients, respectively. In univariate analysis, several clinicopathologic variables displayed a relationship with disease-free survival (DFS) (Table 1). The recurrence of disease was statistically significantly associated with a number of clinicopathological variables including tumor stage (P < 0.001), histologic types (p = 0.008), SKP2 expression level (p < 0.001), serum squamous cell carcinoma (SCC) antigen level (p = 0.015) and carcinoembryonic antigen (CEA) level (p = 0.01) before treatment (Table 1). The SKP2 The expression patterns of SKP2 in cervical cancer tissues were correlated with clinicopathological variables including age at diagnosis, tumor stage, histological type, histological grade, tumor size, percentage of death and recurrence, recurrence site and the levels of SCC and CEA before therapy (Table 2). The high expression of SKP2 was found in 64 patients (43%), whereas low SKP2 expression was detected in 85 patients (57%) (Table 2). Patients with high expression level of SKP2 (≥ 10.1%) www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44048 Table 1: Summary of patient distribution according to disease status Oncotarg 44049 www.impactjournals.com/oncotarget were associated with recurrence (hazard ratios (HRs): 2.52, 95% confidence interval (CI), 1.75–3.62, P < 0.001) and death (HRs: 2.01, 95% CI, 1.46–2.76, P < 0.001). Median DFS of patients whose tumors expressed high level of SKP2 (18.4 months; mean, 31.6 ± 31.4 months) was shorter compared with lower expression patien (83.9 months; mean, 65.5 ± 37.5 months), and th difference was significant (P < 0.001). Patient characteristics with SKP2 as an independent prognostic factor in cervical cancer Similarly, 46 (71.8%) of the 64 patients with high SKP2 expression of tumor and 28 (32.9%) of the 85 lower expression of was shorter compared with lower expression patients (83.9 months; mean, 65.5 ± 37.5 months), and this difference was significant (P < 0.001). Similarly, 46 (71.8%) of the 64 patients with high SKP2 expression of tumor and 28 (32.9%) of the 85 lower expression of were associated with recurrence (hazard ratios (HRs): 2.52, 95% confidence interval (CI), 1.75–3.62, P < 0.001) and death (HRs: 2.01, 95% CI, 1.46–2.76, P < 0.001). Median DFS of patients whose tumors expressed high level of SKP2 (18.4 months; mean, 31.6 ± 31.4 months) Patient characteristics with SKP2 as an independent prognostic factor in cervical cancer Similar 46 (71.8%) of the 64 patients with high SKP2 expressi of tumor and 28 (32.9%) of the 85 lower expression Table 1: Summary of patient distribution according to disease status Status of disease Univariate Multivariate Adjusted Variable Total Recurrence Disease free P value P value All cases 149 68 77 Age ≥ 60 74 (49.7) 31 (43.7) 40 (56.3) < 60 75 (50.3) 37 (50.0) 37 (50.0) 0.445 0.724 Stage (%) lower (I,II) 120 (80.5) 46 (39.3) 71 (60.7) higher (III,IV) 29 (19.5) 22 (78.6) 6 (21.4) < 0.001 0.027 Histologic type (%) squamous cell carcinoma 139 (93.3) 59 (43.7) 76 (56.3) adenocarcinoma 5 (3.4) 5 (100) 0 adenosqumous cell carcinoma 5 (3.4) 4 (80.0) 1 (20.0) 0.008 0.001 Histologic grade (%) G1 10 (6.7) 5 (50.0) 5 (50.0) G2 37 (24.8) 11 (29.7) 26 (70.3) G3 and unknown 102 (68.5) 52 (53.1) 46 (46.9) 0.056 0.109 Tumor size (%) ≥ 4 cm 81 (54.4) 43 (63.2) 38 (49.4) < 4 cm 68 (45.6) 25 (36.8) 39 (50.6) 0.098 0.214 SKP2 expression level (SD) 11.6 (10.6) 17.2 (12.1) 6.8 (6.2) < 0.001 < 0.001 SCC level (SD) 12.5 (26.9) 18.6 (36.3) 6.8 (12.7) 0.015 0.042 CEA level (SD) 14.2 (57.4) 24.7 (82.4) 5.1 (11.7) 0.01 0.027 Chemotherapy (%) 35 (23.5%) 19 (54.3) 16 (45.7) 0.746 0.472 Radiotherapy (%) pelvic only 42 (28.1) 20 (30.8) 20 (28.6) pelvic and brachytherapy 97 (65.1) 45 (69.2) 50 (71.4) 0.606 0.428 dose (SD) 5955.0 (1559.1) 6045.2 (1702.8) 5895.1 (1431.5) 0.576 0.596 Operation pattern (%) no hysterectomy 105 (70.5) 54 (81.8) 51 (67.1) simple hysteretomy 6 (4.0) 0 6 (25.0) radical hysterectomy 31 (20.8) 12 (18.2) 19 (7.9)) 0.030 0.703 Abbreviations: CEA: Carcinoembryonic antigen, G1: grade 1, G2: grade 2, G3: grade 3, SCC: squamous cell carcinom antigen, SD: standard deviation. was shorter compared with lower expression patients (83.9 months; mean, 65.5 ± 37.5 months), and this difference was significant (P < 0.001). Similarly, 46 (71.8%) of the 64 patients with high SKP2 expression of tumor and 28 (32.9%) of the 85 lower expression of was shorter compared with lower expression patients (83.9 months; mean, 65.5 ± 37.5 months), and this difference was significant (P < 0.001). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44049 cell lines, microarray data of SKP2 mRNA expressions of different cervical cancer cell lines were obtained from GEO Datasets database (GDS3233) [16]. We found that SKP2 mRNA expressions were higher in radioresistant cervical cell lines (Figure 2A). To investigate the association of SKP2 expression with radioresistance, we decreased the expression of SKP2 with shRNA (TRCN0000007530 and TRCN0000007534) in HeLa and C33A cells (Figure 2B). Next, Western blotting analysis was performed to investigate the association between SKP2 protein expression and radiosensitivity in HeLa cells and C33A cells that were previously characterized as radioresistant and radiosensitive cervical cancer cells, respectively [17]. Indeed, we also noted a high protein expression of SKP2 in HeLa cells and a weak expression in C33A cells (Figure 2C). Therefore, we transfected the SKP2/scrambled shRNA in HeLa cells to knockdown SKP2 expression and stable clones were selected with puromycin (4μg/mL) (HeLa-scr/HeLa-SKP2(sh)). Empty plasmid or cDNA of SKP2 was transfected in C33A cells tumor died of disease during the period of follow-up. This difference was significant (P < 0.001). Hence, recurrence, locoregional recurrence, death, larger tumor size, higher stage and poor differentiation occurred more frequently when cervical cancer in higher staining group for SKP2 (Table 2). Age, stage, histologic type, tumor size SCC level or CEA level was not associated with expression of SKP2 in the same subset of patients (Table 2). While FIGO stage was shown to be an important factor of recurrence of cervical cancer [7], here we further demonstrated that patients with tumors displaying high staining for SKP2 were more likely to recur at locoregional site (HRs: 3.76, CI, 1.91–7.16, p < 0.001) compared with patients who had lower SKP2 expression after radiation therapy. SKP2 increases the survival of cervical cancer cells after irradiation To determine whether SKP2 is differentially expressed in radioresistant and radiosensitive cervical Figure 1: Immunostaining of SKP2 expression of pretreatment cervical cancer and the association with disease free survival. (A) Weak (a, c) and strong (b, d) staining of SKP2 was shown in adenocarcinoma (bottom row) and squamous cell carcinoma (top row). (x200, size bar: 100 μm); (B) Receiver operator characteristic (ROC) curve was shown for expression of SKP2 in discriminating recurrence. Excellent accuracy was demonstrated (area under the curve, 0.82; standard error, 0.035). The cutoff level of SKP2 expression on the curve was 10.1% of nuclear staining. (C) SKP2 immunohistochemical staining expression status (higher: ≥ 10.1%, lower: < 10.1%) before radiotherapy exhibited a significant (log-rank, p < 0.001) relationship with patient disease free survival. (D) Kaplan-Meier survival curve for cervical cancer patients were classified as higher (III, IV) and lower (I, II) FIGO stage. FIGO stage status exhibited a significant (log-rank, p <0.001) relationship with patient disease free survival. Figure 1: Immunostaining of SKP2 expression of pretreatment cervical cancer and the ass Figure 1: Immunostaining of SKP2 expression of pretreatment cervical cancer and the association with disease free survival. (A) Weak (a, c) and strong (b, d) staining of SKP2 was shown in adenocarcinoma (bottom row) and squamous cell carcinoma (top row). (x200, size bar: 100 μm); (B) Receiver operator characteristic (ROC) curve was shown for expression of SKP2 in discriminating recurrence. Excellent accuracy was demonstrated (area under the curve, 0.82; standard error, 0.035). The cutoff level of SKP2 expression on the curve was 10.1% of nuclear staining. (C) SKP2 immunohistochemical staining expression status (higher: ≥ 10.1%, lower: < 10.1%) before radiotherapy exhibited a significant (log-rank, p < 0.001) relationship with patient disease free survival. (D) Kaplan-Meier survival curve for cervical cancer patients were classified as higher (III, IV) and lower (I, II) FIGO stage. FIGO stage status exhibited a significant (log-rank, p <0.001) relationship with patient disease free survival. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44050 Table 2: Demographic patient distribution according to SKP2 expression before therapy Table 2: Demographic patient distribution according to SKP2 expression before therapy SKP2 expression score before therapy Variable Total ≥ 10.1 % < 10.1% P value All cases 149 64 (43.0) 85 (57.0) Age (%) ≥ 60 74 (49.7) 29 (39.2) 45 (60.8) < 60 75 (50.3) 35 (46.7) 40 (53.3) 0.409 Stage (%) lower (1,2) 120 (80.5) 48 (40.0) 72 (60.0) higher (3,4) 29 (19.5) 16 (55.2) 13 (44.8) 0.029 Histologic type (%) squamous cell 139 (93.3) 58 (41.7) 81 (58.3) adenocarcinoma 5 (3,4) 3 (60.0) 2 (40.0) adenosqumous cell 5 (3.4) 3 (60.0) 2 (40.0) 0.54 Histologic grade (%) G1 10 (6.7) 5 (50.0) 5 (50.0) G2 37 (24.8) 8 (21.6) 29 (78.4) G3 and unknown 102 (68.5) 51 (50.0) 51 (50.0) 0.01 Tumor size (%) ≥ 4 cm 81 (54.4) 41 (50.6) 40 (49.4) < 4 cm 68 (45.6) 23 (33.8) 45 (66.2) 0.047 Death (%) 74 (49.7) 46 (62.2) 28 (37.8) < 0.001 Recurrence (%) 68 (46.9) 46 (67.6) 22 (32.4) < 0.001 Recurrence site (%) locoregional 42 (61.8) 33 (71.7) 13 (28.3) distant 26 (38.2) 9 (40.9) 13 (59.1) 0.014 SCC level before therapy (SD) 12.4 (27.1) 11.9 (20.6) 13.0 (30.9) 0.315 CEA level before therapy (SD) 14.5 (58.2) 14.0 (10.4) 6.1 (3.0) 0.462 Abbreviations: CEA: Carcinoembryonic antigen, G1: grade 1, G2: grade 2, G3: grade 3, SCC: squamous cell carcinoma antigen, SD: standard deviation. Abbreviations: CEA: Carcinoembryonic antigen, G1: grade 1, G2: grade 2, G3: grade 3, SCC: squamous cell carcinoma antigen, SD: standard deviation. for SKP2 overexpression and stable clones were selected with zeocin (C33A-SKP2/C33A-vector). The expression level of SKP2 was confirmed by immunoblotting analysis. Transfection of cDNA with SKP2 increased the expression of SKP2 in C33A-SKP2 cells (Figure 2D). The shRNA of SKP2 decreased SKP2 expression in protein levels of HeLa-SKP2(sh) cells (Figure 2B, 2E). Furthermore, we assessed the effect of SKP2 in the radiation response of cervical cancer cells in vitro. The cell survival efficiency determined by colony-forming assay in C33A-SKP2/C33A-vector cells or HeLa-SKP2(sh)/ scr cells. Demonstrated that SKP2-overexpressing C33A cells exhibited higher colony formation compared with vector control cells (Figure 2D). The survival fraction rate showed a statistically significantly suppressed in SKP2-knockdowned HeLa cells compared with control (Figure 2E). SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation DNA damage clearance is a significant factor of radioresistance in cancer cells [19, 20] and γ-H2AX has g gi radioresistance in cancer cells [19, 20] and γ-H2AX has γ SKP2(sh) cells after exposure to irradiation than in control Figure 2: SKP2 enhanced cervical cancer cell survival upon ionizing radiation in vitro. (A) SKP2 mRNA expressions of different cervical cancer cell lines were obtained from GEO Datasets database (GDS3233). The radioresistant cervical cell lines had significantly higher expression of SKP2 than radiosensitive cell lines (P = 0.007). (B) We decreased the expression of SKP2 of HeLa and C33A cell with lentivirus with pLKO.1-sh-SKP2 (TRCN0000007530 (7530) and TRCN0000007534 (7534)). Real time PCR and western blotting were done for evaluation of the efficiency of the lentivirus. (C) Parental human radioresistant HeLa and radiosensitive C33A cervical cancer cells, (D) C33A cells were overexpressed SKP2 by SKP2 cDNA transfected and compared with empty vectors control. (E) HeLa cells were decreased SKP2 by shRNA and compared with scrambled (Scr) RNA. (F) Control scramble shRNA or SKP2 shRNA- transfected HeLa cells were treated with the indicated doses of SKP2-C25 (SKP2 inhibitor). After irradiation, cell survival was detected by colony formation assay. Cell survival fraction was significantly reduced in dose-dependent manner in HeLa-scr cells treated with the indicated doses of SKP2-C25. However, The inhibitory effect of SKP2 inhibitor on cell survival was not seen in the cells when SKP2 was decreased. C-E. Clonogenic survival rates and expression of SKP2 were detected. Cells were irradiated (6 Gy) and clonogenic assay performed 14 days following irradiation. Clonogenic assays were performed during three independent experiments at each paired condition (with and without SKP2 modulation). (P < 0.005 ). Figure 2: SKP2 enhanced cervical cancer cell survival upon ionizing radiation in vitro. (A) SKP2 mRNA expressions of different cervical cancer cell lines were obtained from GEO Datasets database (GDS3233). The radioresistant cervical cell lines had significantly higher expression of SKP2 than radiosensitive cell lines (P = 0.007). (B) We decreased the expression of SKP2 of HeLa and C33A cell with lentivirus with pLKO.1-sh-SKP2 (TRCN0000007530 (7530) and TRCN0000007534 (7534)). Real time PCR and western blotting were done for evaluation of the efficiency of the lentivirus. (C) Parental human radioresistant HeLa and radiosensitive C33A cervical cancer cells, (D) C33A cells were overexpressed SKP2 by SKP2 cDNA transfected and compared with empty vectors control. www.impactjournals.com/oncotarget We further investigated whether inhibition of SKP2 activity decreased cervical cancer cells survival after irradiation by SKP2-C25, an inhibitor of SKP2 that has been shown to selectively suppress SKP2 E3 ligase activity [18]. As shown in Figure 2F, the survival fraction of HeLa-scr cells treated with SKP2-C25 was lower than DMSO group. Cell survival fraction was significantly reduced in dose-dependent manner in HeLa-scr cell line but not in SKP2-HeLa(sh) cells treated with the indicated doses of SKP2-C25 before irradiation. Together, these www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44051 results suggest that SKP2 overexpressing cells showed increased cell survival and lower expression of SKP2 or loss of SKP2 activity in cervical cancer cells associated with lower survival fraction after irradiation. been employed as a sensor of double-strained DNA breaks (DSBs) after γ-irradiation treatment [21]. Therefore, we investigated the effects of SKP2 expression on DNA damages after irradiation in HeLa and C33A cells which SKP2 expression was stably silenced or over-expressed. The immunofluorescence analysis revealed a much more γ-H2AX foci were noted in the nuclei of HeLa-SKP2(sh) cells than HeLa control cells (Figure 3A) (P < 0.001). More γ-H2AX foci localized in the nuclei of C33A- SKP2(sh) cells after exposure to irradiation than in control SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation Western blots were done to determine the level of γ-H2AX and H2AX. GAPDH was used as the protein-loading control. (A) More γ-H2AX foci were found in HeLa-SKP2(sh) than HeLa-scr cells, after irradiation. The results of western blotting showed greater expression in HeLa-SKP2(sh) than HeLa-scr cells following irradiation. (B) More γ-H2AX foci were found in C33A-SKP2(sh) than C33A-scr cells, after irradiation. The results of western blotting showed greater expression in C33A-SKP2(sh) than C33A-scr cells following irradiation. (C) More γ-H2AX foci were found in C33A-vector than C33A-SKP2 cells, after irradiation. (D) The results of western blotting showed, greater expression of γ-H2AX in HeLa-SKP2(sh), C33A-SKP2(sh) and C33A-vector cells than HeLa-scr, C33A-scr and C33A-SKP2 cells following irradiation, respectively. Lower expression SKP2 of cells associated higher expression of γ-H2AX after irradiation. (P <0.001). amages clearance upon ionizing radiation in human cervical cancer cell lines. C33A- 3A-SKP2, HeLa-scr and HeLa-SKP2(sh) cells were treated with 6-Gy irradiation. After 4 hours ng for γ-H2AX (green) and DNA counterstaining with DAPI (blue). Number of γ-H2AX was ermine the level of γ-H2AX and H2AX. GAPDH was used as the protein-loading control. (A) More 2(sh) than HeLa-scr cells, after irradiation. The results of western blotting showed greater expression following irradiation. (B) More γ-H2AX foci were found in C33A-SKP2(sh) than C33A-scr cells, blotting showed greater expression in C33A-SKP2(sh) than C33A-scr cells following irradiation. C33A-vector than C33A-SKP2 cells, after irradiation. (D) The results of western blotting showed, SKP2(sh), C33A-SKP2(sh) and C33A-vector cells than HeLa-scr, C33A-scr and C33A-SKP2 cells Figure 3: SKP2 increased DNA damages clearance upon ionizing radiation in human cervical cancer cell lines. C33A- scr, C33A-SKP2(sh), C33A-vector, C33A-SKP2, HeLa-scr and HeLa-SKP2(sh) cells were treated with 6-Gy irradiation. After 4 hours incubated, immunofluorescence staining for γ-H2AX (green) and DNA counterstaining with DAPI (blue). Number of γ-H2AX was counted. Western blots were done to determine the level of γ-H2AX and H2AX. GAPDH was used as the protein-loading control. (A) More γ-H2AX foci were found in HeLa-SKP2(sh) than HeLa-scr cells, after irradiation. The results of western blotting showed greater expression in HeLa-SKP2(sh) than HeLa-scr cells following irradiation. (B) More γ-H2AX foci were found in C33A-SKP2(sh) than C33A-scr cells, after irradiation. The results of western blotting showed greater expression in C33A-SKP2(sh) than C33A-scr cells following irradiation. (C) More γ-H2AX foci were found in C33A-vector than C33A-SKP2 cells, after irradiation. SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation (E) HeLa cells were decreased SKP2 by shRNA and compared with scrambled (Scr) RNA. (F) Control scramble shRNA or SKP2 shRNA- transfected HeLa cells were treated with the indicated doses of SKP2-C25 (SKP2 inhibitor). After irradiation, cell survival was detected by colony formation assay. Cell survival fraction was significantly reduced in dose-dependent manner in HeLa-scr cells treated with the indicated doses of SKP2-C25. However, The inhibitory effect of SKP2 inhibitor on cell survival was not seen in the cells when SKP2 was decreased. C-E. Clonogenic survival rates and expression of SKP2 were detected. Cells were irradiated (6 Gy) and clonogenic assay performed 14 days following irradiation. Clonogenic assays were performed during three independent experiments at each paired condition (with and without SKP2 modulation). (P < 0.005 ). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44052 to recover for 2 hours, and then for detection of DNA damage by alkaline comet assay (Figure 4). Cells did not show significant different tail movement before irradiation. After receiving irradiation, C33A-vector cells produced a significant increase in the level tail moment than C33A- SKP2 cells (P < 0.05) (Figure 4A). While HeLa-scr did not show the significant difference of tail movement between before and after irradiation, increase of tail movement was observed in HeLa-SKP2(sh) after irradiation (Figure 4B). Together, these results suggested that SKP2 significantly increased the response of cervical cancer cells to efficiently repair DNA damage by irradiation. cells (Figure 3B) (P < 0.001). Significantly fewer γ-H2AX foci were noted in C33A-SKP2 cells than control cells (P < 0.001) (Figure 3C). The results of western blotting showed, greater expression of γ-H2AX in HeLa-SKP2(sh), C33A-SKP2(sh) and C33A-vector cells than HeLa-scr, C33A-scr and C33A-SKP2 cells following irradiation, respectively. Lower expression SKP2 of cells associated higher expression of γ-H2AX after irradiation (Figure 3D). Since the single cell gel electrophoresis assay (comet assay) has been used as a useful reliable genotoxicity test that detects DNA migration caused by strand breaks [22], we applied the cells to be exposed to irradiation, allowed we applied the cells to be exposed to irradiation, allowed repair DNA damage by irradiation. Figure 3: SKP2 increased DNA damages clearance upon ionizing radiation in human cervical cancer cell lines. C33A- scr, C33A-SKP2(sh), C33A-vector, C33A-SKP2, HeLa-scr and HeLa-SKP2(sh) cells were treated with 6-Gy irradiation. After 4 hours incubated, immunofluorescence staining for γ-H2AX (green) and DNA counterstaining with DAPI (blue). Number of γ-H2AX was counted. SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation (D) The results of western blotting showed, greater expression of γ-H2AX in HeLa-SKP2(sh), C33A-SKP2(sh) and C33A-vector cells than HeLa-scr, C33A-scr and C33A-SKP2 cells following irradiation, respectively. Lower expression SKP2 of cells associated higher expression of γ-H2AX after irradiation. (P <0.001). www.impactjournals.com/oncotarget Oncotarget 44053 SKP2 promotes DNA double-strand breaks repair pathways cell lines and the stable clones were selected under G418 (1 mg/ML). The stable clone cells were transfected with the RFP–ISceI–GR reported plasmid [25] to induce DSBs and showed RFP+ (Figure 5B). Cells that successfully repaired the DSBs showed GFP+ (Figure 5B). The quantitative GFP+/RFP+ ratio represented the efficiency of DSBs repair with HR or NHEJ (Figure 5C). HeLa cells with stable knockdown of SKP2 (HeLa-SKP2(sh)) showed significantly lower efficiency of DSBs repair regardless of whether of HR or NHEJ pathway (Figure 5C). These results suggested that SKP2 may improve the efficiency of DSBs repair, in both HR and NHEJ pathways. DNA double-strand breaks (DSBs) are critical events that can lead to cell death or genomic alterations including deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and dysfunction in these pathways result in genome instability and promote tumorigenesis [23]. To investigate the roles of SKP2 on DSB repair pathways in cervical cells after irradiation, we first generated reporter cell lines stably containing green fluorescent protein (GFP)-based reporter constructs. Induction of DSBs can be induced by the expression of ISceI and either NHEJ or HR pathway can repair DSBs and restore the function of the GFP gene [24]. The reporter plasmids of NHEJ-I, NHEJ-C or HR (Figure 5A) were individually transfected to indicated Discussion Chen TP, et al., reported higher expression of SKP2 in invasive cervical cancer than in cervical intraepithelial neoplasia and in normal cervical epithelium [26], it Figure 4: SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation. Alkaline comet assay was performed to assess DNA damages after irradiation treatment (RT) 2 hours in (A) C33A-vector/ C33A-SKP2 and (B) HeLa-scr / HeLa- SKP2(sh) stable clone cells. The assay was done in cells without irradiation (no RT) as control. Differences of tail movement before and after irradiation 2-hour were compared in (C33A-vector and C33A-SKP2) and (HeLa-scr and HeLa-SKP2(sh)). (P < 0.001). Figure 4: SKP2 enhances the responses of cervical cancer cells to DNA damage after irradiation. Alkaline comet assay was performed to assess DNA damages after irradiation treatment (RT) 2 hours in (A) C33A-vector/ C33A-SKP2 and (B) HeLa-scr / HeLa- SKP2(sh) stable clone cells. The assay was done in cells without irradiation (no RT) as control. Differences of tail movement before and after irradiation 2-hour were compared in (C33A-vector and C33A-SKP2) and (HeLa-scr and HeLa-SKP2(sh)). (P < 0.001). www.impactjournals.com/oncotarget Oncotarget 44054 The important prognostic variables of cervical cancer are represented by FIGO stage, tumor size, depth of invasion, lymph-vascular space involvement and histological type [7]. Locoregional recurrence following radiotherapy is still a challenge because successful salvage treatment for LRR is difficult. Surgical treaments after radiotherapy expected some promising effects in reducing locoregional recurrence. However, clinical studies failed to establish a significant benefit [28–30], probably owing to the small number of patients studied and the lack of selection of patients with high risks of recurrence. While several biomarkers as prognosis factors for radiotherapy have been studied such as survivin, cIAP1 and Galectin-1 [3, 7, 31–35], the role of these molecules in concurrent chemoradiation therapy (CCRT) remains to be validated, because CCRT has been the gold standard treatment for locally advanced cervical cancer since 1999 [36]. Our study in a well-documented series of cervical cancer indicates that SKP2 may play important roles in cervical carcinogenesis and progression. In agreement with previous report [27], we found that a high expression of SKP2 in cervical cancer was correlated with higher stage, poorer differentiation, larger tumor size and deeper of invasion. Discussion Importantly, we identified a correlation between SKP2 expression, disease recurrence and decreased survival after radiotherapy that was not observed in previous report [27], which recruited patients who received surgical treatment, only 38 (11.4%) patients received postoperative radiation. The discrepancy could be in part due to the majority of patients in previous study enrolled 58 cases of cervical intraepithelial neoplasia III (CIN III), 102 cases of microinvasive SCC, 123 cases of invasive SCC, and most subjects were radiotherapy-naive. Conversely, the studied population in our present study was homogenous for cervical cancer patients who received the protocol of radiotherapy. Figure 5: SKP2 increased the efficiency of NHEJ and HR repair of DSBs. (A) The reporter plasmids of NHEJ-I, NHEJ-C and HR were used to generate the reporter cell lines with stable expression of the reporter. (B) The cells were transfected with the RFP–ISceI– GR showed RFP-positive signal. ISceI cut the reporter plasmid ISceI-sites and created DSBs of DNA. Cells that successfully repaired the DSBs showed GFP-positive signal. (C) The ration of GFP+/RFP+ presented the efficiency of DSBs repair with HR or NHEJ. HeLa- SKP2(sh) showed significantly lower efficiency of DSBs repair (both HR and NHEJ) than HeLa-scr. (P < 0.005). Figure 5: SKP2 increased the efficiency of NHEJ and HR repair of DSBs. (A) The reporter plasmids of NHEJ-I, NHEJ-C and HR were used to generate the reporter cell lines with stable expression of the reporter. (B) The cells were transfected with the RFP–ISceI– GR showed RFP-positive signal. ISceI cut the reporter plasmid ISceI-sites and created DSBs of DNA. Cells that successfully repaired the DSBs showed GFP-positive signal. (C) The ration of GFP+/RFP+ presented the efficiency of DSBs repair with HR or NHEJ. HeLa- SKP2(sh) showed significantly lower efficiency of DSBs repair (both HR and NHEJ) than HeLa-scr. (P < 0.005). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44055 patients primarily treatment with radiotherapy reveals that higher SKP2 immunostaining is associated with poor DFS (P < 0.001), especially locoregional recurrence. In our series, FIGO stage of uteri cervical cancer is also an important variable of recurrence; patients with higher FIGO stage had higher recurrent rate and shorter DFS (Table 1 and Figure 1D). Higher stage was not an independent factor associated with locoregional recurrence. It meant response to radiotherapy was a different phenomenon not necessarily related to stage but to a variety of factors, such as cell biological factors [33]. Discussion While higher level of immunostaining of SKP2 has also been shown to be in poor response to radiotherapy in oral, nasopharyngeal and rectal cancers [37–39]. This study is the first to report that higher level of immunostaining of SKP2 predicts poor response to radiotherapy in cervical cancer. The results of this study also showed patients with high expression of SKP2 (> 10.1% nuclear stain) had higher recurrence of cancer after radiotherapy, especially locoregional recurrence (Table 2). The results implied the cervical cancer cells with high expression of SKP2 had high radioresistant ability. i which is critical for facilitating ATM recruitment for DNA damage repair and improve radioresistance of cervical cancer cells. Further studies are of interest to pursue this hypothesis. yp Cisplatin, the most commonly used agent applied to CCRT in cervical cancers, cisplatin mechanism of action is that the drug induces its cytotoxic properties through binding to nuclear DNA and subsequent interference with normal transcription, and/or DNA replication mechanisms [43, 44]. Cisplatin has anti-neoplastic activity and radiosensitizer effects. However, administration of cisplatin may result in hematological, gastrointestinal, renal toxicity and neuropathy. Previous study showed inhibitor of SKP2 displayed potent effects to decrease cancer cell viability, but only slightly decreased cell viability of normal epithelial cells [18]. Due to the side effects of cisplatin, we aimed to investigate whether the SKP2 inhibitor may act as a radiosensitizer in cervical cancer, and with lower toxicity to normal cells. Our data showed the inhibitor of SKP2 (SKP2-C25) displayed potent effects to increase the radiosensitivity of HeLa cells (Figure 2F) suggesting that it may act as a potential radiosensitizer for cervical cancer. However, whether SKP2-C25 can work, as a better radiosensitizer with more specific anti-neoplastic effects and fewer side effects than cisplatin remains to be further investigated. i g y To further extend our findings from the clinical data, SKP2 mRNA expressions between radiosensitive and radioresistant cervical cancer cell lines from GEO Datasets database (GDS3233) [16] were acquired to explore the correlation between SKP2 expression and radioresistance in cervical cancer cell lines. Indeed, we found that SKP2 mRNA expressions were higher in radioresistant cervical cell lines (Figure 2A). Expression of endogenous SKP2 was much weaker in C33A than HeLa cells (Figure 2). Previous study showed C33A cells were more radiosensitive than HeLa cells [17]. In our data, HeLa cells were more resistant to irradiation than C33A cells. Discussion Increased expression of SKP2 promoted the radiation resistance of C33A cells (Figure 2D). Decreased SKP2 level increased the radiation sensitivity of HeLa cells (Figure 2E). These data prove that SKP2 had a significant role in radioresistance. Increased SKP2 level could defend against radiation-induced cell death. The relation between SKP2 and response to radiotherapy might be due to the fact that SKP2 is involved in DNA double- strand break repair [40]. DNA repair is a significant factor of radioresistance and survival of irradiated tumor cells critically depends on DNA repair [41, 42]. SKP2 had been reported that play an important role in homologous recombination repair of DSBs through interaction with NBS1, a key component of the Mre11/Rad50/NBS1 (MRN) complex to recruit ATM to DNA foci for activation [40]. Our results also showed that SKP2 protein increased the ability of clearance of DSBs induced by irradiation in cervical cancer cell lines with decreasing Gamma-H2AX foci number and expression level in SKP2 overexpressing cells after irradiation. We hypothesize that SKP2 may interact with MRN associated protein complex and trigger ubiquitination of these associated proteins upon DSBs, In conclusion, we found for the first time that high expression of SKP2 was correlated with poor response of radiotherapy and prognosis of cervical cancer. High expression of SKP2 of tumor predicts higher locoregional recurrence of cervical cancer after radiotherapy. The patients with high expression of SKP2 cervical cancer are at high risk of locoregional recurrence and might be appropriate for adjuvant hysterectomy after radiotherapy. These results provide insights into the role of SKP2 in cervical cancer and suggest that inhibitor of SKP2 is a good radiosensitizer with potential anti-neoplastic function that may act as novel experimental intervention for developing effective therapeutic strategies against cervical cancer. Colony formation assay HeLa or C33A cells were transfected with the SKP2, sh-SKP2 or the negative control (vector or scramble RNA), followed by selection for stable clones. After irradiation, 2000, 1000, and 500 cells were seeded separately in 6-well plates for colony formation. After 14 days, colonies were fixed and stained with a mixture of 6% glutaraldehyde and 0.5% crystal violet. Only if a single colony contained more than 50 cells was it scored using a microscope [47]. Each assay was performed in duplicate on three independent occasions. Irradiation of cells Cells were transferred to 25-cm2 flasks and incubated in DMEM with 10% FBS at 37ºC with 5% CO2 for 24 hours. The flasks were placed on a linear accelerator Clinac 600C/D (VARIAN, USA) with a fixed source skin distance and X-ray irradiation at 4 Gy/min. Transfection of cervical cancer cell lines The SKP2 expression plasmid, pcDNA3–SKP2 was a gift of Dr. Hui-Kuan Lin [40]. For overexpression of the SKP2, C33A cells were transfected with pcDNA3– SKP2 or pcDNA3 using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. After transfection, the cells were grown in G418 at 800 µg/ml for stable clone selection. Selected stable clones (C33A-SKP2 and C33A-vector) were maintained in the growth medium with G418 at 500 µg/ml. The lentiviral vectors were obtained from Taiwan National RNAi Core Facility (Academia Sinica, Taiwan), including pLKO.1. null-T (5′-TCAGTTAACCACTTTTT-3′) and pLKO.1-sh-SKP2 (TRCN0000007530: 5′-GCCTAAGCT AAATCGAGAGAA-3′ and TRCN0000007534: 5′-GATA GTGTCATGCTAAAGAAT-3′). For viral infection, 3 × 104 HeLa or C33A cells were incubated with lentivirus in the presence of 8 µg/ml polybrene, followed by 4 µg/ml puromycin selection for stable clones (C33A-SKP2(sh), C33A-scr, HeLa-SKP2(sh) and HeLa-scr) of lentivirus- transduced cells. Immunohistochemical staining All tissues were obtained from formalin-fixed, paraffin-embedded tissue blocks. Representative are as of tumor were selected by a pathologist from hematoxylin stained sections. For immunohistochemical staining, sections were deparaffinized with xylene rinse, rehydrated with a graded alcohol series (100%, 95%, 85%, and 75%), and then rinsed with distilled water. Antigen retrieval was enhanced by citrate buffer (10 mM, pH 6.0). Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide solution. The slides were incubated with primary antibodies, SKP2 (Invitrogen, clone 2C8D9, 1:200) overnight at 4ºC and further incubated with a secondary antibody 2 hours at 25ºC. Antigen–antibody complexes were detected by DAB (Dako, Glostrup, Denmark) and counterstained with Gill’s hematoxylin (Merck, Whitehouse, NJ, USA). The expression of SKP2 was considered positive if immunoreactivity was found in at nuclei. Digital image analysis was carried out to determine the nuclear staining levels by using “ImmunoRatio”, a web based analysis software [46]. Minimum of four microscopic field areas were analyzed for each tumor slide and the data were plotted as percent positive cells for SKP2 staining. Patients This is a retrospective study composed of patients treated between January 2004 and December 2006. Tissue samples from all patients showed pathologic proof of cervical cancer (total 334 patients). The stages were assigned according to the International Federation of Gynecology and Obstetrics (FIGO) 2009 system. The inclusion criteria were FIGO stage I–IV, the cell types (squamous, adenosquamous, or adenocarcinoma) and undergoing radiotherapy; this resulted of 202 patients. After excluding the patients, whose tissues were from outside the hospital, were insufficient, or did not receive www.impactjournals.com/oncotarget Oncotarget 44056 regular follow-up, 149 patients were analyzed. All patients underwent external-beam RT to whole pelvis of 39.6-50.4 Gy, with a daily fraction of 1.8–2 Gy, 5 fractions per week. High-dose-rate intracavitary brachytherapy was delivered to 97 patients after the completion of external beam RT. The details of the RT have been described previously [3]. The cumulative biologically effective dose at point A was at least 70 Gy10. Most of them (109 patients) did not receive hysterectomy. Radical and simple hysterectomy were delivered to 31 and 6 patients, respectively. All patients underwent radiotherapy after biopsy of cervix uteri or hysterectomy. This study was approved by the Institutional Review Board in Kaohsiung Chang Gung Memorial Hospital (KCGMH) (102-0084C) and conducted in accordance with approved institutional guidelines. The clinic data were obtained as de-identified data released by KCGMH for research purpose. This study followed the requirement of reporting recommendations for tumor marker prognostic studies [45]. 5% CO2 at 37ºC and passaged for fewer than 6 months after receipt or resuscitation. SKP2-C25 (Xcessbio, San Diego, CA, USA) was prepared 10 mM stock solution in dimethyl sulfoxide. Cell culture and reagents Cervical cancer cell lines, HeLa and C33A cells were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) that cell lines authentication was performed by BCRC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/mL streptomycin and 0.4 mM L-glutamine (Sigma) in a humidified 95% atmosphere with Immunoblotting Cells were lysed in lysis buffer supplemented with 1% protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 44057 NHEJ-C, or HR reporter plasmid (kind gift of Prof. Vera Gorbunova) [24]. G418, at 1mg/ml, was added to the media 1 day post-transfection. Colonies were picked after 7–10 days on selection. Transfection with the RFP–ISceI–GR (from Addgene) was performed. The final concentration of triamcinolone acetonide (Sigma, St Louis, MO) added was 10−7 M [25]. After adding the triamcinolone acetonide, RFP–ISceI–GR chimera protein shifted to nuclear and cut the ISceI site of reporter plasmid. Cells were harvested and washed with cold PBS after adding the triamcinolone acetonide 2 hours. After fixation in 4% buffered paraformaldehyde in PBS for 10 min at room temperature, slides were examined under a Nikon E800 microscope, and images were collected using a SPOT RT3 microscope camera. Captured images were analyzed for foci of GFP and RFP. The numbers of GFP+ and RFP+ cells were determined. The ratio between GFP+ and RFP+ cells was used as a measure of DSB repair efficiency. NHEJ-C, or HR reporter plasmid (kind gift of Prof. Vera Gorbunova) [24]. G418, at 1mg/ml, was added to the media 1 day post-transfection. Colonies were picked after 7–10 days on selection. Transfection with the RFP–ISceI–GR (from Addgene) was performed. The final concentration of triamcinolone acetonide (Sigma, St Louis, MO) added was 10−7 M [25]. After adding the triamcinolone acetonide, RFP–ISceI–GR chimera protein shifted to nuclear and cut the ISceI site of reporter plasmid. Cells were harvested and washed with cold PBS after adding the triamcinolone acetonide 2 hours. After fixation in 4% buffered paraformaldehyde in PBS for 10 min at room temperature, slides were examined under a Nikon E800 microscope, and images were collected using a SPOT RT3 microscope camera. Captured images were analyzed for foci of GFP and RFP. The numbers of GFP+ and RFP+ cells were determined. The ratio between GFP+ and RFP+ cells was used as a measure of DSB repair efficiency. electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with SKP2 (1:1000; Zymed), anti-γ-H2AX antibody (1:1000; Cell Signaling), H2AX (1:1000; Cell Signaling), beta- tubulin (1:500; Santa Cruz Biotechnology) or GAPDH (1:5,000; Chemicon) and then incubated with horseradish peroxidase-conjugated secondary antisera (Amersham, Buckinghamshire, UK). Enhanced chemiluminescence was performed with ECL-Plus (Amersham Pharmacia ). Acknowledgments We thank Dr. Jenq-Lin Yang and the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Taiwan for improving the quality of immunofluorescence staining and imaging of the manuscript. RNAi reagents were obtained from the National Core Facility for Manipulation of Gene Function and CRISPR / Genomic Research Center, Academia Sinica, supported by the National Core Facility Program for Biotechnology Grants of MOST (MOST 104- 2319-B-001-001). Alkaline comet assay Indicated cells were harvested and washed with ice- cold phosphate-buffered saline (PBS) before and 2 hours after irradiation. Alkaline comet assay was performed as previous described [44, 50]. Briefly, the cell suspension (1 × 105 cells/ml) was mixed with low melting point agarose (Sigma) at a final concentration of 1% and cast on microscopic slides. Approximately 50 μL of the mixture of agarose and cells were placed on slides, and the agarose was solidified at 4ºC for 45 min. After solidification, the cover slips were removed and the slides placed in the lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 and 1% Triton X-100) for 1 h at 4 ºC in the dark. Later, the slides were placed in a horizontal gel electrophoresis unit filled with fresh electrophoretic buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13) and left in this buffer for 40 min for DNA unwinding. Without changing the alkali solution the slides were electrophoresed for 30 min at 25 V (1.2 V/cm, 42–44 mA) at 8ºC. After electrophoresis, the slides were rinsed with 0.4 M Tris, pH 7.5 (3 × 5 min) and then stained with 1 μM 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI, 50 μl per slide). Statistical analysis Comparisons between SKP2 high-expression and low-expression groups for clinicopathologic factors were evaluated using the x2 with Fisher exact. The median follow-up duration was 73.1 months (range, 2–113 months) for 149 independently validated cases. The endpoint analyzed was disease-free survival (DFS), last visit or death. The durations were calculated from the date of operation until the occurrence of recurrence or last follow-up appointment. Univariate survival analyses were conducted using Kaplan–Meier plots and the statistical significance of difference between curves was evaluated by the two-sided log-rank test. Furthermore, hazard ratios (HRs) and 95% confidence intervals (CIs), which were computed from univariate and multivariable Cox proportional hazards regression models were used to assess associations between SKP2, recurrence and survival. The two-sided Student t-test was used to analyze clinicopathologic factors, colony formation assay, γ-H2AX foci, tail movement and ratio of GFP+/RFP+ for cell line samples. All of the statistical analyses were performed using the SPSS statistical package. All P values less than .05 were considered statistically significant. Immunofluorescence staining of phospho- (Ser-139)- H2AX (γ-H2AX) Cells with/without irradiation were transferred to slides at a density of 3 × 103 cells using cytospin. Immunofluorescence staining of γ-H2AX was performed as previously described [48]. 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Ferrandina G, Margariti PA, Smaniotto D, Petrillo M, Salerno MG, Fagotti A, Macchia G, Morganti AG, Cellini N, Scambia G. Authors’ contributions Conception and design: H.Y. Kang, H.C. Fu. Development of methodology: H.C. Fu, Y.C. Yang, and H.Y. Kang. Acquisition of data (acquired and managed patients, provided facilities, etc.): H.C. Fu, H. Lin, Y. C. Ou , C.C. Chang Chien, H.Y. Huang , J. Lan. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.C. Fu, Y.J. Chen, H.Y. Kang.Writing, review, and/or revision of the manuscript: H.C. Fu, H.P. Chi, Y.C. Yang, Y.J. Chen, H.Y. Kang. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.E. Huang, Y.C. Yang, Y.J. Chen, E.Y. Huang, and H. Y. Kang. Study supervision: H.Y. Kang 9. Chang L, Graham P, Hao J, Ni J, Deng J, Bucci J, Malouf D, Gillatt D, Li Y. Cancer stem cells and signaling pathways in radioresistance. Oncotarget. 2016; 7:11002–17. doi: 10.18632/oncotarget.6760. 10. Wu CH, Lin YW, Wu TF, Ko JL, Wang PH. Clinical implication of voltage-dependent anion channel 1 in uterine cervical cancer and its action on cervical cancer cells. Oncotarget. 2016; 7:4210–4225. doi: 10.18632/ oncotarget.6704. 11. Frescas D, Pagano M. Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer. Nat Rev Cancer. 2008; 8:438–449. NHEJ and HR assay NHEJ and HR assay was modified the method as previous described [24]. Briefly, we generated reporter cell lines, indicated cells were transfected with NHEJ-I, www.impactjournals.com/oncotarget Oncotarget 44058 Grant support This work was supported by grants, CMRPG 8A1062, 860261-3 and 890911-2, CMRPD 8708431- 3, 8911181-3 from Kaohsiung Chang Gung Memorial Hospital and NMRPD 170341-3 (NSC97-2321-B-182- 004-MY3) from the Ministry of Science and Technology, Taiwan to Dr. Hong-Yo Kang. 7. Gadducci A, Guerrieri ME, Greco C. Tissue biomarkers as prognostic variables of cervical cancer. Crit Rev Oncol Hematol. 2013; 86:104–129. 8. Kim HS, Yoon G, Ryu JY, Cho YJ, Choi JJ, Lee YY, Kim TJ, Choi CH, Song SY, Kim BG, Bae DS, Lee JW. Sphingosine kinase 1 is a reliable prognostic factor and a novel therapeutic target for uterine cervical cancer. Oncotarget. 2015; 6:26746–26756. doi: 10.18632/ oncotarget.4818. conflicts of interest 6. Morice P, Rouanet P, Rey A, Romestaing P, Houvenaeghel G, Boulanger JC, Leveque J, Cowen D, Mathevet P, Malhaire JP, Magnin G, Fondrinier E, Berille J, et al. Results of the GYNECO 02 study, an FNCLCC phase III trial comparing hysterectomy with no hysterectomy in patients with a (clinical and radiological) complete response after chemoradiation therapy for stage IB2 or II cervical cancer. Oncologist. 2012; 17:64–71. The authors declare no competing financial interests. 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https://openalex.org/W2028652322	https://hal.archives-ouvertes.fr/hal-02457008/document	English	null	Maternal Effects on Anogenital Distance in a Wild Marmot Population	PloS one	2,014	cc-by	5,033	To cite this version: Timothée D Fouqueray, Denis Blumstein, Julien G. A. Martin. Maternal Effects on Anogeni- tal Distance in a Wild Marmot Population. PLoS ONE, 2014, 9 (3), pp.e92718. 10.1371/jour- nal.pone.0092718. hal-02457008 Introduction they might contribute to offspring defeminisation or masculinisa- tion. However, the importance of this maternal source has not been studied in the wild. Maternal effects represent the influence of a mother’s genotype or phenotype to her offspring’s phenotype independently of additive genetic effects [1] and have numerous implications in a wide range of species on prenatal (development, immunity, stress level) and postnatal life (social rank, dispersal, reproductive performance, etc.) [2–5]. Early exposure to androgens is morphologically evident by the anogenital distance (AGD), the distance between the anus and the genital papilla. AGD results from the elongation of the perineal tissue, which is triggered by testosterone during the early development, and thus, males have larger AGD than females [13,14]. AGD, at young ages, is thus a proxy of early exposure to androgens [15,16] and is relatively easy to quantify under field conditions. Females (of several species) with larger AGD are masculinized. They are less likely to survive and more likely to disperse [10], are less likely to become pregnant [17], are less preferred by males, and have smaller and male-biased litters [18]. Similar findings have been observed as well in humans, including positive correlations between AGD and fertility in men [19], and ovarian follicles numbers in women [20]. For mammals, the intrauterine environment is a potentially important source of maternal effects on a variety of life history traits [6]. Pre-natal exposure to sex steroid hormones emerges from the complex hormonal environment fetuses are exposed to. Each individual is exposed to androgens produced by its male siblings via transplacental and transamniotic diffusion [7]. Testosterone has organisational and activational effects on the central nervous system, masculinising and defeminising the behavior exhibited later in life [8,9]. For instance, female rats (Rattus norvegicus) exposed to neonatal testosterone engaged in more masculinised impulsive behavior [9]. Similarly, masculinised yellow-bellied female marmots (Marmota flaviventris) had lower survival, were more likely to disperse, and had reduced weaning success compared with females exposed to reduced levels of intrauterine testosterone [10]. Thus, testosterone is an important modulator of life-history traits [10]. We studied a population of individually marked wild yellow- bellied marmots for which litter effects but not maternal effects on anogenital distance were known: female yellow-bellied marmots’ morphology, behavior, survival, and dispersal are influenced by the proportion of males in the litter [4,10]. Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: marmots@ucla.edu Maternal Effects on Anogenital Distance in a Wild Marmot Population Timothe´e D. Fouqueray1,2, Daniel T. Blumstein1,3, Raquel Monclu´ s4, Julien G. A. Martin1,5* 1 Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America, 2 De´partement de Biologie, E´cole Normale Supee´rieure de Lyon, Lyon, France, 3 The Rocky Mountain Biological Laboratory, Crested Butte, Colorado, United States of America, 4 Departamento de Biologı´a, Universidad Auto´noma de Madrid, Madrid, Spain, 5 School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom Abstract In mammals, prenatal exposure to sex steroid hormones may have profound effects on later behavior and fitness and have been reported under both laboratory and field conditions. Anogenital distance is a non-invasive measure of prenatal exposure to sex steroid hormones. While we know that intra-uterine position and litter sex ratio influence anogenital distance, there are other, heretofore unstudied, factors that could influence anogenital distance, including maternal effects. We capitalized on a long-term study of wild yellow-bellied marmots (Marmota flaviventris) to study the importance of maternal effects on explaining variation in anogenital distance and found significant effects. The strength of these effects varied annually. Taken together, our data highlights the strong variability due to environmental effects, and illustrates the importance of additive genetic and maternal genetic effects on neonatal anogenital distance. We suspect that, as others apply recently popularised quantitative genetic techniques to study free-living populations, such effects will be identified in other systems. Citation: Fouqueray TD, Blumstein DT, Monclu´s R, Martin JGA (2014) Maternal Effects on Anogenital Distance in a Wild Marmot Population. PLoS ONE 9(3): e92718. doi:10.1371/journal.pone.0092718 Editor: Maria` Alemany, University of Barcelona, Faculty of Biology, Spain Editor: Maria` Alemany, University of Barcelona, Faculty of Biology, Spain Received December 4, 2013; Accepted February 25, 2014; Published March 20, 2014 Received December 4, 2013; Accepted February 25, 2014; Published March 20, 2014 Copyright: 2014 Fouqueray 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: 2014 Fouqueray et al. This is an open-access article distributed under the terms of the Creative Comm unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: DTB was supported by the National Geographic Society, UCLA (Faculty Senate and the Division of Life Sciences), a Rocky Mountain Biological Laboratory research fellowship, and by the NSF (IDBR-0754247 and DEB-1119660 to DTB, as well as DBI 0242960 and 0731346 to the Rocky Mountain Biological Laboratory). RM was supported by postdoctoral fellowships from the Spanish Ministerio de Innovacio´n y Ciencia and the Fulbright program. JGAM was supported by a FRQNT postdoctoral fellowship and the NSF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. March 2014 \| Volume 9 \| Issue 3 \| e92718 HAL Id: hal-02457008 https://hal.science/hal-02457008v1 Submitted on 27 Jan 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Results : Females taken as reference {: N males: N total DF: numerator, denominator degrees of freedom F-cond: conditional Wald F-test doi:10.1371/journal.pone.0092718.t001 Estimate (SE) DF F-cond P (Intercept) 4.899 (0.808) 1, 33.5 36.750 ,0.001 Sex [Male] 4.205 (0.185) 1, 468.1 514.300 ,0.001 Mass 0.010 (0.001) 1, 388.7 124.100 ,0.001 Litter sex-ratio{ 1.749 (0.452) 1, 243.6 15.000 ,0.001 Litter size 20.058 (0.063) 1, 187.5 0.856 0.356 Days since emergence 20.038 (0.038) 1, 453.7 0.963 0.327 Maternal Effects on Anogenital Distance variance components, to assess how much of the variance in AGD in neonates was due to maternal, genetic and environmental effects. By fitting different random effects that are linked or not linked to a pedigree, it is thus possible to decompose the variance of a trait into its genetic and environmental effects. Random effects linked to the pedigree provide information of additive genetic variance whereas random effects not linked to the pedigree provide environmental variance estimates. Since an animal model is a mixed effect model, additional fixed effects could also be included to correct for potential biases in the variance estimates. By fitting different random effects that are linked or not linked to a pedigree, it is thus possible to decompose the variance of a trait into its genetic and environmental effects. Random effects linked to the pedigree provide information of additive genetic variance whereas random effects not linked to the pedigree provide environmental variance estimates. Since an animal model is a mixed effect model, additional fixed effects could also be included to correct for potential biases in the variance estimates. Results As expected from previous analyses [10], we found a significant effect of litter sex ratio on AGD (Table 1), whereby pups in male- biased litters had greater AGDs. Male AGD was significantly greater than female AGD (Table 1) as previously [10,29]. In addition, larger animals had larger AGDs (Table 1). For our sample of animals trapped within 10 days of emergence, there was no significant effect of the number of days since emergence, nor was there a significant effect of litter size (Table 1). Starting in 2002, we took a hair sample from every individual for DNA parentage assignment using 12 microsatellite loci. This permitted us to assign (with 95% confidence) paternity and maternity (for further details about the procedure and the study population, see [21]). The average litter size for yellow-bellied marmots is about 3–4 pups but varies from 1 to 10 pups. Females give birth at most once a year in the burrows, where the pups stay during lactation. They emerge about 25 days later to start foraging by themselves [22]. Litter sex-ratio was estimated using all pups trapped. For AGD analysis, however, we only used those individuals with full parentage and that were trapped within their first 10 days following natal emergence because AGD at that age is not as biased by morphological differences between individuals as it would be for older individuals [23]. We thus used AGD records from 564 pups, from 183 different litters, that were produced by 91 different mothers between 2002 and 2010. This represented 67% of all pups observed during that period. There were significant maternal effects at the phenotypic level accounting for 5.8% of the remaining variance (Fig. 1, Table 2, model 1, LRT = 3.98, df = 1, P = 0.046). When we decomposed the variance into additive genetic (LRT = 1.51, df = 1, P = 0.219), Table 1. Estimates (with standard error) of the fixed effects on anogenital distance of juvenile yellow-bellied marmots studied at the Rocky Mountain Biological Laboratory. Estimate (SE) DF F-cond P (Intercept) 4.899 (0.808) 1, 33.5 36.750 ,0.001 Sex [Male]* 4.205 (0.185) 1, 468.1 514.300 ,0.001 Mass 0.010 (0.001) 1, 388.7 124.100 ,0.001 Litter sex-ratio{ 1.749 (0.452) 1, 243.6 15.000 ,0.001 Litter size 20.058 (0.063) 1, 187.5 0.856 0.356 Days since emergence 20.038 (0.038) 1, 453.7 0.963 0.327 Estimates significantly different from zero are in bold. Ethics Statement Free-living yellow-bellied marmots were studied under research protocol ARC 2001-191-01 as well as permits issued by the Colorado Division of Wildlife. The research protocol was approved by the UCLA Animal Care Committee on 13 May 2002 and renewed annually. Trapping, measuring, and marking marmots are routine techniques that have been conducted with no deleterious consequences for over 50 years in this population of marmots. By design, marmots were not harmed during the course of this study. As fixed effects, we included mass (to correct for differences in body size), the number of days since emergence at trapping (to take into account the morphological development of the perineal tissue), sex (to control for sexual dimorphism), proportion of males in the litter (to control for litter effects), and litter size (to control for the number of siblings producing androgens). We tested the significance of the fixed effects using conditional Wald tests. For random effects, we first fitted a model including only maternal identity (to estimate between mother variation), year (to assess the inter-annual environmental variation), and the identity of the litter. We then decomposed the maternal effects into its genetic and environmental components. Thus, we included pup identity linked to the pedigree (direct additive genetic effect), maternal identity (maternal environment), maternal identity linked to the pedigree (maternal genetic), year, and the identity of the litter. We tested the significance of random effects using a log- likelihood ratio test comparing the full model to a model without a specific random effect [27,28]. Variance ratios for random effects were estimated using the estimated phenotypic variance from the animal model (i.e., the sum of variance parameters in the model after accounting for the fixed effects [28]). Experimental Subjects Yellow-bellied marmots have been studied since 1962 in and around the Rocky Mountain Biological Laboratory, Gunnison County, CO, USA (elevation approx. 2890 m). Marmots are individually marked and followed throughout their lives. Natal emergence date was determined by daily observations of the colonies during the active season (mid-April to mid-September). As soon as they emerged from their natal burrow, pups were trapped, sexed, ear tagged, and fur marked with Nyanzol fur dye. Assigning marmots to sex is straightforward and was successfully repeated during numerous trapping sessions by different observers. Subjects were weighed using a digital scale (accurate to 25 g) and we measured the AGD using digital callipers (accuracy 1 mm). Every summer, new observers were trained to measure AGD accurately by making multiple AGD measurements on different individuals until they obtained measurements similar to trained observers. This training ensured consistency in our measurements across observers. Since all pups within a litter were not trapped and measured by the same observers, measurement error due to the observer should only increase the noise in the data and make it more difficult to detect significant effects. Introduction Given the strong relation between androgenization and life-history traits later in life, it seems crucial to disentangle the different sources of variation that might affect the offspring phenotype. For that we fitted an animal model, a statistical technique designed to decompose Apart from male siblings’ testosterone, androgens are secreted by the mother’s placenta, adrenal glands, and ovaries [11,12], and PLOS ONE \| www.plosone.org March 2014 \| Volume 9 \| Issue 3 \| e92718 1 March 2014 \| Volume 9 \| Issue 3 \| e92718 Statistical Analyses Using the asreml function [24] in the statistical package R, v.2.14.1 [25], we fitted an animal model [26] to decompose the variance of AGD into its additive genetic, maternal (environment and genetic), litter and year components. An animal model is a particular type of mixed model in which the different individuals are not considered independent but related to each other by a matrix of relatedness (most often obtained from a pedigree) [26]. March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org 2 Maternal Effects on Anogenital Distance Table 2. Estimates (with standard error) of variance components and proportion of variance of anogenital distance in yellow-bellied marmot pups. Variance component Variance ratio Model LL AG MG ME L Y P AG MG ME L Y 1. ME + L + Y 2704.49 - - 0.390 (0.227) 0.311 (0.236) 2.480 (1.387) 6.728 (1.447) - - 0.058 (0.036) 0.046 (0.036) 0.368 (0.220) 2. AG + MG +ME + L + Y 2701.56 0.613 (0.558) 0.287 (0.304) 0.000 (0.000) 0.252 (0.207) 2.450 (1.372) 6.800 (1.572) 0.090 (0.084) 0.042 (0.045) 0.000 (0.000) 0.037 (0.031) 0.360 (0.218) 3. AG +ME + L + Y 2702.14 0.960 (0.518) - 0.029 (0.207) 0.300 (0.236) 2.521 (1.407) 6.847 (1.574) 0.140 (0.082) - 0.004 (0.030) 0.043 (0.035) 0.368 (0.222) 4. MG +ME + L + Y 2702.32 - 0.542 (0.260) 0.000 (0.000) 0.274 (0.208) 2.409 (1.348) 6.742 (1.411) - 0.080 (0.042) 0.000 (0.000) 0.040 (0.032) 0.357 (0.213) AG: additive genetic; MG: maternal genetic; ME: maternal environment; L: litter; Y: year; P: phenotypic; LL: log-likelihood; Variance ratio of AG is the heritability, h2. The raw phenotypic variance of AGD was 13.272. The phenotypic variance of AGD after controlling for the fixed effects was estimated as 6.743 (1.411). Significant random effects are in bold. doi:10.1371/journal.pone.0092718.t002 maternal environment (LRT = 1.18, df = 1, P = 0.277), and maternal genetic (LRT ,0.001, df = 1, P.0.999) components, we found that none of them were significant (Table 2, model 2). However, the inclusion of both additive genetic and maternal genetic effects provided a marginally better fit than when they were excluded (Fig. 1, Table 2, model 1 vs. model 2: LRT = 5.56, df = 2, P = 0.053). Statistical Analyses In addition, additive genetic (LRT = 4.70, df = 1, P = 0.030) and maternal genetic (LRT = 4.39, df = 1, P = 0.036) effects were both found to be significant if only one of them was included in the model and their estimates were larger (Fig. 1, Table 2, models 3 and 4). This suggests that despite nearly a decade’s worth of data, we had insufficient power to conclusively isolate both effects. Nonetheless, it suggests that both effects could be important. When a maternal genetic effect was included in a model, the maternal environment explained ,0.001 of the variance, a finding that suggests that most of the variance between mothers were due to maternal genetic effects. Litter identity effects were not significant in any model (Fig. 1, Table 2, all P.0.141). Year was significant in all models (all P,0.001) and explained 36% of the remaining variance in AGD (Fig. 1, Table 2). References Meaney MJ, McEwen BS (1986) Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats. Brain Research 398: 324–328. 24. Gilmour A, Gogel B, Cullis, Thompson R (2009) ASReml user guide Release 3.0 URL. Available: http://wwwvsnicouk/downloads/asreml/release2/doc/ asreml-Rpdf. Accessed 2014 Feb 27. 9. Bayless DW, Darling JS, Daniel JM (2013) Mechanisms by which neonatal testosterone exposure mediates sex differences in impulsivity in prepubertal rats. Hormones and Behavior 64: 764–769. 25. R Development Core Team (2011) R: A language and environment for statistical computing R Foundation for Statistical Computing. Available: http:// wwwR-projectorg/. Accessed 2014 Feb 27. 10. Monclu´s R, Blumstein DT (2012) Litter sex composition affects life-history traits in yellow-bellied marmots. 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Science 208: 597–599. 29. Monclu´s R, Cook T, Blumstein DT (2011) Masculinized female yellow-bellied marmots initiate more social interactions. Biology Letters 8: 208–210. 30. Chawnshang C (2002) Androgens and Androgen Receptor: Mechanisms, Functions, and Clinical Applications. New York: Springer. 503 p. 14. Zehr JL, Gans SE, McClintock MK (2001) Variation in reproductive traits is associated with short anogenital distance in female rats. Developmental Psychobiology 38: 229–238. 31. vom Saal FS (1989) Sexual differentiation in litter-bearing mammals: influence of sex of adjacent fetuses in utero. Journal of Animal Science 67: 1824–1840. 15. Palanza P, Parmigiani S, vom Saal FS (1995) Urine marking and maternal aggression of wild female mice in relation to anogenital distance at birth. Physiology and Behavior 58: 827–835. 32. References 17. Drickamer LC (1996) Intra-uterine position and anogenital distance in house mice: consequences under field conditions. Animal Behaviour 51: 925–934. 1. Wolf JB, Wade MJ (2009) What are maternal effects (and what are they not)? Philosophical Transactions of the Royal Society Series B 364: 1107–1115. 18. Ba´nszegi O, Szenczi P, Dombay K, Bilko´ A, Altba¨cker V (2012) Anogenital distance as a predictor of attractiveness, litter size and sex ratio of rabbit does. Physiology and Behavior 105: 1126–1230. 2. Ro¨del HG, Starkloff A, Bruchner B, von Holst D (2008) Social environment and reproduction in female European rabbits (Oryctolagus cuniculus): Benefits of the presence of litter sisters. Journal of Comparative Psychology 122: 73–83. 19. Eisenberg ML, Hsieh MH, Walters RC, et al. (2011) The Relationship between Anogenital Distance, Fatherhood, and Fertility in Adult Men. PLoS ONE 6(5): e18973. 3. Duckworth RA (2009) Maternal effects and range expansion: a key factor in a dynamic process? Philosophical Transactions of the Royal Society Series B 364: 1075–1086. 20. Mendiola J, Roca M, Minguez-Alarcon L, Mira-Escolanao M-P, Lo´pez-Espin JJ, et al. (2012) Anogenital distance is related to ovarian follicular number in young Spanish women: a cross-sectional study. Environmental Health 11: 90. 4. Monclu´s R, Tiulim J, Blumstein DT (2011) Older mothers follow conservative strategies under predator pressure: The adaptive role of maternal glucocorticoids in yellow-bellied marmots. Hormones and Behavior 60: 660–665. 21. Blumstein DT, Lea AJ, Olson LE, Martin JGA (2010) Heritability of anti- predatory traits: vigilance and locomotor performance in marmots. Journal of Evolutionary Biology 23: 879–887. 5. Garnier R, Boulinier T, Gandon S (2012) Coevolution between maternal transfer of immunity and other resistance strategies against pathogens. Evolution 66: 3067–3078. 22. Armitage KB (2003) Marmots: Marmota monax and allies. In: Feldhamer GA, Thompson BC, Chapman JA, editors. Wild mammals of North America. The John Hopkins University Press, Baltimore, pp. 188–210. 6. Ryan BC, Vandenbergh JG (2002) Intrauterine position effects. Neuroscience and Biobehavioral Reviews 26, 665–678. 7. Even MD, Dhar MG, vom Saal FS (1992) Transport of steroids between fetuses via amniotic fluid in relation to the intrauterine position phenomenon in rats. Journal of Reproduction and Fertility 96: 709–716. 23. Vandenbergh J (2009) Effects of intrauterine position in litter-bearing mammals. In: Maestripieri D, Mateo JM, editors. Maternal Effects in Mammals. Chicago: The University of Chicago Press. pp. 203–226. 8. Acknowledgments We thank all the members of Team Marmot who helped collect data over the past decade. Maternal Effects on Anogenital Distance Nevertheless, further quantitative measurements of maternal and pup androgen levels, in addition to formal genetic analyses of maternal endocrinological traits are needed to formally evaluate the mechanism underlying the maternal genetic effect on AGD. Such studies, beyond the scope of our study of free-living animals, would greatly improve our understanding of the mechanistic origin of the maternal effect we identified. variation. Despite extensive training, personnel turnover between years could be responsible for some of this variation. However, other biologically relevant factors could also explain annual variation. For instance, the current environment (food availability, predation level, weather conditions) could also explain annual variation through its influence on maternal stress and body condition [4,10]. In conclusion, AGD is a widely used proxy of early exposure to testosterone, and it is related to numerous other life-history traits [4,10,16,38]. We showed that by decomposing sources of variation using the animal model, we were able to identify, for the first time in a wild population, both an additive genetic and a maternal genetic effect on AGD that is expected to also impact future life- history traits and fitness of the pups. When other researchers quantify the magnitude of maternal effects on AGD and its genetic basis, we will be in a better position to understand its general importance. Author Contributions While the existence of a significant litter composition fixed effect agrees with previous findings [10], it is noteworthy that the bulk of the remaining variation in AGD variance was explained by annual Conceived and designed the experiments: JGAM. Performed the experiments: TDF DTB RM JGAM. Analyzed the data: TDF DTB RM JGAM. Wrote the paper: TDF DTB RM JGAM. Conceived and designed the experiments: JGAM. Performed the experiments: TDF DTB RM JGAM. Analyzed the data: TDF DTB RM JGAM. Wrote the paper: TDF DTB RM JGAM. Discussion We have shown that 6% of the remaining variation in the AGD of marmot pups is explained by a maternal effect. Our results also suggest that AGD is heritable and influenced by maternal genetic and non-maternal environmental effects, thus indicating that a mother’s genotype produces specific developmental conditions for her offspring. The presence of a maternal genetic effect on AGD could result from various mechanisms during gestation or lactation. It could be due to genetic differences in maternal care, maternal milk content or maternal stress. A more promising avenue of explanation might reside in utero during development. AGD is widely used as a proxy for prenatal androgen hormone exposure in rodents [14]. Prenatal exposure to androgens may influence vertebrate life-history traits [29–32], because organiza- tional and activational events take place at this stage. This androgen-sensitive prenatal developmental window is thus a key period that may influence life-history traits. Here, a possible Table 2. Estimates (with standard error) of variance components and proportion o Figure 1. Variance ratio and components estimated for random effects from anogenital distance models of juvenile marmots. The random effects were additive genetic (AG), maternal genetic (MG), maternal environmental (ME), litter (L) and year (Y) effects. N indicates that an effect was not fitted in the model and a 0 indicates that the effect was fitted but estimated as zero. * indicates significant effects. doi:10.1371/journal.pone.0092718.g001 Figure 1. Variance ratio and components estimated for random effects from anogenital distance models of juvenile marmots. The random effects were additive genetic (AG), maternal genetic (MG), maternal environmental (ME), litter (L) and year (Y) effects. N indicates that an effect was not fitted in the model and a 0 indicates that the effect was fitted but estimated as zero. * indicates significant effects. doi:10.1371/journal.pone.0092718.g001 March 2014 \| Volume 9 \| Issue 3 \| e92718 March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org 3 Maternal Effects on Anogenital Distance 1. Wolf JB, Wade MJ (2009) What are maternal effects (and what are they not)? Philosophical Transactions of the Royal Society Series B 364: 1107–1115. Maternal Effects on Anogenital Distance explanation of the maternal effects on the AGD would be such a hormonal transfer from the mother to her pups. However, some studies in laboratory rats, Rattus norvegicus, have found no correlation between maternal testosterone levels and fetal testos- terone levels. However, they did find a negative relationship between maternal testosterone levels and the capacity of nutrient transport across the placenta, resulting in reduced fetal growth [33]. Therefore, maternal testosterone might indirectly affect offspring AGD by influencing the offspring mass. In primates, in utero exposure to androgens has been explored mainly through the study of the 2d:4d ratio. There is evidence that up to 66% of the variance is explained by additive genetic effects. In addition, there is evidence of strong environmental effects and for shared environment effects (including maternal environment and part of maternal genetic effects) [34–37]. Our results are thus in line with previous studies suggesting additive genetic, maternal environment and maternal genetic effects on in utero exposition to androgens. Nevertheless, further quantitative measurements of maternal and pup androgen levels, in addition to formal genetic analyses of maternal endocrinological traits are needed to formally evaluate the mechanism underlying the maternal genetic effect on AGD. Such studies, beyond the scope of our study of free-living animals, would greatly improve our understanding of the mechanistic origin of the maternal effect we identified. explanation of the maternal effects on the AGD would be such a hormonal transfer from the mother to her pups. However, some studies in laboratory rats, Rattus norvegicus, have found no correlation between maternal testosterone levels and fetal testos- terone levels. However, they did find a negative relationship between maternal testosterone levels and the capacity of nutrient transport across the placenta, resulting in reduced fetal growth [33]. Therefore, maternal testosterone might indirectly affect offspring AGD by influencing the offspring mass. In primates, in utero exposure to androgens has been explored mainly through the study of the 2d:4d ratio. There is evidence that up to 66% of the variance is explained by additive genetic effects. In addition, there is evidence of strong environmental effects and for shared environment effects (including maternal environment and part of maternal genetic effects) [34–37]. Our results are thus in line with previous studies suggesting additive genetic, maternal environment and maternal genetic effects on in utero exposition to androgens. 38. Kerin TK, Vogler GP, Blizard DA Stout JT, McClearn GE, et al. (2003) Anogenital distance measured at weaning is correlated with measures of blood chemistry and behaviors in 450–day-old female mice. Physiology and Behavior 78: 697–702. 37. Paul SN, Kato BS, Cherkas LF, Andrew T, Spector TD (2006) Heritability of the second to fourth digit ratio (2d:4d): A twin study. Twin Research and Human Genetics 9: 215–219. g ( ) J y p gy 36. Nelson E, Voracek M (2010) Heritability of digit ratio (2D:4D) in rhesus macaques (Macaca mulatta). Primates 51: 1–5. References Field EF, Whishaw IQ, Pellis SM, Watson NV (2006) Play fighting in androgen- insensitive tfm rats: evidence that androgen receptors are necessary for the development of adult playful attack and defense. Developmental Psychobiology 48: 111–120. 16. Vandenbergh JG, Huggett CL (1995) The anogenital distance index, a predictor of the intrauterine position effects on reproduction in female house mice. Laboratory Animal Science 45: 567–573. 33. Sathishkumar K, Elkins R, Chinnathambi V, Gao H, Hankins GD, et al. (2011) Prenatal testosterone-induced fetal growth restriction is associated with PLOS ONE \| www.plosone.org March 2014 \| Volume 9 \| Issue 3 \| e92718 4 March 2014 \| Volume 9 \| Issue 3 \| e92718 yg 35. Voracek M, Dressler SG (2009) Brief communication: Familial resemblance in digit ratio (2D:4D). American Journal of Physical Anthropology 140: 367–380. down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology 9: 110. 34. Gobrogge KL, Breedlove SM, Klump KL (2008) Genetic and Environmental Influences on 2D:4D Finger Length Ratios: A Study of Monozygotic and Dizygotic Male and Female Twins. Archives of Sexual Behavior 37: 112–118. 35. Voracek M, Dressler SG (2009) Brief communication: Familial resemblance in digit ratio (2D:4D). American Journal of Physical Anthropology 140: 367–380. 36. Nelson E, Voracek M (2010) Heritability of digit ratio (2D:4D) in rhesus macaques (Macaca mulatta). Primates 51: 1–5. gy 34. Gobrogge KL, Breedlove SM, Klump KL (2008) Genetic and Environmental Influences on 2D:4D Finger Length Ratios: A Study of Monozygotic and Dizygotic Male and Female Twins. Archives of Sexual Behavior 37: 112–118. down-regulation of rat placental amino acid transport. Reproductive Biology and Endocrinology 9: 110. 37. Paul SN, Kato BS, Cherkas LF, Andrew T, Spector TD (2006) Heritability of the second to fourth digit ratio (2d:4d): A twin study. Twin Research and Human Genetics 9: 215–219. 38. Kerin TK, Vogler GP, Blizard DA Stout JT, McClearn GE, et al. (2003) Anogenital distance measured at weaning is correlated with measures of blood chemistry and behaviors in 450–day-old female mice. Physiology and Behavior 78: 697–702. Maternal Effects on Anogenital Distance March 2014 \| Volume 9 \| Issue 3 \| e92718 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5
https://openalex.org/W2396174284	https://eprints.leedsbeckett.ac.uk/id/eprint/4110/1/Joanne%20Trigwell.pdf	English	null	Effect of a sport-for-health intervention (SmokeFree Sports) on smoking-related intentions and cognitions among 9-10 year old primary school children: a controlled trial	BMC public health	2,016	cc-by	12,953	* Correspondence: c.e.mcgee@ljmu.ac.uk 1Centre for Public Health, Liverpool John Moores University, Henry Cotton Campus, 15-21 Webster Street, Liverpool L3 2AT, UK Full list of author information is available at the end of the article © 2016 McGee et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Effect of a sport-for-health intervention (SmokeFree Sports) on smoking-related intentions and cognitions among 9-10 year old primary school children: a controlled trial Ciara E. McGee1, Joanne Trigwell2, Stuart J. Fairclough3,6, Rebecca C. Murphy4, Lorna Porcellato1, Michael Ussher5 and Lawrence Foweather4 Citation: McGee, CE and Trigwell, J and Fairclough, SJ and Murphy, RC and Porcellato, L and Ussher, M and Foweather, L (2016) Effect of a sport-for-health intervention (SmokeFree Sports) on smoking-related intentions and cognitions among 9-10 year old primary school children: a controlled trial. BMC Public Health. ISSN 1471-2458 DOI: https://doi.org/10.1186/s12889-016-3048-3 Citation: McGee, CE and Trigwell, J and Fairclough, SJ and Murphy, RC and Porcellato, L and Ussher, M and Foweather, L (2016) Effect of a sport-for-health intervention (SmokeFree Sports) on smoking-related intentions and cognitions among 9-10 year old primary school children: a controlled trial. BMC Public Health. ISSN 1471-2458 DOI: https://doi.org/10.1186/s12889-016-3048-3 Link to Leeds Beckett Repository record: Link to Leeds Beckett Repository record: https://eprints.leedsbeckett.ac.uk/id/eprint/4110/ Link to Leeds Beckett Repository record: https://eprints.leedsbeckett.ac.uk/id/eprint/4110/ Document Version: Article (Published Version) Creative Commons: Attribution 4.0 Creative Commons: Attribution 4.0 Creative Commons: Attribution 4.0 The aim of the Leeds Beckett Repository is to provide open access to our research, as required by funder policies and permitted by publishers and copyright law. The Leeds Beckett repository holds a wide range of publications, each of which has been checked for copyright and the relevant embargo period has been applied by the Research Services team. We operate on a standard take-down policy. If you are the author or publisher of an output and you would like it removed from the repository, please contact us and we will investigate on a case-by-case basis. Each thesis in the repository has been cleared where necessary by the author for third party copyright. If you would like a thesis to be removed from the repository or believe there is an issue with copyright, please contact us on openaccess@leedsbeckett.ac.uk and we will investigate on a case-by-case basis. McGee et al. BMC Public Health (2016) 16:445 DOI 10.1186/s12889-016-3048-3 Background g Smoking is an addiction often initiated in childhood, with approximately 207,000 children taking up smoking each year in the UK [1]. The earlier a child starts to smoke, the higher their chances of becoming a regular smoker and the more difficult it becomes to quit [2]. Early onset of smoking and persisting in the habit in adulthood increases the risk of developing lung cancer and other smoking-related diseases [3, 4]. Preventing smoking uptake in children by de-normalising tobacco use is therefore a key public health priority for the UK Government [5], which is aiming for a tobacco-free gen- eration by 2025 [6]. Although only 0.3 % of 8-10 year old UK primary school children have ever smoked [7], some children develop intentions to start smoking [8]. Behavioural intentions to smoke are worth paying spe- cial attention to as they are theorised to be the first step in smoking initiation [9]. Intentions to smoke may be explained by individual smoking-related cognitions such as attitudes towards smoking and cigarette refusal self- efficacy [10–12]. Since children living in socially de- prived areas have a high intention to smoke [13] and harbour misconceptions about the harms of smoking [14], implementing a smoking prevention intervention in primary school may prevent children from starting to smoke. Schools are considered an appropriate setting for smok- ing prevention because they can provide an efficient means of reaching large numbers of children [15] and provide an opportunity to ‘set’ healthy and enduring patterns of behav- iour [16, 17]. Consequently, numerous school-based smok- ing prevention programmes have been developed and implemented over the last decade to discourage smoking uptake and deter regular use [18, 19]. Previous interven- tions have predominantly targeted adolescents, whilst pro- grammes that have been developed for primary school aged children have been implemented outside the UK [18, 19]. Waiting until secondary school to intervene with smoking prevention programmes can be too late, since by then ado- lescents may have developed deep rooted smoking expect- ancies and norms [20–22] and, for some the behaviour is already underway (8 % of 15 year olds smoke) [23]. SmokeFree Sports (SFS) was a sport-for-health smok- ing prevention intervention for youth in Liverpool, which is one of the most deprived communities in England [39] where addressing inequalities in tobacco use is a public health priority. (Continued from previous page) (Continued from previous page) Conclusion: SFS demonstrated short-term positive effects on smoking attitudes among children, and cigarette refusal self-efficacy among girls. Although no effects were observed for non-smoking intentions, children said that SFS made them more determined not to smoke. Most children had strong intentions not to smoke; therefore, smoking prevention programmes should perhaps target early adolescents, who are closer to the age of smoking onset. Keywords: Smoking, Prevention, Children, Intervention, School-based Keywords: Smoking, Prevention, Children, Intervention, School-based Interventions typically use participatory approaches like game-based learning and activities with sport coaches, who represent important role models for youth, to transmit health promotion messages and positively shape attitudes [28, 29]. The use of sport as an educa- tional platform for tobacco control has previously been trialled in the US and Canada with initiatives such as Tobacco Free Sports [30], Tobacco Free Athletes [31] and Play, Live, Be Tobacco Free [32]. Sport-for-health interventions have several potential benefits over trad- itional classroom-based smoking prevention education approaches. First, participation in physical activity improves health not only directly but also through its protective effect against smoking initiation in youth [33, 34]. Second, given that all primary school children are required to participate in physical education, this lesson could provide a forum to integrate smoking edu- cation messages within the primary school curriculum. Third, the use of sport as a smoking prevention strategy encourages active engagement with the intervention as well as interactions with other pupils and teaching staff, and is consistent with National Institute for Health and Care Excellence (NICE) recommendations [35] to de- liver interactive and participatory smoking prevention interventions. Finally, integrating physical activity into the learning process may enable children to efficiently retain and retrieve learned information [28, 36–38]. To the authors’ knowledge, no published study has evalu- ated the use of sport-for-health programmes for smok- ing prevention in the UK. Abstract Background: Preventing children from smoking is a public health priority. This study evaluated the effects of a sport-for-health smoking prevention programme (SmokeFree Sports) on smoking-related intentions and cognitions among primary school children from deprived communities. Methods: A non-randomised-controlled trial targeted 9-10 year old children from Merseyside, North-West England. 32 primary schools received a programme of sport-for-health activities over 7 months; 11 comparison schools followed usual routines. Data were collected pre-intervention (T0), and at 8 months (T1) and one year post-intervention (T2). Smoking-related intentions and cognitions were assessed using an online questionnaire. Intervention effects were analysed using multi-level modelling (school, student), adjusted for baseline values and potential confounders. Mixed-sex focus groups (n = 18) were conducted at T1. Results: 961 children completed all assessments and were included in the final analyses. There were no significant differences between the two study groups for non-smoking intentions (T1: β = 0.02, 95 % CI = -0.08–0.12; T2: β = 0.08, 95 % CI = -0.02–0.17) or for cigarette refusal self-efficacy (T1: β = 0.28, 95 % CI = -0.11–0.67; T2: β = 0.23, 95 % CI = -0.07–0.52). At T1 there was a positive intervention effect for cigarette refusal self-efficacy in girls (β = 0.72, 95 % CI = 0.21–1.23). Intervention participants were more likely to ‘definitely’ believe that: ‘it is not safe to smoke for a year or two as long as you quit after that’ (RR = 1.19, 95 % CI = 1.07–1.33), ‘it is difficult to quit smoking once started’ (RR = 1.56, 95 % CI = 1.38–1.76), ‘smoke from other peoples’ cigarettes is harmful’ (RR = 1.19, 95 % CI = 1.20–2.08), ‘smoking affects sports performance’ (RR = 1.73, 95 % CI = 1.59–1.88) and ‘smoking makes ‘no difference’ to weight’ (RR = 2.13, 95 % CI = 1.86–2.44). At T2, significant between-group differences remained just for ‘smoking affects sports performance’ (RR = 1.57, 95 % CI = 1.43–1.72). Focus groups showed that SFS made children determined to remain smoke free and that the interactive activities aided children’s understanding of smoking harms. (Continued on next page) McGee et al. BMC Public Health (2016) 16:445 Page 2 of 16 Background Established in October 2010, SFS was commissioned as part of the ‘SmokeFree Liverpool’ public health campaign. The intervention was designed in accordance with the NICE guidance [35] and the Medical Research Council (MRC) framework for developing and evaluating complex interventions [40]. Phase one of SFS (February-June 2011) was a commu- nity feasibility trial in five youth clubs, which received 12 weeks of coaching activities (dance, dodge-ball and boxing) delivered by trained sports coaches [29, 41]. A Sport-for-health programmes use sport as a mechan- ism to promote health and prevent disease [24–27]. Page 3 of 16 McGee et al. BMC Public Health (2016) 16:445 Page 3 of 16 McGee et al. BMC Public Health (2016) 16:445 Page 3 of 16 Page 3 of 16 formative evaluation demonstrated that the intervention helped to prevent youth from initiating smoking and had positive benefits on their attitudes and knowledge about smoking [29, 41]. However, coaches reported challenges associated with its delivery in youth clubs and recom- mended that the intervention be trialled in schools. Phase two (February-April 2012) therefore examined the feasibil- ity of a six week intervention in three primary schools [42]. Trained coaches delivered twelve sessions of sports (football and dance). Similar positive benefits for chil- dren were observed, whilst teachers and coaches per- ceived SFS to be acceptable for smoking prevention education [42]. These promising results led to the de- velopment of a larger, controlled trial to investigate the effectiveness of SFS in Liverpool primary schools. Study design A school-based non-randomised controlled trial was con- ducted to evaluate the effect of a sport-for-health smoking prevention intervention, SmokeFree Sports on children’s intentions (not) to smoke and smoking-related cognitions. Due to funding requirements, SFS was offered to all primary schools within the Liverpool City Council ad- ministrative boundaries and therefore an a priori sam- ple size calculation was not undertaken. Schools within Knowsley, another metropolitan borough in Merseyside with similar characteristics to Liverpool in terms of adult smoking rates (Liverpool: 24.2 %; Knowsley: 27.6 %) [44], deprivation levels [45] and ethnic composition [46], were recruited as comparison schools. For logistical reasons, it was not possible to blind the research team to the group- allocation. Schools were clustered into two groups: The present study evaluated whether SFS, a sport- for-health smoking prevention intervention, is effect- ive in increasing non-smoking intentions in 9-10 year old primary school children from Liverpool, immedi- ately post-intervention and at a follow-up one year later. Secondary aims were to investigate the impact of the intervention on children’s attitudes towards smoking and cigarette refusal self-efficacy, termed smoking-related cognitions hereafter. The study also investigated whether sex moderated the intervention effects as differences in cognitive vulnerability towards smoking have been found between preadolescent boys and girls [14]. In addition, focus groups with children were conducted to produce more complete knowledge to inform interpretations of intervention effectiveness. Since sport-for-health interventions are an emergent area of health promotion research where evaluations are sparse and/or have lacked methodological rigour [26, 43], it is recognised that rigorous evaluations of interventions are needed to inform future practice and procedures [25]. 1. Intervention group (Liverpool): Schools received their usual smoking-related education plus SFS g 2. Comparison group (Knowsley): Schools received their usual smoking education only A schematic overview of the intervention and evaluation components is shown in Fig. 1. Data collection occurred over 20 months with measurements at baseline (T0, September and October 2012) and post-intervention (T1, June 2013) whilst children were in Year 5 of primary school, and at one year after the intervention had finished (T2, June 2014; Year 6 of primary school). Ethical approval for the study was granted by Liverpool John Moores University Research Ethics Committee (12/SPS/038). Participants and recruitment p In September 2012, all eligible primary schools (main- stream state schools; n = 154), from Liverpool (n = 104) Timeline Intervention schools Comparison schools Legend Baseline [T0] Sept-Oct 2012 Child smoking questionnaire Child carbon monoxide breath reading Teacher/coach self-efficacy questionnaire Training workshop for teachers/coaches Multi-skill activity session Direct observation of coaching sessions Interviews with teachers Dance session Football session x 2 School assembly with sports star Focus groups with children Teacher evaluation forms Interviews with coaches 0-2 Months Nov-Dec 2012 3-4 Months Jan-Feb 2013 5-7 Months Mar-May-2013 Post-intervention [T1] Jun 2013 One year follow-up [T2] Jun 2014 A B D C C E J F G G H F I L H F I A B M A B A B A B A B K C A B C D E F G H I J K L M Fig. 1 Schematic overview of SmokeFree Sports intervention and evaluation components Fig. 1 Schematic overview of SmokeFree Sports intervention and evaluation components McGee et al. BMC Public Health (2016) 16:445 Page 4 of 16 Page 4 of 16 and Knowsley (n = 50), were invited to participate in the study via email, post and telephone. Once schools had given written informed consent to participate in the study, all Year 5 children (aged 9–10 years; n = 1393) were invited to take part. Parents/guardians received a letter containing a parent and child information sheet and opt-out form. Parents/guardians were given contact information for the research team to discuss the project and could opt their child out of the study by telephone or by signing and returning the opt-out form. At data collection, children were given a verbal explanation of the study and asked to give signed assent. Children could withdraw from the research study at any time. Measures The primary outcome measure was intentions (not) to smoke of the participating children; secondary outcomes included individual smoking-related cognitions (attitudes and refusal self-efficacy). Outcomes were assessed based on a self-reported questionnaire completed by children at T0, T1 and T2, and through focus groups with chil- dren, which were conducted at T1 only due to funding restrictions. Comparison group Children in the comparison group were requested to fol- low their usual smoking education. It is not mandatory to address smoking education in Key Stage 2 (pupils aged 7–11) of the UK National Curriculum [53], and it is at schools’ discretion to include the topic as part of Personal Social Health and Economic education. On completion of data collection at one year follow-up, comparison schools received a copy of the SFS training manual, and children were given a SFS water bottle, drawstring bag and pen for participating in the re- search study. Intervention The SFS intervention was delivered during school hours in Liverpool primary schools between October 2012 and May 2013. The intervention focused on smoking pre- vention and therefore Year 5 children (aged 9-10) were identified as an important cohort to target. Further, NICE [35] guidelines postulate that smoking prevention efforts would be most effective if they began in primary school. A detailed description of the SFS intervention has been published elsewhere [47, 48]. Briefly, the socio- ecological model [49] and cognitive theories including the Health Belief Model [50], Theory of Planned Be- haviour [9] and Social Cognitive/Learning [51], guided the intervention design. The intervention aimed to im- plement a programme of smoking prevention actions through fun, participatory and interactive sports activ- ities delivered by teachers and coaches. Intervention components included provision of professional devel- opment training, a training manual including 10 ses- sion plans, five coaching sessions delivered by SFS coaches, a school assembly with a local sports star, sports equipment as incentives for teachers to deliver and evaluate a minimum of five SFS sessions, a smoke free pledge for children to sign, and incentives for chil- dren for participating in the research (SFS water bottle, drawstring bag and pen). Behaviour change techniques [52] used with children included a behavioural con- tract (smoke free pledge); social support, advice, verbal persuasion and positive reinforcement (from peers, teachers and coaches) on remaining never smokers; in- formation and salience of the social, emotional and health consequences of smoking; an exploration of the pros and cons of smoking; awareness raising of regret children will feel if they smoke; social comparisons with peers to dispel myths that smoking is normative; modelling of never smoking (from elite athletes); cigarette refusal rehearsals, and the promotion of phys- ical activity as a positive distraction to smoking. Focus groups with children intention not to smoke. Cronbach alpha for total intention showed good internal consistency (α = 0.81). Eighteen mixed-sex focus groups with children (n = 95; 45 % boys) were facilitated by trained researchers imme- diately following the intervention [T1]. Focus groups comprised of five to six children, lasted between 30 and 50 min and were audio recorded using a Dictaphone. Children’s perceptions of smoking, appropriateness of the intervention, and improvements for future imple- mentation were explored. Photographs of SFS games were used to help children recall activity type [62, 63]. To aid the credibility of data, facilitators’ reflected inter- pretations back to children during the focus groups. The present study focuses on children’s perceptions sur- rounding the impact of SFS on intentions (not) to smoke and individual smoking-related cognitions, thus other findings are discussed in the process evaluation paper, which has been published elsewhere [64]. Refusal self-efficacy was measured using three items adapted from a nine-item self-efficacy scale in adoles- cents [54]. Items assessed the child’s confidence in their ability to be a non-smoker and refuse cigarettes in dif- ferent situations. Responses consisted of Likert scales ranging from ‘not confident at all’ (1) to ‘very confident’ (5) and were summed to create a total refusal self-effi- cacy score (range 3–15). Cronbach alpha for the combined scale showed good internal consistency (α = 0.81). A high score on the scale indicated a high level of refusal self- efficacy. Attitude structure includes affective, behavioural and cognitive components [60]. For the purpose of this study, the cognitive component of children’s attitudes was ex- plored through items adapted from the Global Youth To- bacco Survey (GYTS) [56] and the Health Survey for England [57], including ‘Do you think smoking is bad for your health?’, ‘Once someone has started smoking, do you think it will be difficult to quit?’, ‘Do you think that it is safe to smoke for only a year or two as long as you quit after that?’, ‘Do you think the smoke from other people’s cigarettes is harmful to you?’. An additional item ‘Do you think smoking effects sport performance?’ was developed by the research team. Responses ranged from ‘definitely not’ (1) to ‘definitely yes’ (4). A sum- mary scale was created but internal consistency was low (α = .49). Focus groups with children Since the data for individual attitude items were positively skewed and distribution was not improved by statistical transformation, responses were collapsed into dichotomous variables for analyses: a definitive negative attitude towards smoking (i.e. ‘defin- itely yes’) was scored 1; the remaining response cat- egories (i.e. ‘probably yes’, ‘probably not’ and ‘definitely not’) indicated a more favourable attitude towards smoking and thus were collapsed into a single group and scored 0. One attitude item (‘Do you think that it is safe to smoke for only a year or two as long as you quit after that?’) was reverse coded in order to main- tain consistent scale direction for all items. An add- itional attitude item, ‘Do you think smoking makes you gain weight?’ was also included from the Health Survey for England [57]. Whilst it is recognised that smoking is associated with weight loss [61], smoking is widely discouraged by public health professionals for weight control. Therefore, a key message included within the curriculum was that regular physical activity and healthy eating, but not smoking, was important for maintenance of a healthy weight. Thus responses for this item were collapsed into a dichotomous variable for analysis with ‘no difference’ scored 1 and the remaining response categories (i.e., ‘lose weight’ or ‘gain weight’) grouped and scored 0 Smoking questionnaire g q A questionnaire was constructed using items adapted from questionnaires previously used with this age group [54–57]. Background demographic information has been described in detail elsewhere [14]. Briefly, demographics assessed included age (years), gender (1 = girl, 0 = boy), ethnicity (1 = white British, 0 = other) and SES (home postcodes were used to generate indices of multiple deprivation (IMD) scores [39]. Children’s enjoyment of physical activity was assessed using the 16 item Physical Activity Enjoyment Scale [58]. Parent, sibling and friend smoking behaviour (1 = smokes (parent/sibling/friend) or tried (friend only), 0 = non-smoker) was assessed using an item from the Health Survey for England [57]. Child smoking behaviour (0 = never smoked, 1 = ever smoked) was also measured for descriptive purposes using a single item from the Health Survey for England [57]. As an indicator of smoking status, expired carbon monoxide (CO) concentrations were taken in private and recorded using a piCOsimple Smokerlyzer (Bedfont Scientific UK, England) with a reading above 10 ppm used as cut-off for defining smokers [59]. Intention (not) to smoke was assessed using two items from the Health Survey for England [57], ‘Do you think you will smoke in the next month/year?’, as well as an item designed by the research team ‘Do you think you will smoke in secondary school?’. Responses ranged from ‘definitely yes’ (1) to ‘definitely not’ (4) and were summed to produce a total intention score (range 3– 12). A high score on total intention indicated a strong McGee et al. BMC Public Health (2016) 16:445 Page 5 of 16 Page 5 of 16 Page 5 of 16 Analyses y Participants with missing data at either post-intervention [T1] or follow-up [T2] were not considered in the analyses (i.e., a complete case analysis). To describe the demo- graphic characteristics of children at baseline [T0] and dif- ferences concerning primary (smoking intentions) and secondary outcomes (attitudes towards smoking and re- fusal self-efficacy), general descriptive analyses were con- ducted. Independent t-tests and chi-square tests were used to assess whether the primary and secondary out- comes differed between the study groups at baseline, and to assess differences between those participants included and excluded from the final analysis. Multilevel linear and logistic regression analyses examined intervention effects on the primary and secondary outcomes. To account for the clustering effect among children being nested in schools, a two-level data structure was conducted. Chil- dren were defined as the first level unit of analysis and schools the second level unit of analysis [65]. Two ana- lyses were conducted for each of the outcome variables to examine the intervention effects. The first analysis deter- mined the difference between the intervention and com- parison group adjusting for baseline value of the outcome measure (‘crude’ analysis). The second analysis determined this effect when the covariates were added to the model (‘adjusted’ analysis); these covariates included age, eth- nicity, deprivation level, mother/father/sibling/friend smoking, intentions to smoke and individual smoking- related cognitions, since these variables may influence each other [9, 51]. Additionally, physical activity enjoy- ment was adjusted for in the analysis because we hypothesised that children who enjoy physical activity may be more amenable to a sport-for-health interven- tion. In addition, separate analyses for boys and girls were performed to assess intervention effects between baseline and post-intervention, and baseline and one McGee et al. BMC Public Health (2016) 16:445 Page 6 of 16 Page 6 of 16 pre-defined from topics covered in the group schedule. Subsequently, broad codes were collapsed into higher and lower order themes and descriptive and interpretive sum- maries were written based on recursive engagement with the data. A combination of inductive analysis and de- ductive techniques were used to generate codes. To aid the credibility and trustworthiness of the results, ana- lyses and interpretations of the data were discussed amongst three members (CM, JT and LF) of the re- search team [67]. year follow-up. To determine whether the intervention effect was different for boys and girls, a dichotomous interaction term (labelled ‘sex’) was constructed. Analyses Regres- sion coefficients in each model were assessed for signifi- cance using the Wald statistic with one degree of freedom. As the prevalence of negative attitudes towards smok- ing was high in both study arms, odds ratios were con- verted to relative risks [66] to avoid overestimation of effects and for ease of interpretation of results. Ana- lyses were performed using IBM SPSS Statistics v.22 and MLwiN 2.30 software (Centre for Multi-level Mod- elling, University of Bristol, UK). Statistical significance was set at p < 0.05, and at p < 0.10 for the sex inter- action term [66]. Results However, the withdrawal of two intervention schools due to internal staffing issues excluded 68 children. Had the schools not withdrawn, assuming all children would have continued through the study, the retention at follow-up would have been 74 %. Compared with interven- tion children included in the analyses, a higher proportion of intervention children that were excluded from the ana- lyses had a sibling that smoked (p < 0.01) and a lower pro- portion believed that smoking is bad for health (p < 0.05). Other baseline values did not differ between those included and excluded. Baseline characteristics for the final sample of child participants (mean age 9.6 ± 0.3 years, 50.4 % female, 98.3 % White British) are shown in Table 1. Over seven in ten (71.9 %) participating children lived within areas Smoking behaviour data is reported for descriptive purposes. Results Figure 2 shows the flow of schools and participants through the trial. In total, 43 schools participated in the study (27.9 % response rate), including 32 (31 %) from Liverpool and 11 (22 %) from Knowsley. Schools that de- clined to participate provided diverse reasons for not tak- ing part (e.g., too busy, key teacher on sick leave, already in Child focus groups were transcribed verbatim, imported into NVivo 10 software, and subjected to thematic analysis [67]. This process involved reading and re-reading text and assigning broad thematic codes, some of which were Fig. 2 Flow of schools and participants through the study. Six children were absent at both post-intervention and follow-up ig. 2 Flow of schools and participants through the study. Six children were absent at both post-intervention and follow-up McGee et al. BMC Public Health (2016) 16:445 Page 7 of 16 Page 7 of 16 ranked within the highest 10 % for deprivation in England. The majority of children reported to have never smoked at T0 (97.5 %: comparison group, 96.3 %; intervention group, 97.9 %). CO readings were recorded for 82 % of participants and confirmed self-reported non-smoking status with all readings below 10 ppm (Mean = 1.3 ± 0.7 ppm). Over half of children (57.3 %) reported that at least one family member was a current smoker, whilst al- most one in five children reported having a friend who smoked. Children generally had strong non-smoking in- tentions and high refusal self-efficacy expectations, though 40-58 % of children displayed more favourable attitudes towards smoking on five of the six attitude items. Children in the intervention group were less likely to be White British and were significantly more deprived (p < 0.01) than children in the comparison group. A higher propor- tion of intervention children, in particular girls, definitely believed that smoking was difficult to quit (p < 0.01) and that smoking affects sports performance (p < 0.05). No other significant group differences were found at T0. receipt of external projects). Of the 1393 potentially eligible children at T0, 1143 completed baseline measures (92 % response rate); 961 children completed assessments at T0, T1, and T2 and were included in the final analyses (84 % participation rate). Participant retention ranged from 80 % (T0) to 79 % (T2) in the comparison group. The interven- tion group’s retention ranged from 83 % at baseline to 68 % at T2. Notes: IMD, Indices of multiple deprivation score; aat least one friend smokes or tried Cigarette refusal self-efficacy The adjusted multilevel linear regression analyses showed no significant intervention effects between base- line and T1 (Table 2), and between baseline and T2 (Table 3), for refusal self-efficacy. Non-smoking intentions The adjusted multilevel linear regression analyses indi- cated no significant intervention effects on non-smoking intentions between baseline and T1 (Table 2), and be- tween baseline and T2 (Table 3). Results Self-reported smoking prevalence at T1 Table 1 Sample characteristics of children at baseline Comparison Intervention All (n = 241) M ± SD or % Boys (n = 114) M ± SD or % Girls (n = 127) M ± SD or % All (n = 720) M ± SD or % Boys (n = 363) M ± SD or % Girls (n = 357) M ± SD or % Demographics Age (years) 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 Ethnicity (White British) 98.3 99.1 97.6 82.1 82.6 81.5 Deprivation level (IMD) 50.9 ± 17.9 49.8 ± 17.7 51.9 ± 18.2 55.7 ± 16.4 55.5 ± 16.5 55.8 ± 16.3 Enjoyment of physical activity PACES enjoyment scale (range 1–5) 4.3 ± 0.7 4.3 ± 0.7 4.3 ± 0.7 4.3 ± 0.7 4.2 ± 0.8 4.4 ± 0.6 Smoking intentions Total non-smoking intentions (range 4–12) 11.7 ± 1.0 11.6 ± 1.1 11.7 ± 0.9 11.8 ± 0.8 11.7 ± 1.0 11.9 ± 0.5 Self-efficacy Total refusal self-efficacy (range 3–15) 13.5 ± 3.2 13.5 ± 3.1 13.5 ± 3.3 13.7 ± 3.0 13.4 ± 3.3 14.0 ± 2.7 Attitudes towards smoking Smoking is bad for health (‘definitely yes’) 88.8 87.7 89.8 90.1 86.2 94.1 Safe to smoke year or two (‘definitely not’) 59.8 60.5 59.1 64.4 63.6 65.3 Difficult to quit once started (‘definitely yes’) 43.2 45.6 40.9 52.8 51.2 54.3 Others smoke harmful to you (‘definitely yes’) 59.3 60.5 58.3 66.1 63.1 69.2 Affects sports performance (‘definitely yes’) 51.0 53.5 48.8 58.3 59.2 57.4 Makes you gain/lose weight (‘no difference’) 42.3 37.7 46.5 42.6 45.2 40.1 Social influences Mother smoking 40.7 39.5 41.7 34.9 32.2 37.5 Father smoking 43.6 47.4 40.2 38.2 38.0 38.4 Sibling smoking 10.8 10.5 11.0 9.2 8.0 10.4 Friend smokinga 18.7 25.4 12.6 17.1 22.9 11.2 Notes: IMD Indices of multiple deprivation score; aat least one friend smokes or tried Table 1 Sample characteristics of children at baseline McGee et al. BMC Public Health (2016) 16:445 Page 8 of 16 Page 8 of 16 and T1 (Table 2) for four attitude towards smoking items. Results At T1, compared with children in the comparison group, children that participated in the SFS intervention were more likely to ‘definitely’ believe that: ‘it is not safe to smoke for a year or two as long as you quit after that’ (RR = 1.19, 95 % CI 1.07 to 1.33, p < 0.001), ‘it is difficult to quit smoking once started’ (RR = 1.56, 95 % 1.38 to 1.76, p < 0.001), ‘smoke from other peoples’ cigarettes is harmful to you’ (RR = 1.19, 95 % CI 1.20 to 2.08, p < 0.001), and ‘smoking affects sports performance’ (RR = 1.73, 95 % CI 1.59 to 1.88, p < 0.001). In addition, a moderate positive intervention effect was observed on the attitude item: ‘smoking makes ‘no difference’ to weight’ (RR = 2.13, 95 % CI 1.86 to 2.44, p < 0.001). Between baseline and T2 (Table 3), significant between-group differences remained on only one of the six attitude items. Com- pared with children in the comparison group, children who received the SFS intervention were more likely to ‘definitely’ believe that ‘smoking affects sports perform- ance’ (RR = 1.57, 95 % CI, 1.43, 1.72, p < 0.001), a small positive intervention effect. (comparison group, 97.5 %; intervention group, 98.6 %) and T2 (comparison group: 97.1 %, intervention group, 98.2 %) remained similar to T0, suggesting that low rates of smoking continued over time. Notes: β = beta coefficient; RR = relative risk; CI = confidence interval, I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention Si ifi i i ff (P ) bAdjusted for group and baseline value of the outcome measure g aUnadjusted within-group mean difference (post-intervention minu b bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity d th /f th / ibli /f i d ki Additionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk p usted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physic er/sibling/friend smoking dβ value eRelative risk bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level sex age ethnicity other individual smoking-related cognitions at baseline enjoyment of physical activity Attitudes towards smoking The adjusted multilevel logistic regression analyses indi- cated small positive intervention effects between baseline Table 2 Multilevel analyses of the effectiveness of the SmokeFree Sports intervention between baseline (T0) and post-intervention (T1) Mean differencea (T1-T0: M ± SD or %) Crude modelb Adjusted modelc Outcome measure β or RR (95 % CI) P β or RR (95 % CI) P Smoking intentions Total non-smoking intentions I 0.03 ± 0.98 0.03d (−0.07, 0.13) 0.51 0.02d (−0.08, 0.12) 0.71 C 0.07 ± 0.95 Cigarette refusal self-efficacy Total refusal self-efficacy I 0.29 ± 3.52 0.28d (−0.10, 0.67) 0.15 0.28d (−0.11, 0.67) 0.17 C 0.15 ± 3.77 Attitudes towards smoking Smoking is bad for health (‘definitely yes’) I 3.8 1.03e (0.99, 1.06) 0.19 1.03e (0.99, 1.06) 0.15 C 2.5 Safe to smoke…year or two (‘definitely not’) I 3.1 1.23e(1.11, 1.36) <0.001* 1.19e (1.07, 1.33) 0.01 C −5.0 Difficult to quit once started (‘definitely yes’) I 15.4 1.58e (1.41, 1.78) <0.001* 1.56e (1.38, 1.76) <0.001* C −0.5 Others smoke is harmful to you (‘definitely yes’) I 2.4 1.19e (1.07, 1.31) <0.01* 1.19e (1.20, 2.08) <0.01* C −2.5 Affects sports performance (‘definitely yes’) I 30.3 1.70e (1.58, 1.85) <0.001* 1.73e (1.59, 1.88) <0.001* C 0.9 Makes you gain or lose weight (‘no difference’) I 26.3 2.11e (1.85, 2.41) <0.001* 2.13e (1.86, 2.44) <0.001* C −9.1 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval, I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention Significant intervention effect (P < 0 05) McGee et al. Attitudes towards smoking BMC Public Health (2016) 16:445 Page 9 of 16 Table 3 Multilevel analyses of the effectiveness of the SmokeFree Sports intervention between baseline (T0) and one year follow-up (T2) Mean differencea (T2-T0: M ± SD or %) Crude modelb Adjusted modelc Outcome measure β or RR (95 % CI) P β or RR (95 % CI) P Smoking intentions Total non-smoking intentions I 0.06 ± 0.97 0.08d (−0.02,0.18) 0.13 0.08d (−0.02,0.17) 0.14 C 0.06 ± 1.18 Cigarette refusal self-efficacy Total refusal self-efficacy I 0.56 ± 3.23 0.27d (−0.02,0.56) 0.07 0.23d (−0.07,0.52) 0.13 C 0.45 ± 3.73 Attitudes towards smoking Smoking is bad for health (‘definitely yes’) I 5.5 0.98e (0.96,1.01) 0.28 0.99e (0.97,1.01) 0.42 C 8.3 Safe to smoke…year or two (‘definitely not’) I −0.6 1.05e (0.95,1.16) 0.42 1.03e (0.93,1.15) 0.65 C 0.4 Difficult to quit once started (‘definitely yes’) I 5.8 1.15e (1.02,1.29) 0.05 1.15e (1.02,1.3) 0.06 C 6.6 Others smoke is harmful to you (‘definitely yes’) I 0.0 1.14e (1.08,1.81) 0.03 1.13e (1.05,1.79) 0.05 C −2.0 Affects sports performance (‘definitely yes’) I 23.2 1.55e (1.42,1.69) <0.001 1.57e (1.43,1.72) <0.001* C 1.3 Makes you gain or lose weight (‘no difference’) I −2.0 1.05e (0.90,1.22) 0.60 0.98e (0.83,1.16) 0.84 C −3.7 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (one year follow-up minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (one year follow-up minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk in the future because of social norms, and or using smoking as a coping mechanism for managing stress: Sex interaction effects Tables 4 and 5 show the results of the sex interaction analyses between baseline and T1, and between base- line and T2, respectively. Between baseline and T1, sex moderated the intervention effects for cigarette refusal self-efficacy; a significant small positive inter- vention effect was found in girls (β = 0.72, 95 % CI 0.21 to 1.23, p < 0.01) but not boys (β = -0.18, 95 % CI -0.77 to 0.41, p = 0.54). No other sex interactions were observed. “I don’t want to smoke when I’m older but I’ll probably end up changing my mind because I want to be like one of my sisters who smokes” (Girl, School 1). “I’m not saying I definitely won’t [smoke] because it’s just something that might happen if something stressful happens” (Boy, School 2). Qualitative findings During group discussions children were able to recall the health messages delivered during the intervention, particularly in relation to the health implications associ- ated with smoking, its impact on sport performance, the chemical properties in a cigarette and its addictive na- ture, and its impact on weight (see Table 7). During focus groups the majority of children articu- lated that SFS made them more determined not to smoke in the future. Children’s reasons for not smoking surrounded some of the key messages received and or activities played during the intervention (see Table 6 for a summary of children’s reasons for their non-smoking intentions). During group discussions some children expressed a level of uncertainty regarding their future smoking behaviour and verbalised that they may smoke Discussion This study examined the short and medium term effects of a sport-for-health intervention (SFS) on 9-10 year old McGee et al. BMC Public Health (2016) 16:445 Page 10 of 16 Table 4 Multilevel analyses exploring interaction effects by sex between baseline (T0) and post-intervention (T1) Mean differencea (T1-T0: M ± SD or %) Intervention* sexb (crude model) Boysc Girlsc Outcome measure Boys Girls β or RR (95 % CI) P β or RR (95 % CI) P β or RR (95 % CI) P Smoking intentions Total non-smoking intentions I 0.10 ± 1.2 −0.04 ± 0.7 0.10d (−0.10, 0.30) 0.32 n/a − n/a - C 0.16 ± 1.0 0.00 ± 0.9 Cigarette refusal self-efficacy Total refusal self-efficacy I 0.32 ± 3.9 0.25 ± 3.2 0.87d (0.09, 1.64) 0.03* −0.18d (−0.77, 0.41) 0.54 0.72d (0.21, 1.23) <0.01* C 0.39 ± 3.6 −0.07 ± 3.8 Attitudes towards smoking Smoking is bad for health (‘definitely yes’) I 5.5 2.0 1.05e (1.00, 1.11) 0.12 n/a - n/a - C 4.4 0.8 Safe to smoke…year or two (‘definitely not’) I 0.9 5.3 1.16e (0.95, 1.42) 0.23 n/a - n/a - C −4.4 −5.6 Difficult to quit once started (‘definitely yes’) I 17.4 13.5 1.19e (0.90, 1.57) 0.30 n/a - n/a - C 2.6 −3.1 Others smoke harmful to you (‘definitely yes’) I 3.3 1.4 1.14e (0.84, 2.23) 0.29 n/a - n/a - C −0.9 −4.0 Affects sports performance (‘definitely yes’) I 30.3 30.3 1.16e (0.90, 1.50) 0.34 n/a - n/a - C 5.3 −3.1 Makes you gain or lose weight (‘no difference’) I 23.4 29.1 1.21e (0.79, 2.31) 0.36 n/a - n/a - C −2.6 −15.0 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyses were significant, adjusted analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (post-intervention minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk ses exploring interaction effects by sex between baseline (T0) and post-intervention (T1) Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Discussion BMC Public Health (2016) 16:445 Page 11 of 16 Table 5 Multilevel analyses exploring interaction effects by sex between baseline (T0) and one year follow-up (T2) Mean differencea (T2-T0: M ± SD or %) Intervention sexb (crude model) Boysc Girlsc Outcome measure Boys Girls β or RR (95 % CI) P β or RR (95 % CI) P β or RR (95 % CI) P Smoking intentions Total non-smoking intentions I 0.10 ± 1.2 0.00 ± 0.7 −0.01d (−0.21, 0.19) 0.89 n/a - n/a - C 0.06 ± 1.4 0.06 ± 0.9 Cigarette refusal self-efficacy Total refusal self-efficacy I 0.84 ± 3.4 0.27 ± 3.0 −0.20d(−0.78, 0.39) 0.51 n/a - n/a - C 0.37 ± 3.8 0.53 ± 3.7 Attitudes towards smoking Smoking is bad for health (‘definitely yes’) I 8.3 2.5 1.02e (1.00, 1.04) 0.17 n/a - n/a - C 10.5 6.3 Safe to smoke…year or two (‘definitely not’) I −0.5 −0.9 0.94e (0.76, 1.17) 0.64 n/a - n/a - C −2.6 3.1 Difficult to quit once started (‘definitely yes’) I 7.8 4.0 1.03e (0.81, 1.31) 0.84 n/a - n/a - C 6.2 7.1 Others smoke harmful to you (‘definitely yes’) I 1.6 −1.7 1.11e (0.77, 2.15) 0.41 n/a - n/a - C −0.9 −3.2 Affects sports performance (‘definitely yes’) I 25.1 21.3 1.05e (0.83, 1.32) 0.73 n/a - n/a - C 5.3 −2.3 Makes you gain or lose weight (‘no difference’) I −2.5 −1.7 0.99e (0.26, 3.70) 0.99 n/a - n/a - C 1.8 −8.7 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyses were significant, adjusted analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown aUnadjusted within-group mean difference (one year follow-up minus baseline) bAdj sted for gro p and baseline al e of the o tcome meas re Table 6 Children’s reasons for their non-smoking intentions Health messages Quotes Health implications of smoking “I won’t [smoke] because if you smoke you will damage your heart and if you don’t smoke you can live a long and healthy life” (Girl, School 7). “There’s a reason why I wouldn't smoke because your lungs wouldn't be in the best condition” (Boy, School 10). Impact on sports performance “I wouldn’t [smoke] because It’s harder to breathe and harder to do exercise” (Boy, School 9). Discussion Where crude analyses were significant, adjusted analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown *Significant intervention effect (P < 0 05) eRelative risk indicated that the SFS intervention did not significantly impact on children’s smoking intentions; children in both the intervention and comparison group reported a strong intention not to smoke in the future and thus a ceiling effect limited our ability to detect between- group differences. These findings are comparable to other smoking prevention programmes targeting pri- mary school aged children [19, 69], but are inconsistent with two school-based interventions that reported a posi- tive effect on intentions to smoke among elementary school children from the USA [70–72]. A further study examined the immediate and long term effects of a smok- ing education programme implemented in Dutch elemen- tary schools [73]. The study reported no short term effects on intention to smoke during elementary school. How- ever, when children were followed up at secondary school children’s intentions (not) to smoke and smoking- related cognitions (attitudes and refusal self-efficacy) using a controlled trial. The SFS intervention had no effect on children’s ratings of non-smoking intentions, though qualitative data suggested that participation in SFS made children more determined not to smoke. In addition, a small positive short term effect was found for refusal self-efficacy among girls in the intervention group. Participation in SFS also increased the likelihood of having negative attitudes towards smoking immedi- ately after the intervention, with children stating that the intervention reinforced non-smoking opinions, though limited effects were found one year after the intervention. Smoking intentions are precursors to and predictive of smoking initiation in youth [8, 68]. Quantitative data McGee et al. cient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyses were significant, adjusted on term included in adjusted multilevel models) were conducted with results for each sex shown d for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical ac bling/friend smoking roup mean difference (one year follow-up minus baseline) and baseline value of the outcome measure Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyse analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown Discussion “If you smoked you wouldn’t be able to run as long or play as long as other [non-smoking] people” (Boy, School 3). Cigarette contents and addiction “There’s over 4,000 chemicals in a cigarette and they’re not nice, rat poison, nicotine, rocket fuel” (Girl, School 6). “I wouldn’t [smoke] because it’s addictive and you won’t be able to stop cos of the nicotine” (Girl, School 5). Impact on weight “People think when you smoke you lose weight but you really don’t” (Boy, School 5). “It’s healthier not to smoke and there’s no difference in your weight, you’re just better off not smoking”(Boy, School 6). “It’s healthier not to smoke and there’s no difference in your weight, you’re just better off not smoking”(Boy, School 6). one year after the intervention, children who received the intervention had significantly higher non-smoking inten- tions and smoked less than the control group [73]. The above mentioned studies might suggest that smoking pre- vention interventions may be more effective if imple- mented in secondary school, as children may be more likely to have developed intentions to smoke if they are closer to the actual age of smoking onset (i.e., age 14-16 years [19, 73]. Nevertheless, given that qualitative data suggested that SFS had strengthened children’s non- smoking intentions, a longer term follow-up study is war- ranted to investigate whether implementing SFS during primary school is effective at reducing smoking behaviour and smoking intentions in adolescence, following the tran- sition to secondary school. by long term follow-up. It is possible, therefore, that additional ‘booster’ sessions may be necessary to sustain attitude changes in preadolescent children, particularly those residing in deprived communities. However, the evidence on the effectiveness of booster sessions is lim- ited and inconsistent [79]. The likelihood of starting to smoke increases in ado- lescence [80, 81] and so enhancing skills to resist social pressures to smoke is important for smoking prevention [82]. Whilst a small positive effect was observed on cigarette refusal self-efficacy among girls in the interven- tion group at post-intervention, no group differences were apparent at one-year follow-up. It is possible that short term intervention effects on refusal self-efficacy were not found in boys because efficacy levels increased from baseline to post-intervention among boys in both the intervention and comparison groups. Discussion “Because I like doing lots of sports and if I smoked in the future it would be difficult to do [sports]” (Boy, School 8). Cigarette contents and addiction “Because I’ve learnt [in SFS] how many chemicals are in a cigarette and what goes into them, that’s why I wouldn’t smoke” (Boy, School 8). I’m very confident that I’m not going to smoke cos they’ve [SFS] told us how bad it [smoking] is and there are over four thousand chemicals [in a cigarette] and it can be hard to quit” (Girl, School 1). Financial costs of smoking “I wouldn’t [smoke] because it costs you loads of money”(Girl, School 5). Table 6 Children’s reasons for their non-smoking intentions “I wouldn’t [smoke] because it costs you loads of money”(Girl, School 5). Financial costs of smoking McGee et al. BMC Public Health (2016) 16:445 Page 12 of 16 Table 7 Children’s’ understanding of the health messages received during the intervention Health messages Quotes Health implications of smoking “The [SFS] games show you the damage that [smoking] does to your arteries and lungs” (Boy, School 3). “A smoker would get more phlegm and a non-smoker would get less phlegm” (Boy, School 4, Gp 1). Impact on sports performance “Your heart beats faster when you’re doing exercise when you’re a smoker” (Boy, School 5). “If you smoked you wouldn’t be able to run as long or play as long as other [non-smoking] people” (Boy, School 3). Cigarette contents and addiction “There’s over 4,000 chemicals in a cigarette and they’re not nice, rat poison, nicotine, rocket fuel” (Girl, School 6). “I wouldn’t [smoke] because it’s addictive and you won’t be able to stop cos of the nicotine” (Girl, School 5). Impact on weight “People think when you smoke you lose weight but you really don’t” (Boy, School 5). “It’s healthier not to smoke and there’s no difference in your weight, you’re just better off not smoking”(Boy, School 6). Table 7 Children’s’ understanding of the health messages received during the intervention Health messages Quotes Health implications of smoking “The [SFS] games show you the damage that [smoking] does to your arteries and lungs” (Boy, School 3). “A smoker would get more phlegm and a non-smoker would get less phlegm” (Boy, School 4, Gp 1). Impact on sports performance “Your heart beats faster when you’re doing exercise when you’re a smoker” (Boy, School 5). Conclusions h In summary, the results of this study indicate that SFS was effective at changing attitudes towards smoking, and increasing the level of individual self-efficacy to refuse ciga- rettes among girls immediately post-intervention. Although no quantitative intervention effects were observed for non- smoking intentions, children articulated that SFS made them more determined not to smoke. Overall, these find- ings may suggest that sport-for-health interventions offer a promising strategy for smoking prevention efforts, though a long term follow-up study is needed to determine whether the SFS intervention is effective at preventing smoking in secondary school. Moreover, further evidence is needed from randomised controlled-trials. The fact that almost all children had not developed an intention to smoke might indicate that smoking prevention programmes should tar- get early adolescents (aged 11-13 years), who are closer to the actual age of smoking onset. The present study has several strengths. First, in ac- cordance with MRC guidance for the development of complex interventions, SFS was designed following ex- tensive formative work, school and community feasibility studies [29, 41, 42]. Second, this study adopted a mixed- methodology approach consistency with the Standard Evaluation Framework for physical activity interventions [85]. Third, this study followed children one year after the end of the intervention. Fourth, the study had a large sample size and reasonably low attrition rates were observed. Fifth, process evaluation measures were used to explore the implementation of SFS [64]. Finally, to the date, the results of this study provide the first globally published evidence for the effectiveness of a large scale school-based sport-for-health smoking pre- vention intervention. Several limitations should be acknowledged. First, of the 154 schools approached, only 43 agreed to participate. Previous research has shown that some parents and school officials may be concerned that exposing preado- lescent children to smoking prevention programmes may stimulate their interest and curiosity about smoking [86]. However, the primary reason given by schools for non- participation was limited time, which might be related to the fact that smoking prevention is not mandatory in Key Stage 2 of the UK National Curriculum [53]. Fur- ther, it was encouraging to note that participation in SFS did not increase rates of smoking initiation. Sec- ond, the reliance on self-report in the assessment of outcome variables carries a risk of measurement error due to inaccurate recall, literacy issues and social desir- ability bias [82, 87]. Acknowledgements Th f di f thi Acknowledgements The funding for this project was provided by Liverpool City Council and Liverpool Primary Care Trust. The authors are grateful to all steering group members including; Helen Casstles (Liverpool Community Health NHS Trust); Claire Spencer (Liverpool City Council); Matthew Philpott (Health Equalities Group); Andrew Wileman (Merseyside Sports Partnership); Julie McCann (Healthy Schools); and Aaron Leach (Florence Melly Primary School) for their guidance and support in designing the SFS intervention. The authors would like to extend their thanks to Helen Casstles and Katie Dougherty (Liverpool Community Health NHS Trust) for co-delivering the bespoke training workshop and to the sports coaches from Liverpool City Council, Liverpool FC Foundation and Everton in the Community for delivering SFS activity sessions. Many thanks also go to the sports athletes Natasha Jonas (Boxing), Matthew Lee (Handball) and Tom Wolfenden (Badminton) for co-delivering the SFS assembly. Finally, we would like to thank all the primary schools and children who participated in this study. Conclusions h However, self-reports have been demonstrated to be accurate provided confidentially is assured [88]. Third, it was not possible to blind study Discussion Though not directly com- parable due to methodological differences, these results are in accord with Crone and colleagues [72], who also noted a number of short term positive between-group effects on attitudes towards smoking that had reduced Page 13 of 16 McGee et al. BMC Public Health (2016) 16:445 Page 13 of 16 participants or the research team to the intervention because of the practical nature of the intervention. Fourth, primary and secondary outcomes focused on intentions to smoke and smoking-related cognitions, respectively, which may or may not result in smoking initiation at a later age [13]. Fifth, given that the major- ity of children were White British and from one of the most deprived local authorities in England, these results may not generalise to other racial and socio-economic child populations. Sixth, focus groups were only con- ducted with children from intervention schools to in- form interpretations of intervention effectiveness and therefore it is unknown if children from comparison schools would have conveyed similar perspectives. Fi- nally, the study did not include a cost-effectiveness evaluation, thus it is unknown whether SFS is a cost- effective smoking prevention initiative. facilitate children in making a rational and logical deci- sion not to smoke during a period when smoking is more age-related and considered as accepted behaviour [84]. To the authors knowledge SFS was the first sport-for- health intervention to engage children in smoking pre- vention. A recently published process evaluation of the intervention suggests that this unique approach was well-received by children, and was considered accept- able to coaches and teachers as intervention deliverers [64]. However, there were variations in intervention fi- delity and teachers’ implementation of intervention ac- tivities that may have reduced the potency of the intervention and the ability to sustain short term effects one year after the intervention [64]. Nevertheless, the limited intervention effects are more likely attributed to children at this age having strong intentions not smoke in the future, though important lessons have been learned that can inform the design of a rando- mised controlled trial [64]. Discussion It is also pos- sible that short term effects on girls’ refusal self-efficacy were not maintained at one-year follow-up because girls’ in the comparison groups ratings increased and they ap- peared to ‘catch up’. These mixed findings likely reflect that self-efficacy is not a static concept [83] and levels of self-efficacy fluctuate over time [80]. The findings are similar to those reported by Isensee et al. [78], who also noted a lack of medium term effects and documented increases in refusal self-efficacy among control group participants. Further, the absence of intervention effect could again be attributed to a ceiling effect; children in the intervention and comparison groups both reported high refusal self-efficacy, reducing the power to detect noticeable effects. It is also worth noting that most chil- dren in the current study did not have friends who smoke, and so have yet to be put to the test of resisting social influences to smoke. Given that self-efficacy is subject to change over time, it has been recommended that smoking prevention programmes are implemented annually in preadolescence and throughout adolescence until the completion of secondary school [35, 77, 80]. Long-term research is required to determine if the SFS primary school smoking prevention intervention can Children’s intentions to smoke can be shaped by their attitudes towards smoking and their self-efficacy expec- tations [9, 10, 74, 75]. Smoking-related knowledge and attitudes are frequently measured and have a propensity to increase following smoking prevention interventions [72, 76–78]. Consistent with these studies, children who participated in the SFS intervention were more likely to develop negative attitudes towards smoking immediately following the intervention than children in the comparison group. Focus group data with children supported these findings and revealed that the SFS games and smoke free messages positively influenced children. 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Researchers who wish to access the data for non-commercial purposes can make a request to the Principal Investigator, Dr Lawrence Foweather. Page 14 of 16 McGee et al. BMC Public Health (2016) 16:445 Received: 1 August 2015 Accepted: 20 April 2016 Received: 1 August 2015 Accepted: 20 April 2016 22. Vitoria PD, Salgueiro MF, Silva SA, de Vries H. Social influence, intention to smoke, and adolescent smoking behavior longitudinal relations. British J Health Psyc. 2011;16(4):779–98. Authors’ contributions primary school children from deprived neighbourhoods: a cross-sectional study. BMC Public Health. 2015;15:225. primary school children from deprived neighbourhoods: a cross-sectional study. BMC Public Health. 2015;15:225. CEM and LF drafted the manuscript and together with JT were responsible for data collection and analyses. LF conceived and managed the project as principal investigator. CM, JT, SF, RM, LP, MU and LF made substantial contributions to the study design, analysis, interpretation of data and editing the manuscript. All authors read and approved the final manuscript. 15. Wen X, Chen W, Gans KM, Colby SM, Lu C, Liang C, Ling W. Two-year effect of a school-based prevention programme on adolescent cigarette smoking in Guangzhou, China: a cluster randomized trial. Int J Epidemiol. 2010;39(3):860–76. 16. Pommier J, Guevel MR, Jourdan D. Evaluation of health promotion in schools: a realistic evaluation approach using mixed methods. BMC Public Health. 2010;10:43. Competing interests Th h d l h 17. Pearson M, Chiton R, Woods HB, Wyatt K, Ford T, Abraham C, Anderson R. Implementing health promotion in schools: protocol for a realist systematic review of research and experience in the United Kingdom (UK). Systematic Reviews. 2012. doi:10.1186/2046-4053-1-48. The authors declare that they have no competing interests. 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https://openalex.org/W3202847747	https://europepmc.org/articles/pmc8498822?pdf=render	English	null	Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability	Frontiers in microbiology	2,021	cc-by	9,954	ORIGINAL RESEARCH published: 24 September 2021 doi: 10.3389/fmicb.2021.697351 Edited by: Sangeeta Tiwari, The University of Texas at El Paso, United States Edited by: Sangeeta Tiwari, The University of Texas at El Paso, United States Reviewed by: Bini Wang, Shaanxi Normal University, China Fengjie Cui, Jiangsu University, China Correspondence: Minseok Seo mins@korea.ac.kr Heebal Kim heebal@snu.ac.kr Reviewed by: Bini Wang, Shaanxi Normal University, China Fengjie Cui, Jiangsu University, China Correspondence: Minseok Seo mins@korea.ac.kr Heebal Kim heebal@snu.ac.kr Correspondence: Minseok Seo mins@korea.ac.kr Heebal Kim heebal@snu.ac.kr Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 19 April 2021 Accepted: 30 August 2021 Published: 24 September 2021 Received: 19 April 2021 Accepted: 30 August 2021 Published: 24 September 2021 Received: 19 April 2021 Accepted: 30 August 2021 Published: 24 September 2021 Soomin Jeon1, Hyaekang Kim1, Youngseok Choi1, Seoae Cho2, Minseok Seo3 and Heebal Kim1,2,4* 1 Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea, 2 eGnome, Inc., Seoul, South Korea, 3 Department of Computer Convergence Software, Korea University, Sejong, South Korea, 4 Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, South Korea Lactobacillus acidophilus (L. acidophilus) is a representative probiotic and is widely used in many industrial products for its beneﬁcial effects on human and animal health. This bacterium is exposed to harsh environments such as high temperatures for manufacturing industrial products, but cell yield under high temperatures is relatively low. To resolve this issue, we developed a new L. acidophilus strain with improved heat resistance while retaining the existing beneﬁcial properties through the adaptive laboratory evolution (ALE) method. The newly developed strain, L. acidophilus EG008, has improved the existing limit of thermal resistance from 65◦C to 75◦C. Furthermore, we performed whole-genome sequencing and comparative genome analysis of wild- type and EG008 strains to unravel the molecular mechanism of improved heat resistance. Interestingly, only two single-nucleotide polymorphisms (SNPs) were different compared to the L. acidophilus wild-type. We identiﬁed that one of these SNPs is a non- synonymous SNP capable of altering the structure of MurD protein through the 435th amino acid change from serine to threonine. We believe that these results will directly contribute to any industrial ﬁeld where L. acidophilus is applied. In addition, these results make a step forward in understanding the molecular mechanisms of lactic acid bacteria evolution under extreme conditions. Keywords: adaptive laboratory evolution (ALE), Lactobacillus acidophilus, heat resistance, whole-genome sequencing, bacterial evolution INTRODUCTION Lactic acid bacteria (LAB) is a gram-positive bacteria that produces lactic acid as a fermentation product (Makarova et al., 2006; Zheng et al., 2020). Since LAB has been mainly applied in dairy products or fermented foods for humans and animals, it has been domesticated toward proﬁtable ends (Steensels et al., 2019). For example, these bacteria are highly resistant to acids and bile salts, making them widely used for industrial purposes such as food manufacturing (Menconi et al., 2014). Functionally, they play a beneﬁcial role in inhibiting the growth of pathogens by producing antimicrobial compounds such as lactic acid, hydrogen peroxide, and bacteriocin (Mohankumar and Murugalatha, 2011). Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability Soomin Jeon1, Hyaekang Kim1, Youngseok Choi1, Seoae Cho2, Minseok Seo3* and Heebal Kim1,2,4* Citation: Jeon SM, Kim HK, Choi YS, Cho SA, Seo MS and Kim HB (2021) Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability. Front. Microbiol. 12:697351. doi: 10.3389/fmicb.2021.697351 September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org Heat-Adapted Strain Development by ALE Jeon et al. 2013). Research to increase the heat resistance of bacteria by applying the ALE method has been conducted in various species such as Escherichia coli (Riehle et al., 2003; Rudolph et al., 2010) and E. faecium (Min et al., 2020). Likewise, studies to improve the thermal resistance of LAB have been steadily carried out over the past 20 years, but most of the studies have only conﬁrmed improved cell viability below 65◦C. For example, survival rates of Lacticaseibacillus paracasei DPC1919, DPC2102, and DPC2013 strains were evaluated at 56–67.5◦C (Jordan and Cogan, 1999). In another study, the thermal resistance of L. acidophilus LA1- 1 was measured at 37–58◦C (Kim et al., 2001). There have also been attempts to develop L. acidophilus EG008 strains using ALE such as L. acidophilus NCFM at 65◦C (Kulkarni et al., 2018) and Lactiplantibacillus plantarum Lp 998 at 45–55◦C (Ferrando et al., 2015). Lactobacillus acidophilus (L. acidophilus) is a representative LAB species that has been well studied in its physiology and functionality. The beneﬁcial health eﬀects of L. acidophilus have been shown in studies of various diseases such as innate immunity (Klein et al., 2008; Foysal et al., 2020), inﬂammatory bowel disease (Peran et al., 2007; Park et al., 2018), and colon cancer (Zhuo et al., 2019). In addition, as scientiﬁc proof comes to light about various eﬀects such as skin wrinkle improvement (Chahuki et al., 2019), skin moisturization (Im et al., 2018), and vaginal cleansing (Bertuccini et al., 2017), the scope of utilizing L. acidophilus in the industrial ﬁeld is gradually expanding. Based on the scientiﬁc evidence of these health beneﬁts, L. acidophilus holds an important position in the probiotic market, and a variety of commercial strains have been discovered such as L. acidophilus NCFM (Altermann et al., 2005), L. acidophilus LA-1 and LA-5 (Schillinger et al., 2003; Matijaši´c et al., 2016), and L. acidophilus DDS-1 (Dash, 2004). L. acidophilus is known to have the same high acid, bile salt, and osmotic resistance as the common LAB (Hutkins et al., 1987; Chou and Weimer, 1999). Citation: Based on these rationales, the primary goal of this study is to develop a strain of L. acidophilus that can withstand conditions of 65◦C or higher through the ALE method. The secondary goal is to minimize changes in the genetic background to maintain the functional advantages of the existing L. acidophilus strain as much as possible. We conﬁrmed this through complete genome analysis using long-read sequencing. When LAB are used for manufacturing industrial products, the bacterial strain is often exposed to extreme environments. Among various environmental factors, high temperature has a major inﬂuence on bacterial survival. For example, LAB are used as an animal feed additive, and they are usually manufactured in pellet form. Pellets are made by compressing ground feed and supplements including LAB by applying air above 80◦C (Skoch et al., 1981). Some feed mills have compression temperatures that can reach 90◦C to destroy feed-borne pathogens such as Salmonella (Jones and Richardson, 2004). After this process, the bacteria are useful to animals only if it survives in the pellet. Thermal and mechanical treatments have physiological and biological eﬀects on living cells, such as denaturing proteins and altering enzymatic activity (Belhadj Slimen et al., 2016), which appears as a decrease in cell viability. Therefore, strains such as Saccharomyces cerevisiae and Bacillus subtilis with high survival rates under heat treatment are mainly used (Haldar et al., 2011; Nguyen et al., 2015). Among the lactic acid strains, Enterococcus faecium is mainly utilized (Boney et al., 2018). L. acidophilus has been suggested as an antibiotic alternative by improving growth performance and nutrient utilization in animal intestines, but their low thermal stability is a limitation for use as feed additives (Simon et al., 2005; Lan et al., 2017). Furthermore, in addition to direct heat treatment to LAB, a thermostable bacterial strain can help protect against heat-induced death or accidental thermal management defects during fermentation, thereby increasing cooling cost-eﬀectiveness (Matsushita et al., 2016). Therefore, it is expected that improving the heat resistance of L. acidophilus will not only increase the industrial utility but also contribute to the expansion of the application range. Frontiers in Microbiology \| www.frontiersin.org Adaptive Laboratory Evolution and Screening a Thermal-Adapted Strain ﬁnal result of ALE is presumed to be a group of individuals with random mutations rather than a single individual. The evaluation temperature was determined as the temperature above the critical point of EG004 strain. Five diﬀerent colonies were picked from A001F8-72. A hundred microliters of cells were injected in 900 µl of pre-heated saline at 66◦C by heat block, and cells were heated for 1 min. After cooling down, the diluted cells were plated on MRS agar to determine viable cell count. The cell survival ratio was expressed by dividing the viable cell count after heat stress by the initial viable cell count. The strain that had the most improved resistance to high temperature was named as “EG008” strain and used for the comparative analysis. To induce the newly improved thermal tolerance of the strain, an ALE experiment at high temperature was applied. The development of a heat-adapted strain consisted of two steps: heat adaptation to the highest survival temperature and single strain selection with thermotolerance (Figure 1). L. acidophilus EG004 strain was used as wild-type, cultured for 16 h, and prepared for heat adaptation to the highest temperature. Ten microliters of cells were injected into 990 µl of pre-heated MRS broth at 60◦C by heat block (ALB64; FINEPCR, Gyeonggi-do, South Korea). Heat treatment was applied for 1 min. After heat treatment, it was allowed to cool down at room temperature for 5 min and cultivated at 37◦C for 24 h. After two iterations, an identical process was repeated with increased temperature by 3◦C until incubation was impossible. All strains of each step were stored with 25% glycerol at −80◦C. Next, we performed a single strain selection from the microbial population obtained through the ﬁnal ALE treatment (A001F8-72). It was performed because the Strain Identiﬁcation and Bacterial Culture A probiotic colony was isolated from a fermented dairy food. To identify bacterial species, 16S rRNA genes were sequenced by Macrogen Inc. (Seoul, South Korea). Sequencing reactions were performed in the DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad, Hercules, CA, United States) using the ABI BigDye (R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Beverly, MA, United States). Primers used for single-pass sequencing were as follows: forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). To remove the unincorporated terminators and dNTPs, the ﬂuorescent-labeled fragments were puriﬁed by the method that Applied Biosystems recommends. The samples were injected to electrophoresis in an ABI 3730xl DNA Analyzer (Applied Biosystems). Using BLAST, 16S rRNA sequences were compared with the NCBI database (Johnson et al., 2008). For bacterial culture, all strains were propagated statically in deMan Rogosa Sharpe broth (MRS broth; Difco Laboratories, Detroit, MI, United States) or on MRS agar [1.5% (wt/vol)] under aerobic condition at 37◦C without shaking. Gram staining was performed using the BD BBLTM Gram stain kit following the manufacturer’s protocol. For viable cell counting, serially diluted cultures were poured into the MRS plates and inoculated at 37◦C for 48 h. The viability of the cell was counted in the colony-forming unit per milliliter (CFU/ml). Cell density was measured by the absorbance at 600 nm (OD600; optical density spectrophotometrically measured at 600-nm wavelength) using an OPTIZEN POP UV-visible spectrophotometer (KLAB, Daejeon, South Korea). Encapsulation and heat pretreatment for heat-shock protein expression have been the main methods studied for improving the heat stress resistance of diverse bacterial strains, but these methods are not cost-eﬀective (Xu et al., 2016; Chen et al., 2017). On the other hand, adaptive laboratory evolution (ALE) artiﬁcially stimulates natural evolution in a laboratory setting, making it relatively easy to improve the desired phenotype of targeted strain (Portnoy et al., 2011; Dragosits and Mattanovich, September 2021 \| Volume 12 \| Article 697351 2 Heat-Adapted Strain Development by ALE Jeon et al. Adaptive Laboratory Evolution and Screening a Thermal-Adapted Strain Bacterial Kinetics The biomass was measured by dried cell weight. Cultured bacteria in 1,000 ml of MRS broth were centrifuged at 4,000 rpm for 10 min, washing twice, and then drying at 60◦C for 3 days. All measurements were repeated three times. The concentration of glucose was measured by HPLC in the culture solution ﬁltered through a 0.45-um membrane ﬁlter [HPLC machine: Dionex Ultimate 3000 (Thermo Dionex, United States/pump, autosampler, and oven), detector: Shodex RI-101 (Shodex, Japan), and column: Sugar-Pak (Waters, 300 × 6.5 mm, United States)]. Glucose (Junsei Chem, 98%) was used as glucose standard. Since batch culture was performed in an Erlenmeyer ﬂask, the Monod equation was used to calculate the factors (Okpokwasili and Nweke, 2006). Assessment of Phenotypical Changes In order to assess improved thermal capacity, cell viability at 55–75◦C was assessed. Cells in the mid-exponential phase were prepared to OD600 at 1.3, corresponding to approximately 1 × 109 CFU/ml. A hundred microliters of cells were injected into 900 µl of pre-heated saline by heat block. After heat was applied FIGURE 1 \| Schematic diagram of the study to develop L. acidophilus EG008 strain. This diagram shows the overall process for developing an EG008 strain of L. acidophilus. The ﬁrst step is the thermal adaptation of L. acidophilus EG004 strains through adaptive laboratory evolution. The second step is the process of selecting the colonies that have the single strain with the highest heat resistance among the various strains that have been thermally adapted. The ﬁnal step is the in silico analysis step to identify and compare the whole genome sequence of L. acidophilus EG004 and EG008 strains, respectively. FIGURE 1 \| Schematic diagram of the study to develop L. acidophilus EG008 strain. This diagram shows the overall process for developing an EG008 strain of L. acidophilus. The ﬁrst step is the thermal adaptation of L. acidophilus EG004 strains through adaptive laboratory evolution. The second step is the process of selecting the colonies that have the single strain with the highest heat resistance among the various strains that have been thermally adapted. The ﬁnal step is the in silico analysis step to identify and compare the whole genome sequence of L. acidophilus EG004 and EG008 strains, respectively. FIGURE 1 \| Schematic diagram of the study to develop L. acidophilus EG008 strain. This diagram shows the overall process for developing an EG008 strain of L. acidophilus. The ﬁrst step is the thermal adaptation of L. acidophilus EG004 strains through adaptive laboratory evolution. The second step is the process of selecting the colonies that have the single strain with the highest heat resistance among the various strains that have been thermally adapted. The ﬁnal step is the in silico analysis step to identify and compare the whole genome sequence of L. acidophilus EG004 and EG008 strains, respectively. September 2021 \| Volume 12 \| Article 697351 3 Frontiers in Microbiology \| www.frontiersin.org Heat-Adapted Strain Development by ALE Jeon et al. for 1 min, cells were plated on MRS agar to quantify the viable cell count. Thermal resistance was presented as the survival ratio. Statistical Analysis All experiences were performed with three replications to check if there is any experimental bias except single strain selection from A001F8-72. Student’s t-test was used to compare assessments of thermal, acidic, and salinity tolerance. We considered a 5% signiﬁcance level. The FDR correction method was used for multiple-testing issues. Comparative Genomic Analysis With Lactobacillaceae Family y To identify closeness between our strains and the Lactobacillaceae family, 22 genomes were used and compared with EG004 and EG008 strains. Twenty-two genomes of Lactobacillaceae and a genome of B. subtilis were downloaded from the NCBI website. The genome of B. subtilis was used as an outgroup to make the phylogenetic root of Lactobacillaceae family. Average nucleotide identity (ANI) was calculated by JSpecies1.2.1 (Richter and Rosselló-Móra, 2009). The 16S rRNA sequences were investigated by RNAmmer and aligned by ClustalW2.1 (Lagesen et al., 2007; Larkin et al., 2007). The phylogenetic trees were generated by MEGA X with 1,000 bootstrap using the neighbor-joining method (Kumar et al., 2018). To compare functional gene contents, protein prediction of 22 Lactobacillaceae genomes was performed by the RAST server. We certiﬁed whether detected variants in EG008 strain existed in other Lactobacillaceae. In the case of genic single-nucleotide polymorphism (SNP), the gene including SNP was used as a blast query for comparison. In the case of intergenic SNP, a sequence with a length of 301 base pairs including the variant was used. Multiple- sequence alignment by ClustalW2.1 was used to conﬁrm sequence detection. In comparison to singleton, BLAST was utilized, and identiﬁcation of gene location was employed by Artemis (Rutherford et al., 2000). The pan-genome analysis was conducted using PGAP program with the cut-oﬀﬁltering of E-value (<1e-10) (Zhao et al., 2012). Annotation of Genomic Information Annotation of Genomic Information The genomes of L. acidophilus EG004 and EG008 strains were annotated, and genes were categorized by protein functions using Rapid Annotations using Subsystems Technology (RAST) server with version 2.0 (Aziz et al., 2008). To identify its functionality and safety as a probiotic, several genetic factors were detected. Antibiotic-resistant genes were inspected by NCBI BLAST with ARG-ANNOT and CARD databases (Gupta et al., 2014; Jia et al., 2016). Virulence factor and prophage gene were identiﬁed by VirulenceFinder 2.0 (Joensen et al., 2014) and PHASTER (Arndt et al., 2016), respectively. IslandView4 discovered genomic islands (Bertelli et al., 2017). Identiﬁcation of bacteriocin was carried out using BAGEL4 (van Heel et al., 2018). To detect variants in the EG008 strain, a comparative analysis was conducted. To ﬁnd a singleton in each strain, orthologous gene and singleton deﬁnition were conducted with OrthoVenn2 software (Xu et al., 2019). Identiﬁed singletons were double checked by the NCBI BLAST. Single-nucleotide variants were detected by nucmer including MUMmer 3.23 (Kurtz et al., 2004). Assessment of Phenotypical Changes To check other phenotypical changes induced by heat stress, acid and salinity tolerance were measured. Inoculated cells were harvested by centrifugation at 12,000 rpm for 10 min at 4◦C. After the supernatant was removed, the pellet was washed two times with 1X phosphate buﬀer (1X PBS). It was resuspended into 1 ml of PBS buﬀer adjusted to acidiﬁed solutions (up to pH 2.0 and 7.0) or salted solutions (7.5 and 15.0%). Cells were exposed to the acidic solution for 2.5 h and the high salt solution for 3.5 h at 37◦C. The viable cell count was measured in order to assess cell survival, and it was calculated by the same method as thermal resistance. SMRT portal system and HGAP4 tool assembled. Genome assembly was conducted with genome size parameter set to 3 Mb. Assembled contig with low quality such as a short length (<20,000 bp) and low coverage (<50×) was eliminated. To correct assembly errors in the assembled genome sequence, the polishing process was repetitively conducted with a quiver algorithm until genomic variants were not found. Assembled genome was circularized by Circlator (Hunt et al., 2015). Complete Genomic Analysis for L. acidophilus EG004 and EG008 Strains L. acidophilus EG004 and EG008 Strains We completed the whole genome sequences from EG004 and EG008 strains through long-read sequencing technology (Supplementary Table 1) to further reveal genomic characteristics to conﬁrm that the newly developed strain is L. acidophilus. Constructed phylogenetic tree based on the 16S rRNA from 22 publicly available Lactobacillaceae whole-genome sequences conﬁrmed that the developed strains were one of the L. acidophilus species (Figure 3A). Of a total of 24 Lactobacillaceae families, full-length sequences of 10 L. acidophilus strains used in the analysis showed a very high degree of similarity to each other (avg. 99.835%) and relatively low similarity (avg. 93.992%) to other Lactobacillaceae families (Figure 3B). This result conﬁrms once again that the heat-resistant strain we developed is L. acidophilus and suggests that strains of L. acidophilus species have a speciﬁc genetic background in common, which is distinct from other Lactobacillaceae families. Likewise, in the functional comparison based on gene annotation, no signiﬁcant diﬀerences were observed between L. acidophilus strains (Figure 3C), but statistically signiﬁcant diﬀerences were found in nine SEED terms at 5% signiﬁcance level (Figure 3D) between L. acidophilus and other Lactobacillaceae. These results are ideal considering that our secondary goal is to maximize heat resistance while maintaining desirable properties such as the antimicrobial properties of L. acidophilus. We conﬁrmed that the genome of the L. acidophilus EG008 strain retained various antibacterial- related genes such as bacteriocin (Supplementary Figure 2). Furthermore, examining the features of the full-length genome, seven genomic islands, and two prophage regions were found identically in both EG004 and EG008 strains (Supplementary Figure 3). In addition, the pan and core genome analysis revealed that both strains are open pan-genome. Antibiotic resistance-related genes and a virulence factor were not found As a result of conducting the experiment to determine the limit of heat resistance of the L. acidophilus EG004 strain, a signiﬁcant decrease in survival rate was observed at 66◦C (Figure 2A). We hypothesized that the L. acidophilus EG008 strain developed by the ALE method would have a signiﬁcantly improved survival rate at such thermal limits. In the case of the newly developed L. acidophilus EG008 strain, the survival rate decreased as the temperature exceeded 66◦C, but a statistically signiﬁcant improvement in the survival rate was observed compared to the EG004 strain at a 5% signiﬁcance level (Figure 2B). In particular, at 66◦C, the identiﬁed limit of heat resistance of L. Development of Heat-Resistant L. acidophilus Strain Based on Adaptive Laboratory Evolution Method y We isolated L. acidophilus EG004 strain from fermented dairy food and identiﬁed it using 16S rRNA sequencing (Supplementary Figure 1). First, in an experiment to ﬁnd out the limit of heat resistance of the wild-type strain, we found out that it was unculturable at 75◦C for 99 generations. To overcome this limit of heat resistance, we developed the EG008 L. acidophilus strain by applying ALE with diﬀerent temperatures from 60◦C to 72◦C (Figure 1). After the application of the ALE method, preliminary heat screening was conducted to select strains with identical genotype that can signiﬁcantly improve heat resistance in the wild-type population. As a result, L. acidophilus cultured in EG008 colony showed the highest possible improvement in heat resistance (47.768%) than others (Table 1). Whole-Genome Sequencing g Whole-genome sequencing was served by Macrogen Inc., using SMRT sequencing. Samples were prepared according to a guide for preparing SMRTbell template for PacBio sequencing. NanoDrop spectrophotometer (Thermo Fisher Scientiﬁc, Waltham, MA, United States) and PicoGreen quantiﬁed the concentration of gDNA. All samples passed screening QC criteria. For PacBio Sequel sequencing, 5 µg of gDNA was served for 10-kb library preparation. For gDNA less than 17 kb, the actual size distribution was evaluated by 2100 Bioanalyzer (Agilent). Sheared gDNA using g-TUBE (Covaris Inc., Woburn, MA, United States) was puriﬁed by AMPure PB magnetic beads (Beckman Coulter Inc., Brea, CA, United States) if the apparent size was greater than 40 kb. A total of 10-µl library was arranged using PacBio DNA Template Prep Kit 1.0. SMRTbell templates were annealed using Sequel Binding and Internal Ctrl Kit 3.0. The Sequel Sequencing Kit 3.0 and SMRT cells 1M v3 Tray were conducted for sequencing. The PacBio Sequel platform captured SMRT cells (Paciﬁc Biosciences, Menlo Park, CA, United States). The next steps were followed as the PacBio Sample Net-Shared Protocol. Raw data from PacBio RS II was assembled by PacBio September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 4 Heat-Adapted Strain Development by ALE Jeon et al. Frontiers in Microbiology \| www.frontiersin.org RESULTS In previous studies that have developed heat-resistant strains in diverse species, it has been reported that the developed heat-resistant strain is additionally endowed with resistance to other types of stress such as acid and high salt concentration simultaneously (Min et al., 2020). Based on this fact, we expected that the newly developed L. acidophilus EG008 strain, which was endowed with improved thermal resistance, may also have cross- resistance. However, no statistically signiﬁcant improvement in survival rate was observed under strongly acidic conditions (Figure 2C) and salt concentration (Figure 2D), which suggests that genes related to heat resistance could be independent of genes related to acid and salt resistance in L. acidophilus. In addition, in order to verify whether the probiotic functionality and fermentation performance were maintained, a comparative evaluation was performed with Lacticaseibacillus rhamnosus GG (LGG), which has well-proven functionality. As a result, the two experimental strains showed higher acid resistance compared to the LGG stain, and the salt-resistance and bile salt resistance were similar (Supplementary Figure 4). In an assessment of fermentation performance, all estimated summary statistics of both strains were similar (Supplementary Table 2). Complete Genomic Analysis for L. acidophilus EG004 and EG008 Strains (C) Investigation of survival rate in acidic conditions. (D) Measurement result of change in survival rate according to salt concentration two SNPs is located in the murD gene, which synthesizes UDP- N-acetylmuramoyl-L-alanine–D-glutamate ligase (Figure 4A), while the other SNP is located intergenically between the galT gene (1,854,039–1,854,833 bp) and the IdtD gene (1,854,986– 1,856,194 bp) (Figure 4B). Both mutations may be strong candidates for conferring heat resistance, but we further investigated for non-synonymous SNPs that are easy to be interpreted biologically. A variant in the murD gene of L. acidophilus EG008 strain causes substitution of nucleotide from T (thymine) to A (adenine). Subsequently, this substitution induces changes in amino acid from S (serine) to T (threonine) at 435th amino acid residue of UDP-N-acetylmuramoyl-L- alanine–D-glutamate ligase (Figure 4C). Through a protein structure analysis, we found that this substitution can trigger a change from hydroxyl to acyl group in the coil part of the protein (Figure 4D), which is located in the extracellular matrix (Figure 4E). These results provide an insight into the mechanism in both genomes. These results are one of the pieces of evidence showing that the newly developed EG008 strain has improved heat resistance while maintaining the basic functional advantages of the existing wild-type L. acidophilus. Complete Genomic Analysis for L. acidophilus EG004 and EG008 Strains acidophilus EG004 strain, an average of 39.11% improvement in survival rate was observed (P = 0.029). At temperatures above 68◦C, the tendency to decrease with a single-digit survival rate was similar to that of EG004, but a statistically signiﬁcant improvement pattern of heat resistance was conﬁrmed. TABLE 1 \| Results of preliminary investigations on heat resistance improvement for a single strain of wild-type L. acidophilus A001F8-72. Colony Before (CFU/ml) After heat shock (CFU/ml) Survival rate (%) SD A001F8-72-1 4.10e+08 1.67e+08 40.899 0.036 A001F8-72-2 4.57e+08 1.13e+08 25.213 0.065 A001F8-72-3 5.17e+08 1.47e+08 28.855 0.034 A001F8-72-4 3.47e+08 1.65e+08 47.768 0.014 A001F8-72-5 4.67e+08 1.81e+08 39.413 0.058 The survival rate (%) was calculated by repeating the preliminary heat resistance improvement experiment three times from each colony containing a single strain. In general, L. acidophilus has a sharp decrease in cell viability at a high temperature of 65◦C or higher. This phenomenon was observed in our experimental results (Figure 2A). The evaluation was conducted at 66◦C, where the survival rate rapidly decreased at. TABLE 1 \| Results of preliminary investigations on heat resistance improvement for a single strain of wild-type L. acidophilus A001F8-72. September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 5 Jeon et al. Heat-Adapted Strain Development by ALE FIGURE 2 \| Experiment to conﬁrm improved thermal resistance at critical temperature above 66◦C. (A) Survival curve of L. acidophilus EG004 strain under different temperature conditions. The survival rate was calculated after 1-min exposure at each condition, and three repeated trials were performed. At 66◦C, a sharp drop in survival was observed. (B) The newly developed L. acidophilus EG008 strain showed an improved survival ratio compared to the EG004 at critical point above 66◦C. P-value is the result of the two-group t-test. (C) Investigation of survival rate in acidic conditions. (D) Measurement result of change in survival rate according to salt concentration. FIGURE 2 \| Experiment to conﬁrm improved thermal resistance at critical temperature above 66◦C. (A) Survival curve of L. acidophilus EG004 strain under different temperature conditions. The survival rate was calculated after 1-min exposure at each condition, and three repeated trials were performed. At 66◦C, a sharp drop in survival was observed. (B) The newly developed L. acidophilus EG008 strain showed an improved survival ratio compared to the EG004 at critical point above 66◦C. P-value is the result of the two-group t-test. Potential Genetic Factor Related to Heat Resistance Improvement Through Comparative Genome Analysis We hypothesized that the genetic factors to improving heat resistance could be found by comparing the whole genome sequences of EG004 and EG008 strains. Interestingly, only two nucleotides were diﬀerent across the two genomes. As a result of performing multiple-sequence alignment including other L. acidophilus strains whose full-length genome sequences were published, a diﬀerent genotype of these two SNPs was observed only in our newly developed EG008 strain. One of the September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 6 Heat-Adapted Strain Development by ALE Jeon et al. FIGURE 3 \| A comparative genome analysis for Lactobacillaceae based on the complete genome sequence of the newly developed L. acidophilus strain. (A) Neighbor-joining tree based on 16S rRNA sequences for 24 Lactobacillaceae families including the strain newly developed in this study. Bootstrapping was performed 1,000 times, and Bacillus subtilis 168 was considered as an outgroup. (B) Hierarchical clustering results based on average nucleotide identity among 24 Lactobacillaceae families. (C) A comparison of predictive SEED ratios between 24 Lactobacillaceae strains. (D) SEEDs with statistically signiﬁcant differences between L acidophilus and the rest of the Lactobacillaceae strains at 5% signiﬁcance level after multiple testing adjustments P < 0 01 and P < 0 001 in FIGURE 3 \| A comparative genome analysis for Lactobacillaceae based on the complete genome sequence of the newly developed L. acidophilus strain. (A) Neighbor-joining tree based on 16S rRNA sequences for 24 Lactobacillaceae families including the strain newly developed in this study. Bootstrapping was performed 1,000 times, and Bacillus subtilis 168 was considered as an outgroup. (B) Hierarchical clustering results based on average nucleotide identity among 24 Lactobacillaceae families. (C) A comparison of predictive SEED ratios between 24 Lactobacillaceae strains. (D) SEEDs with statistically signiﬁcant differences between L. acidophilus and the rest of the Lactobacillaceae strains at 5% signiﬁcance level after multiple testing adjustments. P < 0.01 and **P < 0.001 in comparison of L. acidophilus and other strains. 7 September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org Jeon et al. Heat-Adapted Strain Development by ALE FIGURE 4 \| Two single-nucleotide polymorphisms (SNPs) were found by comparing the complete genome sequences of L. acidophilus EG004 and EG008 strains. (A) A non-synonymous SNP is located at 790,957 bp of genic region in EG008 strain. This genotype change causes the serine amino acid to be transformed into threonine. Potential Genetic Factor Related to Heat Resistance Improvement Through Comparative Genome Analysis (B) A SNP found in the intergenic region of the EG008 strain. (C) Prediction of 3D structural change of MurD protein by the identiﬁed non-synonymous SNP. The serine-to-threonine changes trigger a change from hydroxyl to acyl group (red) in the side chain. (D) The 435th amino acid of the secondary structure is predicted to be the coli part by PSIPRED tool. (E) It is the part of the coil that connects the beta sheet of the third domain and the alpha helix, which is located on the extracellular matrix. FIGURE 4 \| Two single-nucleotide polymorphisms (SNPs) were found by comparing the complete genome sequences of L. acidophilus EG004 and EG008 strains. (A) A non-synonymous SNP is located at 790,957 bp of genic region in EG008 strain. This genotype change causes the serine amino acid to be transformed into threonine. (B) A SNP found in the intergenic region of the EG008 strain. (C) Prediction of 3D structural change of MurD protein by the identiﬁed non-synonymous SNP. The serine-to-threonine changes trigger a change from hydroxyl to acyl group (red) in the side chain. (D) The 435th amino acid of the secondary structure is predicted to be the coli part by PSIPRED tool. (E) It is the part of the coil that connects the beta sheet of the third domain and the alpha helix, which is located on the extracellular matrix. of the cell wall, which is termed as cross protection (Kim et al., 2001; Meena et al., 2016). Based on this fact, we expected that the L. acidophilus EG008 strain would exhibit the same phenomenon as well as a signiﬁcant improvement in heat resistance. However, contrary to expectations, no cross- protection eﬀects were observed, and only thermal resistance was found to be improved (Figures 2C,D). This suggests that the increased heat resistance may be due to reasons other than changes in fatty acid composition. Another possibility is that the three biologically replicated samples were not quantitatively suﬃcient in their sample size to conﬁrm such diﬀerence in cross resistance, which can be a limitation of the study. Further research will be needed to elucidate the mechanisms for the stress adaptation in the strain. of L. acidophilus strains with improved heat resistance at the molecular biology level. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION synthesis in emergency situations such as heat stress and amino acid shortage (Jain et al., 2006). It is known to be a gene involved in the Cpx stress response, one of the well-known envelope damage systems in E. coli, and is upregulated with YgaU when subjected to external stress such as high temperature or high osmotic pressure (Bernal-Cabas et al., 2015; Ultee et al., 2019). Based on these evidences, although it is an intergenic variant, there may be a mechanism that aﬀects the expression level of adjacent genes and ultimately contributes to imparting heat resistance. All of the SNPs found in this study were related to cell wall synthesis, speciﬁcally the peptidoglycan layer. The cell wall maintains the bacterial cell structure, is responsible for the movement of substances, and interacts with host cells or pathogens (Sequeira et al., 1977; Sengupta et al., 2013). Thus, since the cell wall is directly aﬀected by various environmental stresses, including thermal stress, the evolutionary response to external stimuli may take precedence. Evidence for this speculation can be found in previous studies investigating changes in cell walls when external physical stresses such as pH, temperature, osmotic pressure, and high bile salt concentration are applied in L. acidophilus (Khaleghi et al., 2010; Grosu-Tudor et al., 2016; Palomino et al., 2016). Putting all of this together, it indicates that EG008 strain may have evolved to survive at higher temperatures by making cell walls more rigid. We found two SNPs in the genome of EG008 strain, one of which was a non-synonymous SNP located in murD gene encoding UDP-N-acetylmuramoyl-L-alanine–D-glutamate ligase (Figure 4A). This enzyme is involved in the synthesis of peptidoglycan, a component that strengthens the bacterial cell wall (Bertrand et al., 1997). When the synthesis of this enzyme is not performed normally or the integrity of the enzyme is destroyed, the cell is dissolved by the turgor pressure inside the cell. Previous studies have shown that the expression of this protein increased when heat stimulation was applied in various strains such as Staphylococcus aureus and Streptococcus thermophilus (Mengin-Lecreulx et al., 1999; Li et al., 2011). This suggests that the expression of UDP- N-acetylmuramoyl-L-alanine–D-glutamate ligase could be the defense mechanism of bacteria against external heat stress. The non-synonymous SNP found in our results changes the 435th amino acid residue of this enzyme from serine to threonine, causing hydrogen loss and acyl gain in the side chain (Figures 4A,C). DISCUSSION We suspected that this change caused a number of changes, such as the volume of the molecule and the location of the hydroxyl groups, aﬀecting the three- dimensional structure and hydrophilicity, thereby altering the resistance to thermal stimulation. In addition, we conﬁrmed that the genotype of these SNPs was speciﬁcally found only in L. acidophilus EG008 strain (Figures 4A,B). This is one of the evidences that these two SNPs, artiﬁcially evolved by the ALE method, can be associated with the improved thermal adaptability. This study has some practical limitations. The ﬁrst experimental limitation is that the heat-adapted strain selected after ALE treatment may not be the population of strains with the best heat resistance. This occurs by physical restriction in the screening process to isolate single colonies from the population generated through ALE. There are about 109 CFUs randomly mutated strains in the population generated after ALE treatment, but it was not possible to separate the number of all cases into single colonies due to the loss in the dilution process and experimental limitations on culturable colonies on the plate. As a second limitation, although the whole genome sequences were generated using PacBio long-read sequencing technology, the technical limitation for genotyping error still remained. However, the whole genome we completed had depth coverage of 341.59X for the EG008 strain and 255.60X for the EG004 strain, and the possibility of genotyping errors was slim. Thirdly, gene expression analysis was not considered for the comparison of wild-type and heat-adapted strains. Since the genetic variation found in this study can aﬀect the expression level of the transcript, it is expected that this fact will be further revealed if a comparison of the whole transcriptome through RNA sequencing is conducted in the near future. Finally, this study did not cover the experimental validation of the two SNP candidates that were supposed to confer heat resistance to the L. acidophilus EG008 strain. Although many gene manipulations using the CRISPR/cas9 system have been reported, many technical diﬃculties remain in applying CRISPR/cas9 technology to gram-positive bacteria such as LAB (Leenay et al., 2019). It is our ultimate goal to experimentally verify the mutations detected after ALE and to reveal a direct relationship between phenotypes and genetic factors. We expect that, in the near future, if Another SNP was found in the non-coding region, and there were two genes nearby this SNP (Figure 4B). DISCUSSION In this study, we achieved the primary goal for improving the thermal resistance at 65◦C or more to secure the ease of industrial use of the L. acidophilus strain through stepwise ALE method (Figure 1). We have demonstrated that the newly developed L. acidophilus EG008 strain had better thermal resistance than wild type at high temperatures of 65–75◦C (Figure 2B). Recently, studies on the development of EG008 strains using ALE have been conducted (Kulkarni et al., 2018; Min et al., 2020), but most of the experiments were performed at temperatures below 65◦C to ensure applicability in LTST pasteurization (Vaudagna et al., 2002). Therefore, the EG008 strain developed in this study is expected to be utilized for HTST pasteurization, which sterilizes at a relatively high temperature. The second goal of this study was to maintain the beneﬁcial functions of the existing L. acidophilus wild type as much as possible while increasing heat resistance. To investigate this, we constructed complete genome sequences of the developed (EG008) and the wild-type (EG004) strains using third- generation sequencing technology and performed a comparative genome analysis. As a result, only two SNPs were found (Figures 4A,B) between the sequences (Figures 3A,B). In It is well known that repetitive heat stimulation employed in the ALE process can also induce resistance to other stresses such as acid or osmotic pressure by changing fatty acid composition September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 8 Heat-Adapted Strain Development by ALE Jeon et al. addition, there was no diﬀerence between the two strains in the gene annotation and functional analysis, suggesting that the characteristics of the L. acidophilus EG004 are maintained. We found three regions encoding bacteriocin, acidocin, enterolysin A, and helveticin J, in both genomes, which is an important feature for probiotic eﬃcacy. Bacteriocin is a proteinaceous or peptidic toxin secreted by bacteria with antibacterial activity against other strains except for itself (Riley, 1998; Gálvez et al., 2007). It is well known for its function in the gastrointestinal tract to inhibit the invasion of pathogens or competitors and aﬀect the host’s immune system (Dobson et al., 2012; Hegarty et al., 2016). As these substances are widely used as biological preservatives due to their high stability in animals including humans, we believe the ability to produce bacteriocin is considered a beneﬁcial property for industrial use of probiotics. Frontiers in Microbiology \| www.frontiersin.org REFERENCES Chen, M.-J., Tang, H.-Y., and Chiang, M.-L. (2017). Eﬀects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of keﬁr- isolated probiotic Lactobacillus keﬁranofaciens M1. Food Microbiol. 66, 20–27. doi: 10.1016/j.fm.2017.03.020 Altermann, E., Russell, W. M., Azcarate-Peril, M. 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Agents Chemother. 58, 212–220. doi: 10.1128/aac.01310-13 Chahuki, F. F., Aminzadeh, S., Jafarian, V., Tabandeh, F., and Khodabandeh, M. (2019). Hyaluronic acid production enhancement via genetically modiﬁcation and culture medium optimization in Lactobacillus acidophilus. Int. J. Biol. Macromol. 121, 870–881. doi: 10.1016/j.ijbiomac.2018.10.112 Haldar, S., Ghosh, T., and Bedford, M. (2011). Eﬀects of yeast (Saccharomyces cerevisiae) and yeast protein concentrate on production performance of broiler chickens exposed to heat stress and challenged with Salmonella enteritidis. Anim. Feed Sci. Technol. 168, 61–71. ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (Nos. 2020R1F1A1073431 and 2021R1A5A8032895) and a Korea University (Grant No. K2017991). SUPPLEMENTARY MATERIAL The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, BioProject PRJNA657145. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.697351/full#supplementary-material AUTHOR CONTRIBUTIONS technologies such as genetic scissors for microorganisms become common, the results of this study can be veriﬁed. MS and HBK designed the study. SJ and MS wrote the manuscript. SJ analyzed the data. SC generated sequencing data. HKK and YC reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version. Despite these technical limitations, we have succeeded in improving our primary target, the heat-resistant limit temperature of 75◦C, to maximize the industrial usability of L. acidophilus strain. One step further, biomarkers associated with improved thermal resistance were identiﬁed through whole genomic analysis. We anticipate the L. acidophilus strain developed in this study to be directly helpful in industrial sites where a stronger heat-resistant bacterial strain is required. We also believe that the identiﬁed biomarkers provided insights into the mechanisms of heat resistance and evolution of bacteria, including L. acidophilus. DISCUSSION One of them was a gene encoding galactose-1-phosphate uridylyltransferase, which is 2-bp away from the SNP located between the core promoter and the ORF starting point. The other was a gene that synthesizes L,D-transpeptidase. This peptidase uses peptidoglycan or its precursor as a substrate to form 3- 3 peptidoglycan crosslinks in Gram-positive bacteria (Gupta et al., 2010; Peltier et al., 2011). Therefore, there may be mechanisms to regulate the rigidity of the bacterial cell wall by regulating the level of expression of this gene (Brammer et al., 2015). 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Eﬀects of synbiotic fermented milk containing Lactobacillus acidophilus La-5 and Biﬁdobacterium animalis ssp. lactis BB-12 on the fecal microbiota of adults with irritable bowel syndrome: a randomized double-blind, placebo- controlled trial. J. Dairy Sci. 99, 5008–5021. doi: 10.3168/jds.2015-10743 Hutkins, R. W., Ellefson, W. L., and Kashket, E. R. (1987). Betaine transport imparts osmotolerance on a strain of Lactobacillus acidophilus. Appl. Environ. Microbiol. 53, 2275–2281. doi: 10.1128/aem.53.10.2275-228 1.1987 rolled trial. J. Dairy Sci. 99, 5008–5021. doi: 10.3168/jds.2015-107 Matsushita, K., Azuma, Y., Kosaka, T., Yakushi, T., Hoshida, H., Akada, R., et al. (2016). Genomic analyses of thermotolerant microorganisms used for high- temperature fermentations. Biosci. Biotechnol. Biochem. 80, 655–668. doi: 10. 1080/09168451.2015.1104235 Im, A.-R., Lee, B., Kang, D.-J., and Chae, S. (2018). Skin moisturizing and antiphotodamage eﬀects of tyndallized Lactobacillus acidophilus IDCC 3302. J. Med. Food 21, 1016–1023. doi: 10.1089/jmf.2017.4100 Jain, V., Kumar, M., and Chatterji, D. (2006). ppGpp: stringent response and survival. J. Microbiol. 44, 1–10. Meena, S., Mehla, J., Kumar, R., and Sood, S. (2016). REFERENCES Eﬀects of Lactobacillus acidophilus supplementation on growth performance, nutrient digestibility, fecal microbial and noxious gas emission in weaning pigs. J. Sci. Food Agric. 97, 1310–1315. doi: 10.1002/jsfa.7866 Portnoy, V. A., Bezdan, D., and Zengler, K. (2011). Adaptive laboratory evolution— harnessing the power of biology for metabolic engineering. Curr. Opin. Biotechnol. 22, 590–594. doi: 10.1016/j.copbio.2011.03.007 Richter, M., and Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the prokaryotic species deﬁnition. Proc. Natl. Acad. Sci. U.S.A. 106, 19126– 19131. doi: 10.1073/pnas.0906412106 Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., Mcgettigan, P. A., Mcwilliam, H., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. doi: 10.1093/bioinformatics/btm404 Leenay, R. T., Vento, J. M., Shah, M., Martino, M. E., Leulier, F., and Beisel, C. L. J. B. J. (2019). Genome editing with CRISPR−Cas9 in Lactobacillus Riehle, M. M., Bennett, A. F., Lenski, R. E., and Long, A. D. (2003). Evolutionary changes in heat-inducible gene expression in lines of Escherichia coli adapted to September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 11 Jeon et al. Heat-Adapted Strain Development by ALE high temperature. Physiol. Genomics 14, 47–58. doi: 10.1152/physiolgenomics. 00034.2002 Xu, L., Dong, Z., Fang, L., Luo, Y., Wei, Z., Guo, H., et al. (2019). OrthoVenn2: a web server for whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Res. 47, W52–W58. high temperature. Physiol. 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Bioinformatics 28, 416–418. doi: 10.1093/bioinformatics/ btr655 Schillinger, U., Yousif, N. M., Sesar, L., and Franz, C. M. (2003). Use of group- speciﬁc and RAPD-PCR analyses for rapid diﬀerentiation of Lactobacillus strains from probiotic yogurts. Curr. Microbiol. REFERENCES 47, 453–456. Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M., Harris, H., Mattarelli, P., et al. (2020). A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 70, 2782–2858. doi: 10.1099/ijsem.0.004107 Sengupta, R., Altermann, E., Anderson, R. C., Mcnabb, W. C., Moughan, P. J., and Roy, N. C. (2013). The role of cell surface architecture of lactobacilli in host-microbe interactions in the gastrointestinal tract. Mediators Inﬂamm. 2013:237921. 2782–2858. doi: 10.1099/ijsem.0.004107 Zhuo, Q., Yu, B., Zhou, J., Zhang, J., Zhang, R., Xie, J., et al. (2019). Lysates of Lactobacillus acidophilus combined with CTLA-4-blocking antibodies enhance antitumor immunity in a mouse colon cancer model. Sci. Rep. 9:20128. Sequeira, L., Gaard, G., and De Zoeten, G. A. (1977). Interaction of bacteria and host cell walls: its relation to mechanisms of induced resistance. Physiol. Plant Pathol. 10, 43–50. Frontiers in Microbiology \| www.frontiersin.org September 2021 \| Volume 12 \| Article 697351 Conﬂict of Interest: SC and HBK were employed by the company eGnome, Inc. Conﬂict of Interest: SC and HBK were employed by the company eGnome, Inc. Simon, O., Vahjen, W., and Scharek, L. (2005). Micro-organisms as feed additives- probiotics. Adv. Pork Prod. 16, 161. doi: 10.1016/0377-8401(88)90097-1 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Skoch, E., Behnke, K., Deyoe, C., and Binder, S. (1981). The eﬀect of steam- conditioning rate on the pelleting process. Anim. Feed Sci. Technol. 6, 83–90. doi: 10.1016/0377-8401(81)90033-x Steensels, J., Gallone, B., Voordeckers, K., and Verstrepen, K. J. (2019). Domestication of industrial microbes. Curr. Biol. 29, R381–R393. 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. Ultee, E., Ramijan, K., Dame, R. T., Briegel, A., and Claessen, D. (2019). Stress- induced adaptive morphogenesis in bacteria. Adv. Microb. Physiol. 74, 97–141. doi: 10.1016/bs.ampbs.2019.02.001 van Heel, A. J., De Jong, A., Song, C., Viel, J. H., Kok, J., and Kuipers, O. P. (2018). BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Res. 46, W278–W281. Copyright © 2021 Jeon, Kim, Choi, Cho, Seo and Kim. 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. Vaudagna, S. R., Sánchez, G., Neira, M. S., Insani, E. M., Picallo, A. B., Gallinger, M. M., et al. (2002). Sous vide cooked beef muscles: eﬀects of low temperature– long time (LT–LT) treatments on their quality characteristics and storage stability. Int. J. Food Sci. Technol. 37, 425–441. doi: 10.1046/j.1365-2621.2002. 00581.x September 2021 \| Volume 12 \| Article 697351 Frontiers in Microbiology \| www.frontiersin.org 12
https://openalex.org/W3133444323	https://repository.publisso.de/resource/frl:6427110/data	English	null	Recent Advances on Nanomaterials to COVID‐19 Management: A Systematic Review on Antiviral/Virucidal Agents and Mechanisms of SARS‐CoV‐2 Inhibition/Inactivation	Global challenges	2,021	cc-by	14,061	1. Introduction Carvalho, Prof. C. A. Conte-Junior Nanotechnology Network Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (FAPERJ) Rio de Janeiro 20020-000, Brazil Prof. C. A. Conte-Junior Graduate Program in Veterinary Hygiene (PPGHV) Faculty of Veterinary Medicine Fluminense Federal University (UFF) Niterói 24230-340, Brazil Prof. C. A. Conte-Junior Graduate Program in Sanitary Surveillance (PPGVS) National Institute of Health Quality Control (INCQS) Oswaldo Cruz Foundation (FIOCRUZ) Rio de Janeiro 21040-900, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior Graduate Program in Chemistry (PGQu) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) Rio de Janeiro 21941-909, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior Graduate Program in Food Science (PPGCAL) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) Rio de Janeiro 21941-909, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior Nanotechnology Network Carlos Chagas Filho Research Support Foundation of the State of Rio de Janeiro (FAPERJ) Rio de Janeiro 20020-000, Brazil Prof. C. A. Conte-Junior Graduate Program in Veterinary Hygiene (PPGHV) Faculty of Veterinary Medicine Fluminense Federal University (UFF) Niterói 24230-340, Brazil Prof. C. A. Conte-Junior Graduate Program in Sanitary Surveillance (PPGVS) National Institute of Health Quality Control (INCQS) Oswaldo Cruz Foundation (FIOCRUZ) Rio de Janeiro 21040-900, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior COVID-19 Research Group Technological Development Support Laboratory (LADETEC) Department of Biochemistry Federal University of Rio de Janeiro (UFRJ) UFRJ, Rio de Janeiro 21941-909, Brazil E-mail: annacarvalho@iq.ufrj.br; conte@iq.ufrj.br Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior COVID-19 Research Group Laboratory of Advanced Analysis in Biochemistry and Molecular Biology (LAABBM) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) UFRJ, Rio de Janeiro 21941-909, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior COVID-19 Research Group Technological Development Support Laboratory (LADETEC) Department of Biochemistry Federal University of Rio de Janeiro (UFRJ) UFRJ, Rio de Janeiro 21941-909, Brazil E-mail: annacarvalho@iq.ufrj.br; conte@iq.ufrj.br Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior COVID-19 Research Group Laboratory of Advanced Analysis in Biochemistry and Molecular Biology (LAABBM) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) UFRJ, Rio de Janeiro 21941-909, Brazil The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/gch2.202000115. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH. www.global-challenges.com www.global-challenges.com Review 1. Introduction 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. Recent Advances on Nanomaterials to COVID-19 Management: A Systematic Review on Antiviral/Virucidal Agents and Mechanisms of SARS-CoV-2 Inhibition/Inactivation Anna Paula A. Carvalho* and Carlos A. Conte-Junior* 1. Introduction The current pandemic of coronavirus disease 2019 (COVID-19) is recognized as a public health emergency of worldwide concern. Nanomaterials can be effectively used to detect, capture/inactivate or inhibit coronavirus cell entry/ replication in the human host cell, preventing infection. Their potential for nanovaccines, immunoengineering, diagnosis, repurposing medication, and dis- infectant surfaces targeting the novel coronavirus (SARS-CoV-2) is highlighted. In this systematic review the aim is to present an unbiased view of which and how nanomaterials can reduce the spread of COVID-19. Herein, the focus is on SARS-CoV-2, analyzing 46 articles retrieved before December 31, 2020. The inter- face between nanomaterials is described, and the main mechanisms to inhibit SARS-CoV-2 pathogenesis and viral inactivation are also discussed. Nanocar- bons, biopolymeric, copper, and silver nanoparticles are potential antiviral and virucidal agents toward self-cleaning and reusable filter media and surfaces (e.g., facial masks), drug administration, vaccines, and immunodiagnostic assays. Trends in toxicology research and safety tests can help fill the main gaps in the literature and overcome health surveillance’s challenges. Phytochemicals delivery by nanocarriers also stand out as candidates to target and bio-friendly therapy. Nanocellulose might fill in the gaps. Future research using nanomate- rials targeting novel therapies/prophylaxis measures to COVID-19 and future outbreaks is discussed. A novel beta-coronavirus, the novel coro- navirus (SARS-CoV-2), or the severe acute respiratory syndrome coronavirus 2, was described as the causative agent of the pneumonia outbreak of coronavirus dis- ease 2019 (COVID-19), first reported in December 2019 in a local seafood market of Wuhan, Hubei province, China.[1,2] The same beta-coronavirus genus of SARS- CoV-2 was earlier the causative of viral pneumonia pandemics caused by severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respira- tory syndrome coronavirus (MERS-CoV) that emerged in 2002 in China and 2012 in the Arabian Peninsula, respectively.[3] The World Health Organization reported, globally, as of 30 January 2021, more than 101 million confirmed cases of COVID-19 and 2 196 944 deaths.[4] Due to lockdown and quarantine, humanity is now facing its worst economic crisis since World War Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior Graduate Program in Chemistry (PGQu) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) Rio de Janeiro 21941-909, Brazil Dr. A. P. A. Carvalho, Prof. C. A. Conte-Junior Graduate Program in Food Science (PPGCAL) Institute of Chemistry (IQ) Federal University of Rio de Janeiro (UFRJ) Rio de Janeiro 21941-909, Brazil Dr. A. P. A. g g Researchers have shown an increased interest in nanoma- terials for biomedicine, as antiviral drug delivery, due to their unique physical properties such as the small size (offering bio- availability and control of time release), tunable surface charge (for encapsulation of various drug types), and the large surface area to volume ratio (improving the solubility).[14] Nanomate- rials-based platforms with nanoemulsions, carbon nanotubes, gold, and cobalt nanoparticles have been reported in several efforts against viruses outside and inside the host cells.[15–17] For example, recent studies have demonstrated successful attempts with nanomaterials to control other viral human pathogens as human immunodeficiency virus type 1 (HIV-1),[18,19] herpes sim- plex,[16] hepatitis,[20] influenza,[21] and Zika virus.[22] On the other hand, the increased novel variants and class of different viruses with antiviral drug resistance is now a challenge faced by the pharmaceuticals.[23–25] Hence, broad-spectrum antiviral com- pounds are needed. Thus, there has been a growing number of publications focusing on nanomaterials as antivirals by them- selves. www.global-challenges.com Figure 1. Publications indexed on Web of Science, PubMed, Embase, and Scopus. Keywords: “nano OR nanomaterials OR nanoparticle” and “coro- navirus.” Search period: between January 1, 2004 and December 31, 2020. II,[5] with a critical impact on healthcare and socioeconomic costs. Thus, there is a high demand to design tools to enhance antiviral strategies to control the spread of COVID-19. II,[5] with a critical impact on healthcare and socioeconomic costs. Thus, there is a high demand to design tools to enhance antiviral strategies to control the spread of COVID-19. Viruses are considered a natural occurrence of nanoparticles due to their nanometric size. The genus beta-coronavirus (as well as alpha-, gamma-, and delta-coronavirus) is a member of the Coronaviridae family enveloped by spike (S) glycoproteins, positive-sense, single-stranded RNA, with virions of 118–140 nm essential for viral replication.[6] The spike glycoprotein medi- ates the receptor-binding spike protein and membrane fusion, responsible for viral cell entry in coronavirus infections.[7,8] Due to the small size and tunable surface charge, nanoscale mate- rials have great potential to interact with spike protein to block viral infection initiation. Hence, it could be useful to design nanomedicine-based strategies as novel antigens for nanovac- cines, antiviral drugs, and immunomodulatory therapies for COVID-19 management.[9] If a nanomaterial can be modified with specific antiviral ligands (e.g., copper, zinc, silver (Ag)), these nanoantivirals could benefit COVID-19 management.[10] Besides, nanoparticle-based antiviral drug delivery with high specificity can benefit the distinguishment of infected cells from healthy cells.[11] An infection-sensitive drug release based on nanoparticles could also minimize the premature drug release and loss drug before reaching the specific infection sites to overcome the challenge reported in the drug delivery design targeting intracellular infections.[11–13] Figure 1. Publications indexed on Web of Science, PubMed, Embase, and Scopus. Keywords: “nano OR nanomaterials OR nanoparticle” and “coro- navirus.” Search period: between January 1, 2004 and December 31, 2020. repurposing antimicrobial drugs,[35–37] including therapeu- tics approaches against viruses[38–40] and and coronaviruses (CoVs).[41,42] However, only after the COVID-19 emergence, a consider- able number of papers has been published on nanomaterials- based platforms and coronaviruses, although the first studies initiate after the first coronavirus pandemic, the 2002–2004 severe acute respiratory syndrome (SARS)—Figure 1 shows the number of publications retrieved in a simple search in some databases related to the topics “nano” and “coronavirus” between January 1, 2004 and December 31, 2020. Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH DOI: 10.1002/gch2.202000115 2000115 (1 of 18) 2000115 (1 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 2.4. Data Extraction Process For the data extraction of articles included in qualitative syn- thesis, the authors independently extracted and summarized the following information: nanomaterial type, size and shape, preparation strategy, coronavirus specie, significant results, interactions type between nanomaterial and coronavirus, mech- anism of action, and potential application field. • Search component 1: nanoparticle* OR nanomaterial* OR nanostructure* OR CNT OR graphene OR graph* OR “silver nanoparticle” OR AgNp OR liposome OR “gold nanoparti- cles” OR silica OR “self-assembly” OR nanocellulose OR hy- drogel OR “nanoparticle-based RNA” OR “copper nanopar- ticle” • Search component 1: nanoparticle* OR nanomaterial* OR nanostructure* OR CNT OR graphene OR graph* OR “silver nanoparticle” OR AgNp OR liposome OR “gold nanoparti- cles” OR silica OR “self-assembly” OR nanocellulose OR hy- drogel OR “nanoparticle-based RNA” OR “copper nanopar- ticle” www.advancedsciencenews.com www.global-challenges.com • Search component 3: antiviral OR virucide Table 1. Inclusion/exclusion criteria on paper retrieving in this SR. Order Step in PRISMA Inclusion criteria Exclusion criteria On the basis of 1 Identification Studies published in English Reviews, letters to the editor, or editorials Title, keywords and abstracts 2 Screening Studies with anti-coronavirus activity No coronavirus was investigated. 2 At least one nanostructured were studied No nanoscale materials strategy was presented 3 At least one segment of viral capture/inactivation was studied Studies were genome sequencing 4 Eligibility – Studies were preprints, not peer- reviewed processed 5 – Studies did not match the purposes of our study Full-text reading Table 1. Inclusion/exclusion criteria on paper retrieving in this SR. and knowledge gaps on the design of antiviral nanoagents, in which the purposes are therapy, disinfectants, and antiviral sur- faces (e.g., masks and coatings, for public uses), remain.[53,54] Nanomaterials′ modes of action to inhibit infection/inactivate SARS-CoV-2 have yet to be understood. Therefore, our study also contributes to understanding the interactions, types, and mechanisms of action associated between nanomaterials and SARS-CoV-2 inside and outside host cells. 2.2. Search Sources and String Definition Our search protocol strategy used search strings constructed and adapted for six electronic databases: Web of Sciences, PubMed, Embase, Scopus, SciFinder, and Science Direct. Besides, we performed additional searching on the reference list of relevant articles/reviews identified through the initial screening. The recovered papers of search sources were per- formed through a search string that summarizes the ques- tions researched. The string was based on pre-determined groups of keywords related to coronaviruses, nanoscale- based structures, and their use as antiviral/virucide agents accordingly: 2. Systematic Search Methods This SR recovered and assessed all the data available in lit- erature databases about nanomaterials′ antiviral potential to manage the COVID-19 pandemic, from other coronaviruses to the novel SARS-CoV-2. To improve our SR quality, we followed a four-phase flow diagram and the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) statement guidelines[55] supported by the SR management StArt tool.[56] 5 2.3. Search Strategy, Selection Process, and Study Selection Criteria The advanced search in the database was carried out consid- ering research articles published in English between 2000 and 2020. The search started on July 14, 2020 and was updated from August 3 to December 31, 2020 to cover the maximum peer- reviewed papers available, focusing directly on the novel SARS- CoV-2. The results of the screening were uploaded to the StArt tool. The authors first conducted the preliminary selection and extraction of data independently. Table 1 summarizes the inclu- sion/exclusion criteria adopted for the eligibility of studies. Fur- ther details can be seen in our previous work.[57] Silver nanoparticles were studied as antiviral agents for adenoviruses[26] and media filter for airborne viruses.[27] Silica and zinc oxide nanoparticles were reported as antiviral agents for agriculture.[28] Furthermore, non-toxic nanocellulose, extracted from biomass material cellulose, was reported as an ideal and green platform for drug delivery because of its safety, availability, and tunable surface chemistry.[29,30] Since several studies are still undergoing, some overviews discussed nanomaterials′ potential as antiviral candidates for COVID-19, but most took into account studies toward other CoVs.[43–45] A meta-analysis study has recently demonstrated nanoscale materials′ overall efficacy against other coronavi- ruses that appeared before the SARS-CoV-2 virus.[45] The inter- actions between several viruses and graphene have prospected this nanostructure as a future possibility against COVID-19[46,47] as disinfectants and antiviral coatings for personal protective equipment (PPE) for health workers.[46] Once copper (Cu)- based surfaces have also inactivated coronaviruses as human coronavirus 229E (HCoV-229E)[48] and SARS-CoV-2 in a short time compared to other surface materials,[49] nanostructured containing copper, silver, and zinc have also been prospected to inactivate SARS-CoV-2 and manage COVID-19.[10,50,51] Moreover, due to its potent antiviral activity, the enrichment of plasma copper levels was recently hypothesized to boost innate and adaptive human immunity to prevent and treat COVID-19.[52] The primary aim of this systematic review (SR) is i) to iden- tify, through a rigorous literature search analysis with a pre- defined strategy protocol, research using nanomaterials-based approaches against the Coronaviridae family and the latest finds until December 31, 2020 toward the novel SARS-CoV-2 virus. Another objective of our study ii) is to critically analyze nano- materials′ role as both a primary strategy (when the nanomate- rials interact directly with the virus) and a secondary approach (when the nanomaterials improve the efficacy of another antiviral agent) against coronaviruses. Thus, this review also addresses the main finds in the anti-coronavirus activity pro- moted by nanocarriers to identify potential candidates with min- imum off-target effects for COVID-19 control. Some challenges Moreover, studies with phytochemicals derived from nat- ural sources showed that bioactive compounds have already been effective against coronaviruses[31] by increasing the immunity system or destroying the pathogens, which has received attention as an alternative source of drugs with fewer side effects.[32–34] In this context, nanoflora—nanopar- ticles improving bioactive compounds′ bioavailability—was reported to enhance the delivery of insoluble phytochemicals 2000115 (2 of 18) 2000115 (2 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 2.1. Research Question The focus question agrees to the problem, intervention, com- parison, outcome, and study type (PICOS) strategy. As study type, we considered theoretical, hypothetical, computational, pre-clinical, and clinical research. The research questions focused on as follows: Which nanoscale structures showed anti-coronavirus effect potential for COVID-19 control? Which nanocarrier has been reported to improve the use of antivirals agents against coronaviruses? What are the modes of action to inhibit viral infection and types of interactions between the viral surface and the nanosystem hypothesized/purposed to viral inactivation? • Search component 3: antiviral OR virucide • Search component 3: antiviral OR virucide 2.5. Sources of Bias We did not consider the draft/final form of papers deposited directly online at preprint servers that are not peer reviewed. Besides, the eligibility criteria and the impact of missing data might be considered sources of bias. • Search component 2: “SARS-CoV-2” OR CoV OR “nCoV-2019” OR “COVID-19” OR “enveloped viruses” OR virus* OR coro- navirus OR Coronaviridae OR “SARS-CoV” OR SARS • Search component 2: “SARS-CoV-2” OR CoV OR “nCoV-2019” OR “COVID-19” OR “enveloped viruses” OR virus* OR coro- navirus OR Coronaviridae OR “SARS-CoV” OR SARS 2000115 (3 of 18) 2000115 (3 of 18) Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH Global Challenges 2021, 2000115 www.global-challenges.com Figure 2. PRISMA flow diagram with results of the systematic search between 2000 and 2020. Figure 2. PRISMA flow diagram with results of the systematic search between 2000 and 2020. Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 3. Main Findings in animals, were found 12 articles reporting nanomaterials against transmissible gastroenteritis virus (TGEV),[58] avian infectious bronchitis virus (IBV),[59] feline coronavirus (FCoV),[60] murine hepatitis virus (MHV),[61] type II feline infectious peritonitis virus (FIPV),[62,63] and porcine epidemic diarrhea virus (PEDV).[64–69] Concerning the 6 types of corona- virus cause illnesses in people were found 33 different studies reporting the role of nanomaterials against human corona- virus NL63 (HCoV NL63), human coronavirus OC43 (HCoV OC43),[61,70] human coronavirus 229E modified containing a renilla luciferase reporter gene (HCoV-229E-Luc),[71] human MERS-CoV,[72,73] human SARS-CoV,[74] and novel human SARS-CoV-2.[52,75–100] One article was a meta-analysis reporting the overall inhibition efficacy of nanomaterials against FIPV, F-CoV, TGEV, H-CoV, avian coronavirus (AvCoV), SARS-CoV, and MERS-CoV.[45] The results of our systematic review were registered in a PRISMA flow diagram illustrated in Figure 2. This systematic search identified a total of 744 papers at Web of Science, 485 at PubMed, 451 at Embase, 404 at Scopus, 22 at SciFinder, and 4 at Science Direct. Besides, we manually added a further 31 articles updating the search on the databases totaling 2141 papers. Of these, 992 were duplicates/triplicates and were excluded. A total of 1149 remained after the exclusion of repeated articles. After reading the titles, abstracts, and full-text, only 46 papers were adequate for the current study purposes since they matched the eligibility criteria. Of the 46 research papers retrieved for qualitative synthesis, several investigated nanomaterials potential against animal models and cell lines of 3 genera of Coronaviridae family causa- tive illness in animals (n = 6) and humans (n = 6), including alpha-coronavirus (n = 6), beta-coronavirus (n = 5), and gamma- coronavirus (n = 1). Related to 6 types of coronavirus originated We collected data from several nanostructures’ types studied against the coronavirus cited above as nanotubes, nanorods, nanoparticles, nanostars, nanowires, nanocrystals, nanosheets, 2000115 (4 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH www.global-challenges.com In some papers, we identified the role of nanomaterials as a secondary approach against coronaviruses (n = 11), such as those which addressed active phytochemicals delivered by spherical nanoparticles as a natural, less toxic, antiviral agent against coronaviruses, which were categorized to be discussed in a separate section of this SR.[61–64,66–68,74,80,91,93] We summarize in Figure 3 the main types of nanomate- rials with anti-coronavirus properties studies against animal and human coronaviruses found in papers retrieved by this SR. 4. Review of Literature Data Recovered 4.1. Nanomaterials against Causative Agents of Animal Coronaviruses Global Challenges 2021, 2000115 y Although this approach presented a poor control of polydis- persity (i.e., Ag nanowires varied from 20 to 400 nm), direct con- tact between Ag nanowires, Ag nanoparticles, and TGEV virus (a high mortality virus in seronegative suckling piglets) caused an inhibitory effect on viral infection and replication because of the interference with viral infection during attachment and entry (Figure 4B).[58] The mechanism of action was studied and cor- related with i) the inhibition of host cell apoptosis upregulation of p38/mitochondria-caspase-3 signaling route by Ag nanomate- rials; ii) inhibition of the infection initiation through the direct interaction between Ag nanomaterials and the spike glycopro- tein; and iii) Ag nanomaterials may alter the structure of surface proteins of TGEV/PEDV, inhibiting its identification and adhe- sion of the cellular receptor porcine aminopeptidase N.[58] The insights provided by Ag nanomaterials into the antiviral therapy of coronaviruses inspired the development of a nanocomposite by anchoring spherical silver nanoparticle (AgNPs) with thin layers of GO sheets as an antiviral against the lipid enveloped FCoV, with 26% of viral inhibition at the minimum concen- tration, at a non-cytotoxic level.[60] Regarding the mechanism, in a general way, it was suggested that the unique structure of graphene oxide contribute as follow (Figure 4C): i) negatively charged GO might interact with positively charged lipid mem- branes inducing its rupture; ii) then, the lipid tail exposed could strongly bind with the aromatic plane of GO sheets; and finally iii) this GO sheets–lipid membrane interaction can attract more lipid membranes.[60,102] More recently, core–shell bimetallic nanorods were obtained by deposition of silver shell on gold nanorods (Au-AgNRs) followed by releasing of Ag+ and exposure of Ag nanowires after endogenous reactive oxygen species (ROS) stimulation.[69] The system inhibited PEDV replication by a mul- tisite mechanism: i) inhibition of PEDV entry with decreasing This SR presented in the next section the review of our main finds with coronavirus and nanomaterial types, focusing on antiviral properties, as well as the chemical interface and cel- lular mechanisms associated with them. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH www.advancedsciencenews.com www.advancedsciencenews.com nanoparticles;[73] bovine serum albumin (BSA)-coated tellurium nanoparticles (Te/BSA NPs) with a unique triangular star shape (Te/BSA nanostars);[65] GSH-modified zinc-sulfide nanopar- ticles (ZnS NPs);[66] glycyrrhizic-acid-based carbon dots (Gly- CDs);[67] multi-walled carbon nanotubes (MWCNTs) with target functions;[83] metal-decorated single-wall carbon nanotubes (SWCNTs);[100] NLCs;[88] graphene oxide;[60,82,92] iron oxide nano- particles (IONPs),[75] functionalized graphene sheets;[84] bime- tallic nanorods (golden-silver);[69] titania nanoparticles (TNP);[70] polyphosphate (Polyp) nanoparticle;[91] nanosized formazans;[78] copper nanoparticles/nanowires;[52,81,92,94,95] silica nanoparticles (SiNPs);[81] manganese nanodepot;[89] decoy nanoparticles;[90] besides nanocomposites between several of them. nanogels, nanospheres, nanocapsules, nanoclusters, and nano- structured lipid carriers (NLCs). Of these, were identified the following nanotechnology-based approaches: bioconjugated car- boxyl quantum dots (QDs);[74] silver nanomaterials;[58,60,69,80,96,97] synthetic virus-like particles (sVLPs);[77] gold nanoparticles;[59,77] biopolymeric/biodegradable polymeric nanoparticles and hydrogels (e.g., chitosan, collagen, poly(ethylene glycol) (PEG), poly lactic-co-glycolic acid (PLGA), and poly(hydroxyethyl) methacrylate);[61,62,63,79,85–87,91,93] carbon quantum dots (CQDs)[71] and cationic carbon dots based on curcumin (CCM-CDs);[68] glutathione(GSH)-capped silver-sulfide nanoclusters (GSH- capped Ag2S NCs);[64] gold nanorod-based heptad repeat 1 (HR1) peptide;[72] antigen and adjuvant-loaded hollow polymeric 2000115 (5 of 18) © 2021 The Authors Global Challenges published by Wiley VCH GmbH Figure 3. The main types of nanomaterials with antiviral properties studied against animal/human coronaviruses, including the novel SARS-CoV-2, found in the papers retrieved by this review. Global Challenges 2021 2000115 Figure 3. The main types of nanomaterials with antiviral properties studied against animal/human coronaviruses, including the novel SARS-CoV-2, found in the papers retrieved by this review. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 2000115 (5 of 18) Global Challenges 2021, 2000115 Global Challenges 2021, 2000115 www.advancedsciencenews.com www.global-challenges.com www.advancedsciencenews.com Table 2. Nanomaterials approach with anti-coronavirus activity against animal coronaviruses found in the retrieved papers. Nanomaterial Average size Shape Strategy Coronavirus Application Ref. Aga) nanomaterials Colloids: 10 nm Nanowires: 60–400 nm Nanoparticles: <20 nm Spherical and wires Silver (colloids, nanowires, and nanoparticles) TEGVb) (PUR46-MAD) Antiviral therapy [58] GOc) GO-Agd) GOc): 0.6–9 nm GO-Agd): 5–25 nm GOc): layers GO-Agd): spherical Silver anchored to graphene oxide sheets FCoVe) (NTU-156) PPEf) [60] Gold nanoparticles- based sVLPsg) 100–140 nm Like natural viral particles Synthetic Av-CoV-IBVh) spike protein antigen with 100 nm AuNPsi) AvCoV-IBVh) (2575/98) Vaccine immunology [59] Tellurium nanostars 57 nm Triangular star shape MESj)-modified BSAk)-coated tellurium nanostars PEDVl) Antiviral agents [65] Bimetallic nanorods – Rod like (core—shell) Gold nanorods coated by Ag shell deposition PEDVl) Antiviral therapies [69] a)Silver; b)Transmissible gastroenteritis virus; c)Graphene oxide; d)Silver/graphene oxide nanocomposite; e)Feline coronavirus; f)Personal protection equipment; g)Synthetic virus- like particles; h)Avian coronavirus infectious bronchitis virus; i)Gold nanoparticles; j)Mercaptoethane sulfonate; k)Bovine serum albumin; l)Porcine epidemic diarrhea virus. omaterials approach with anti-coronavirus activity against animal coronaviruses found in the retrieved papers. a)Silver; b)Transmissible gastroenteritis virus; c)Graphene oxide; d)Silver/graphene oxide nanocomposite; e)Feline coronavirus; f)Personal protection equipment; g)Synthetic virus- like particles; h)Avian coronavirus infectious bronchitis virus; i)Gold nanoparticles; j)Mercaptoethane sulfonate; k)Bovine serum albumin; l)Porcine epidemic diarrhea virus. a)Silver; b)Transmissible gastroenteritis virus; c)Graphene oxide; d)Silver/graphene oxide nanocomposite; e)Feline coronavirus; f)Personal p like particles; h)Avian coronavirus infectious bronchitis virus; i)Gold nanoparticles; j)Mercaptoethane sulfonate; k)Bovine serum albumin; l)P obtained from the recombinant protein expression of IBV, as model antigens.[59] The core–shell morphology binds with bul- bous surface projections to mimetic natural viral particles and can be applied as a vaccine against AvCov-IBV with an improved humoral and cell-mediated immune response (Figure 4A). Compared to the free antigen protein, the mechanism of the potent immunity response of sVLPs against AvCov-IBV was attributed to primary lymphatic delivery and the multivalent antigen display, the major antibody titers, and the minor infec- tion-like symptoms.[59] In some papers, we identified the role of nanomaterials as a secondary approach against coronaviruses (n = 11), such as those which addressed active phytochemicals delivered by spherical nanoparticles as a natural, less toxic, antiviral agent against coronaviruses, which were categorized to be discussed in a separate section of this SR.[61–64,66–68,74,80,91,93] We summarize in Figure 3 the main types of nanomate- rials with anti-coronavirus properties studies against animal and human coronaviruses found in papers retrieved by this SR. Figure 4. Main nano-based approaches against animal coronaviruses found in this SR: A) sVLPs based on gold nanoparticles and incubation of IBV spike protein; B) inhibitory effects of silver nanomaterials on TGEV-induced host cell infection and p38-MAPK signaling activation; and C) interactions between Ag/GO nanocomposites and FCoV. mitochondrial membrane potential and caspase-3 activity, and (ii) apoptosis induced by virus infection.[69] Therefore, those attempts using silver nanomaterials provide insights into novel therapeutic strategies to prevent coronavirus replication. against animal coronaviruses, which we decided to discuss later (see Section 4.4). Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 4.1. Nanomaterials against Causative Agents of Animal Coronaviruses Several emerging viruses have been known to be transmitted from animals to humans. The scientific community argues that the most probable cause of the COVID-19 pandemic started in the South China seafood market (Wuhan, Hubei) is zoonotic spillover.[1,2] Despite are still unclear which animals transmitted COVID-19 to humans, recent findings show that SARS-CoV-2 has 96% genomic similarity with a bat coronavirus.[101] Thus, these facts motivate us to choose articles and discuss the approaches with the anti-coronavirus properties of nanomaterials of this section, given the possibility of extending the SARS-CoV-2 virus strategies to overcome the challenges of COVID-19 control. The type of nanomaterials found included nanoparticles, nanowires, nanostars, nanospheres, nanocapsules, nanoclus- ters (Table 2). Most of them are inorganic, polymeric, and carbon-based nanomaterials with varied morphology. These include spherical particles, rods, layers, triangular star shape, spherical particles capped with a corona-like structure. The average size ranged from 5 to 140 nm. The high energy surfaces of synthetic nanoparticles were exploited to induce protein corona formation in sVLPs based on gold nanoparticles and incubation of IBV spike protein, 2000115 (6 of 18) 2000115 (6 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH www.advancedsciencenews.com www.global-challenges.com Figure 4. Main nano-based approaches against animal coronaviruses found in this SR: A) sVLPs based on gold nanoparticles and incubation of IBV spike protein; B) inhibitory effects of silver nanomaterials on TGEV-induced host cell infection and p38-MAPK signaling activation; and C) interactions between Ag/GO nanocomposites and FCoV. ww.advancedsciencenews.com www.global-challenges.com www.advancedsciencenews.com www.global-challenges.com Figure 4. Main nano-based approaches against animal coronaviruses found in this SR: A) sVLPs based on gold nanoparticles and incubation of IBV ww.advancedsciencenews.com www.global-challenges.com www.advancedsciencenews.com www.global-challenges.com www.global-challenges.com www.advancedsciencenews.com Table 3. Nanomaterials approach with anti-coronavirus activity as potential candidates against human coronaviruses identified before SARS-CoV-2 found in the retrieved papers. Table 3. Nanomaterials approach with anti-coronavirus activity as potential candidates against human coronaviruses identified before SARS-CoV-2 found in the retrieved papers. Table 3. Nanomaterials approach with anti-coronavirus activity as potential candidates against human coronaviruses identified before SARS-CoV-2 found in the retrieved papers. Nanomaterial Average size Shape Strategy Coronavirus Potential application Ref. Chitosan nanospheres 10 nm–10 µm Spherical Genipin-crosslinked chitosan HCoV NL63a), HCoV OC4b) Adsorbents [61] CQDsc) nanocrystals 4–9 nm Spherical Boronic acid-functionalized CQDs HCoV-229E-Lucd) Antiviral drugs [71] Gold nanorods 18–54 nm Rod Gold nanorods-based HR1e) peptides MERS-CoVf) Antiviral drugs [72] PLGAg) hollow nanoparticles 114 nm Spherical Viral antigens and STINGh) agonists-loaded hollow nanoparticles MERS-CoVf) (EMC 2012) Vaccine [73] TiO2 Nanoparticles (Not reported) Predominantly spherical TNPsi)-coated glass coverslips UVC radiation HCoV NL63a) Self-cleaning surfaces [70] a)Human coronavirus NL63; b)Human coronavirus OC4; c)Carbon quantum dots; d)Modified human coronavirus 229E containing a renilla luciferase reporter gene; e)HR1; f)Middle East respiratory syndrome coronavirus; g)Poly (lactic-co-glycolic acid); h)Stimulator of interferon genes protein; i)Titanium dioxide nanoparticles. a)Human coronavirus NL63; b)Human coronavirus OC4; c)Carbon quantum dots; d)Modified human coronavirus 229E containing a renilla luciferase reporter gene; e)HR1; f)Middle East respiratory syndrome coronavirus; g)Poly (lactic-co-glycolic acid); h)Stimulator of interferon genes protein; i)Titanium dioxide nanoparticles. navirus OC4; c)Carbon quantum dots; d)Modified human coronavirus 229E containing a renilla luciferase reporter gene; e)HR1; virus; g)Poly (lactic-co-glycolic acid); h)Stimulator of interferon genes protein; i)Titanium dioxide nanoparticles. (see Section 4.4), the tremendous adsorptive capacity was attrib- uted to HTCC-virus electrostatic interactions and the high ionic strength caused by the HTCC cationization process. vaccine inhibited viral infection, was immunogenic and pre- vented the induction of undesirable lung disease in immunized human DPP4 enzyme transgenic mice.[73] Recently, preliminary results with the virucidal effect of TNP against the HCoV-NL63 and HCoV-OC43 by photoactive TNP deposited on glass cov- erslips using UVC radiation. The authors also mentioned the potential to control the spread of COVID-19 by self-cleaning surfaces, and hence, they are extending the concept to SARS- CoV-2 in work currently underway.[70] Three types of CQDs were synthesized containing boronic acid groups, which proved to be essential, in a dose-dependent manner, to the higher antiviral activity presented against the human coronaviruses HCoV-229E-Luc.[71] These nanomate- rials were suggested as potential candidates to replace the standard ribavirin/α-interferon (α-IFN), with fewer side effects. Concerning the mechanism of action, it was suggested that CQDs act by multistep at i) the initial stages of viral infection, inhibiting the viral entry, which probably is due to the inhibi- tion of the interactions between the spike protein receptor and host cell membrane, caused by interactions between functional groups of CQDs with viral entry receptors; and ii) an equivalent extensive inhibition activity at the viral replication step.[71] 4.3. Nanomaterials and the Novel SARS-CoV-2: Recent Advances to Reduce the Spread of COVID-19 Table 4 displays computational approaches recently pub- lished repurposing medications, therapies, antiviral and immunologic agents to combat COVID-19 patients. By mole- cular docking, the United States Food and Drug Admin- istration (FDA)-approved magnetic nanoparticles (Fe2O3 and Fe3O4) are repurposed to treat and control COVID-19, based on its efficient hydrophobic interactions and hydrogen bonding with the chimeric S-receptor-binding domain (S1-RBD) of SARS-CoV-2, to form a more stable complex[75] (Figure 5A). Likewise, novel gold nanoparticles functionalized Aiming to develop an antiviral drug and vaccine against MERS-CoV, gold nanorods[72] and biopolymeric hollow nano- particles made from poly (lactic-co-glycolic acid) (PLGA) with core–shell morphology[73] were studied based on HR1 peptides and viromimetic STING agonists, respectively. In the first study, a series of HR1 peptides was developed by molecular docking to inhibit HR1/HR2-mediated membrane fusion between the MERS-CoV and host cells.[72] Further, the nanoparticle-based Table 4. Computational approaches to predict ligand–receptor binding and structure-based drug design for COVID-19 management. Nanomaterial Size Strategy Ligand–receptor binding results Potential application Ref. Iron oxide nanoparticles N/ra) Nano-mineral structure of Fe2O3b) and Fe3O4c) Interactions with S1-RBDd) of SARS-CoV-2e) Repurposing medication [75] PolyPf)/Silica nanoparticles 210 ± 40 nm Optimized polyPf) encapsulated by SiNPsg) Inhibition of binding of ACE2h) to S-protein SARS-CoV-2e), at a physiological solution Immunologic agents [76] Gold nanoparticles N/ra) Peptide-functionalized gold nanoparticles More stable complex with RBDd) of SARS-CoV-2e) than ACE2h). Antiviral agents [77] Nano-sized formazans 23.75 ± 7.16 nm Formazan analogs by dithizone and α-haloketones reaction Inhibition of SARS-CoV-2e) chymotrypsin-like protease, at a physiological solution Antiviral agents [78] L-PLGA NPsi) N/ra) Optimized Remdesivir-loaded L-PLGA NPsi) Interactions Lisinopril-ACE1g) and remdesivir- intracellular targeting protein RdRpj) Antiviral therapy [79] Silver nanoparticles Artemisinin, Artemether, and Artesunate delivery by silver nanoparticles Interactions between negative charges of oxygen atoms of drugs with Ag surface Antiviral drugs [80] a)Not reported; b)Iron(III) oxide or magnetite; c)Iron(II,III) oxide or hematite); d)Chimeric spike-receptor-binding domain; e)Novel coronavirus; f)Ployp; g)Silica nanoparticle; h)Angiotensin-converting enzyme inhibitor 1 or 2; i)Lisinopril covalently grafted onto poly(lactic-co-glycolic acid) nanoparticles; j)RNA-dependent RNA polymerase. Table 4. Computational approaches to predict ligand–receptor binding and structure-based drug design for COVID-1 a)Not reported; b)Iron(III) oxide or magnetite; c)Iron(II,III) oxide or hematite); d)Chimeric spike-receptor-binding domain; e)Novel coronavirus; f)Ployp; g)Silica nanoparticle; h)Angiotensin-converting enzyme inhibitor 1 or 2; i)Lisinopril covalently grafted onto poly(lactic-co-glycolic acid) nanoparticles; j)RNA-dependent RNA polymerase. © 2021 The Authors. 4.2. Nanomaterials against Causative Agents of Human Corona- viruses Identified before SARS-CoV-2 4.2. Nanomaterials against Causative Agents of Human Corona- viruses Identified before SARS-CoV-2 Heparan sulfate (HS) proteoglycans are often used as a cellular attachment receptor to mediate viral infections′ adhesion and internalization. Inspired in this mechanism and in view to mimic the cell surface receptor HS, its analog mercaptoethane sulfonate (MES) was employed as the chemical modifier to synthesize Te/BSA nanostars with the advantage of upregulating its high antiviral activity against PEDV.[65] This inorganic nanomaterial mechanism was correlated with ROS generation inhibition, high- lighting its potential as a broad-spectrum antiviral agent.[65] Table 3 shows the articles retrieved in this SR targeting the anti- viral properties of CQDs, gold nanoparticles, titania oxide nano- particles, and biopolymeric nanoparticles against HCoV-NL63, HCoV-OC43, SARS-CoV, and MERS-CoV. Cationic chitosan nanospheres crosslinked by natural genipin (HTCC) presented high affinity with spike protein of coronaviruses and shown a more significant adsorbent effect against HCoV-NL63 than HCoV-OC43.[61] This biopolymeric nanosphere was appointed as convenient candidates to remove coronaviruses from biological matrices and water with higher selectivity. Among other details that will be discussed later Additionally, a meta-analysis in a series of nanoscale mate- rials in publications retrieved demonstrated a positive inhi- bition efficacy against animal coronaviruses in vitro and in vivo.[78] We retrieved the other six original research articles with nanomaterials and natural compounds for treatment purposes 2000115 (7 of 18) a)Not reported; b)Iron(III) oxide or magnetite; c)Iron(II,III) oxide or hematite); d)Chimeric spike-receptor-binding domain; e)Novel coronavirus; f)Ployp; g)Silica nanoparticle; h)Angiotensin-converting enzyme inhibitor 1 or 2; i)Lisinopril covalently grafted onto poly(lactic-co-glycolic acid) nanoparticles; j)RNA-dependent RNA polymerase. 4.3. Nanomaterials and the Novel SARS-CoV-2: Recent Advances to Reduce the Spread of COVID-19 Global Challenges published by Wiley-VCH GmbH 2000115 (8 of 18) Global Challenges 2021, 2000115 2000115 (8 of 18) Global Challenges 2021, 2000115 www.global-challenges.com www.advancedsciencenews.com Figure 5. Nano-based approaches against SARS-CoV-2 reviewed: A) docking interactions between FDA-approved IONPs with spike RBD od SARS- CoV-2; B) functionalized CNTs with target functions; C) FET-sensor with graphene sheets conjugated to SARS-CoV-2 spike antibody; D) pulmonary drug delivery system using NLCs to treat lung infected by SARS-CoV-2. Figure 5. Nano-based approaches against SARS-CoV-2 reviewed: A) docking interactions between FDA-approved IONPs with spike RBD od SARS- CoV-2; B) functionalized CNTs with target functions; C) FET-sensor with graphene sheets conjugated to SARS-CoV-2 spike antibody; D) pulmonary drug delivery system using NLCs to treat lung infected by SARS-CoV-2. with peptides formed a more stable complex with RBD than angiotensin-converting enzyme 2 (ACE2).[77] A non-toxic approach with SiNPs showed that SiNPs-encapsulated PolyP (to stabilize PolyP against alkaline phosphatase) inhibited the binding of ACE2 to S1-RBD of SARS-CoV-2 at physiological concentration due to interactions between PolyP nanoparti- cles and amino acids on the surface of S1-RBD.[76] Moreover, this strategy was suggested to prevent and treat SARS-CoV-2 infection in the oropharyngeal and boost the immune system of thrombocytopenic COVID-19 patients.[76] Another study reported the potential of synthesized nano-formazans as anti- viral agents to manage COVID-19 infection by docking simu- lations at a physiological solution: the results showed that formazan analogs could bind the active site 3CL protease of SARS-CoV-2, inhibiting the viral replication.[78] Polymer nan- oparticles-optimized Remdesivir to repurpose it as antiviral therapy associated with lisinopril (a molecule of the thera- peutic and lung-protective effect of ACE) by Remdesivir-loaded lisinopril-functionalized PLGA.[79] Thus, the potential of Rem- desivir-optimized nanoparticles was reported by a docking study, which confirmed interactions between lisinopril and ACE; and the binding of Remdesivir and RNA-dependent RNA polymerase (RdRp), an enzyme involved in replication and transcription of the SARS-CoV-2 genome.[79] Therefore, all these nanotechnologies approaches were predicted to inter- fere with viral adhesion to human host cell receptors and viral replication, thus inhibiting the viral infection. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH SWCNTsk) Cylinder Metal-decorated SWCNTsk) PPEl) design [100] GOb) Layers GOb)-PMMAm) film PPEl) design [82] MWCNTsn) Multi-walled cylinder Acidizing and RNA Lyase-modified carbon nanotubes Vaccines and drugs [83] Copper (Not reported) Enrichment of plasma copper levels Immunotherapy [52] Silica-copper nanoparticles Spherical Silica-copper/polymer (silicone or epoxy) nanocoating Superhydrophobic self-cleaning surfaces [81] CuNPsa)/GOb) nanocomposite Nanosheets, nanofibers Electrospinning: multilayers of CuNPsa)/GOb)-PLAc) and CuNPsa)/GOb)-CAd) nanofibers Respirator filter to antiviral face mask [92] Cu-ZIF-8e)/copolymer nanocapsule Core–shell Stabilizing CuNRsf) by Pluronic F-127g), growth of ZIF-8e) to form a uniform core–shell structure Filter media (reusable facial mask) [94] Shellac/CuNPsa) nanohybrid Spherical, ≈100 nm Dual-channel spray-assisted nanocoating of shellac/CuNPsa) to a nonwoven surgical mask Photoactive antiviral mask (self-cleaning) [95] AgNCsh)/silica nanocomposite Predominantly spherical, <200 nm AgNCsh)/silica-based sputtered coating deposited on an FFP3 mask Respirator facial masks, air filter [96] Silver nanoparticles Spherical Colloidal coating: silver nanoparticles dispersed in PVPi) Virucide coating [97] Aluminum nanofibers Fibers Nanostructured aluminum alloy surface by wet etching Antiviral surfaces [98] Gold nanoparticles Spherical, 2.4 nm Sulfonated gold nanomaterials: coating with MUSj) ligands Antiviral agents [99] Graphene sheets Layers PBASEo)-modified graphene sheets FETp) sensor with graphene sheets conjugated to SARS-CoV-2q) spike antibody Immunodetection [84] Polymeric nanoparticles Spherical Bioinspired DNase-Ir)-coated melanin-like nanospheres: recombinant DNase-Ir)/PEGs) coating Therapy for with ARDSt) or sepsis in severe COVID-19 patients [85] Polymeric nanoparticles Spherical, 220 nm DNase-Ir)-coated polydopamine-PEGs) nanoparticles (exogenous administration) Therapy for with ARDSt) or sepsis in severe COVID-19 patients [86] Polymeric nanoparticles (Not reported) Ivermectin-delivery by (PLGA-b-PEG-Mal)u) copolymer nanoparticles (orally administrable) Antiviral drug [87] Nanostructured lipid carriers Spherical Pulmonary delivery of Salinomycin by nanostructured lipid carriers Drug delivery [88] Manganese nanodepot (Not reported) Droplet-confined nanoprecipitation in water-in-oil micro-emulsion + thin-film dispersion method Vaccine adjuvant [89] Decoy nanoparticles (Not reported) Fusing genetically engineered cell membrane nanovesicles (293T/ACE2 and THP-1cells) Therapeutic vaccines [90] a)Copper nanoparticles; b)Graphene oxide; c)Polylactide (as matrix); d)Cellulose acetate (as matrix); e)Copper2+-doped zeolitic imidazolate framework-8; f)Copper nano- wires; g)Block copolymer of PEG-PPG-PEG structure (PEG: poly(ethylene glycol); PPG: poly(propylene glycol)); h)Silver nanoclusters; i)Polyvinylpyrrolidone; j)Decanesulfonic acid; k)Single-walled carbon nanotubes; l)Personal protection equipment; m)Poly(methyl methacrylate (as matrix); n)Multi-walled carbon nanotubes; o)1-Pyrenebutyric acid N-hydroxysuccinimide ester; p)Field-effect transistor; q)Novel coronavirus; r)Deoxyribonuclease I; s)Poly-(ethylene glycol); t)Acute respiratory distress syndrome; u)Poly(lactide-co-glycolide)-block-poly-(ethylene glycol)-maleimide nanoparticles. www.global-challenges.com www.global-challenges.com www.advancedsciencenews.com www.advancedsciencenews.com nd applied research based on nanomaterials approaches to manage COVID-19, found between 2000 and December Table 5. Theoretical, hypothesis and applied research based on nanomaterials approaches to manage COVID-19, found between 2000 and December 31, 2020. Nanomaterial Shape, size Strategy Potential application Ref. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH Global Challenges 2021, 2000115 4.3. Nanomaterials and the Novel SARS-CoV-2: Recent Advances to Reduce the Spread of COVID-19 with peptides formed a more stable complex with RBD than angiotensin-converting enzyme 2 (ACE2).[77] A non-toxic approach with SiNPs showed that SiNPs-encapsulated PolyP (to stabilize PolyP against alkaline phosphatase) inhibited the binding of ACE2 to S1-RBD of SARS-CoV-2 at physiological concentration due to interactions between PolyP nanoparti- cles and amino acids on the surface of S1-RBD.[76] Moreover, this strategy was suggested to prevent and treat SARS-CoV-2 infection in the oropharyngeal and boost the immune system of thrombocytopenic COVID-19 patients.[76] Another study reported the potential of synthesized nano-formazans as anti- viral agents to manage COVID-19 infection by docking simu- lations at a physiological solution: the results showed that formazan analogs could bind the active site 3CL protease of SARS-CoV-2, inhibiting the viral replication.[78] Polymer nan- oparticles-optimized Remdesivir to repurpose it as antiviral therapy associated with lisinopril (a molecule of the thera- peutic and lung-protective effect of ACE) by Remdesivir-loaded lisinopril-functionalized PLGA.[79] Thus, the potential of Rem- desivir-optimized nanoparticles was reported by a docking study, which confirmed interactions between lisinopril and ACE; and the binding of Remdesivir and RNA-dependent RNA polymerase (RdRp), an enzyme involved in replication and transcription of the SARS-CoV-2 genome.[79] Therefore, Table 5 displays 19 recently published articles research focusing on novel coronavirus by theoretical, hypothesis, and applied research based on nanomaterials approaches to manage COVID-19. Of these were found metal nanoparticles, carbon nanomaterials (e.g., carbon nanotubes, graphene layers, and graphene oxide), inorganic/organic polymeric nanoparticles, nanolipids, and nanodecoys purposing antiviral agents and sur- faces, virucide agents, drug delivery, vaccines, immunological agents, and viral detection. Carbon nanotubes were present in an innovative theoretical proposal to develop vaccines and drugs for COVID-19, exploiting coronavirus physical–chemical properties and constructing a target acidification environment (Figure 5B). MWCNTs with target functions, RNA lyase- and acid-functionalized combined with photothermal effects, were hypothesized to block viral infection and replication routes in the host cells.[83] Regarding modes of actions, acidizing the cell environment, generating a photodynamic thermal effect by irradiation of functionalized- carbon nanotubes, and smart drug-delivery of a viral RNA lyase destruction were proposed.[83] Free-energy calculations by DFT were performed on carbon-based nanomaterials and 2000115 (9 of 18) Global Challenges 2021, 2000115 www.advancedsciencenews.com Other research that addressed the antiviral and virucide effect of silver discussed the interaction between coating-based col- loidal AgNPs and SARS-CoV-2 by Luciferase-based pseudovirus entry assay and revealed that Ag nanomaterials potently block viral entry step via disrupting viral integrity.[97] Furthermore, SARS-CoV-2 was wholly inactivated after 6 h of exposure to a nanostructured aluminum alloy surface obtained by wet- etching technique.[98] A recent study hypothesized that excessive neutrophil extracellular traps (NETs) and extracellular DNAs (eDNAs) could activate NETosis, neutrophil-specific programmed cell death might be associated with COVID-19 pathogenesis.[85,105] Nowadays, there are no FDA-approved antiviral medications that can effectively suppress the SARS-CoV-2-mediated neu- trophil activities, cytokine storm, acute respiratory distress syndrome (ARDS), and sepsis, thus, promoting widespread patient improvement.[85,86] Therefore, different strategies using polymeric nanoparticles to deliver antiviral agents have been evaluated to drug repurpose.[85–87] An in vivo study with a septic mouse model showed the potential of bioinspired DNase-I-coated melanin-like nanospheres using PEG to reduce neutrophil counts and modulate sepsis-associated NETosis dysregulation in the plasma of COVID-19 patients alleviating inflammation and mortality.[85] Further research showed an exogenous administration of a long-acting DNase-1, a recombi- nant DNase-1-coated polydopamine-PEG nanoparticulated, can reduce SARS-CoV-2-mediated neutrophil activities and cytokine storm as a potential treatment to COVID-19-related illnesses.[86] Gold nanoparticles coated by decanesulfonic acid ligands inhibited the activity of authentic SARS-CoV-2 in a nanomolar range, and contrarily to most of the strategies that targeted the inhibition of SARS-CoV-2 cell entry by blocking spike pro- tein-ACE2 receptor binding, this sulfonated nanomaterial can inhibit SARS-CoV-2 attachment by blocking spike protein-HS receptors binding and was suggested as simply reversible and potent antiviral agents.[99] Manganese nanodepot (nanoMn) and decoy nanoparticles were proposed as simple, safe, and robust vaccine adjuvants and antiviral agents to manage COVID-19.[89,90] Although man- ganese can reduce IFN response—a central host response against viruses—there is a challenge in its applicability due to non-specific distribution and neurotoxicity.[89] Thus, manga- nese was repurposed in nanoMn with enhanced cell uptake and persisted release of Mn2+ in a pH-sensitive manner, boosted IFN response, broad-spectrum in vitro and in vivo antiviral effects and macrophage polarization; no neuroinflammation effects were observed; nanoMn acted as a vaccine adjuvant to boost host adaptive immunity.[89] Otherwise, a decoy nanopar- ticle made by genetic engineering can protect host cells against COVID-19 infection by a two-step neutralization approach: i) first, virus neutralization followed by ii) inflammatory cytokine neutralization in the second step (e.g., interleukin 6 and gran- ulocyte-macrophage colony-stimulating factor).[90] Thus, the authors reported stabilization of ACE2 expression; protection of host cell against infection by competition between nanodecoy and host cell to SARS-CoV-2 binding; and suppressing immune A simple, highly selective, sensitive, and rapid method for detecting the SARS-CoV-2 virus in nasopharyngeal swab sam- ples from COVID-19 patients, without sample pre-treatment/ labeling, was performed on a field-effect transistor (FET)-based biosensor using functionalized-graphene sheets as a receptor[84] (Figure 5C). www.advancedsciencenews.com A filtration system from a nanofibrous respirator facial mask containing multilayers of Cu nanoparti- cles/GO nanosheets was dispersed in a nanofibrous matrix of biodegradable polylactic acid (PLA) or cellulose acetate (CA).[92] Interestingly, the use of thermoplastic polymers such as PLA and CA can provide a stable fit with face anatomy.[92] Likewise, low-cost scalable synthesis of Cu nanowires/ZIF-8 stabilized by an amphiphilic block copolymer (Pluronic F-127) in a core– shell structure was direct deposited onto a reusable face mask system and produced 55% inhibition of SARS-CoV-2 replication after 48 h at a concentration of 1 µg.[94] A dual-channel spray- assisted nanocoating hybrid of shellac/CuNP to a photoacti- vated antiviral facial mask with self-sterilizing and reusability was reported with virucide effects.[95] Another nanotechnology- based filter air was reported with silver nanoclusters/silica com- posite sputtered coating applied on FFP3 mask with virucide effect, and completely reduced SARS-CoV-2 titer to zero on tested conditions to the sample with the highest content of Ag. www.advancedsciencenews.com www.advancedsciencenews.com sounds like a potential strategy to increasing its absorption at the local infection due to the good aerodynamical properties of NLCs, which could be aerosolized by droplets as antiviral drugs. Besides, the hypothesis was based on pieces of evidence that SAL has the potential to prevent the viral entry into the cytosol, prevent membrane fusion in a pH-dependent way, interact with spike protein, inducing the ACE2 binding, and preventing the release of viral acid nucleic into the cytoplasm.[88,103,104] Iver- mectin is a clinically approved antiviral drug and was repur- posed against SARS-CoV-2, using orally administering PLGA- grafted-PEG-maleimide nanoparticles, an amphiphilic and biodegradable block copolymer system, which was capable of delivering a more potent therapeutic dose.[87] The system dem- onstrated potential for the therapeutic drug to COVID-19 by multisite inhibition into decreasing the viral uptake and trans- mission by i) inhibition of viral spike protein level and its entry rate by downregulation of ACE2 expression, and ii) possibly, inhibition of nuclear transport activities mediated by proteins (e.g., importin α/β1 heterodimer).[87] hypothesized to the self-cleaning surface for implementa- tion in public and healthcare work environments to eradicate SARS-CoV-2 spread and protect against COVID-19 by three-step strategy: i) virus encapsulation, ii) contamination suppression, and iii) virus elimination.[81] Although face masks can limit transmission, the increased demand for disposable masks also demand many resources and has raised concerns about the generation of waste.[94] Thus, some strategies using sustain- able polymers have been studied to reduce transmission and the impact of waste. Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH were proposed to develop nanodevices useful in COVID-19 and future pandemics management.[82,100] Exploiting ROS molecules′ harmfulness to coronavirus, nanofilters based on metal-decorated (Pt, Cu, Rh, and Ru)-SWCNTs, combined with H2O2, were presented as an effective platform for future experi- ments in SARS-CoV-2 absorption.[100] The nanocomposite use for elastomeric respirators and self-cleaning surfaces could be used in hospitals during future pandemics.[100] Further experi- ments combined electrostatic composite films (coagulated GO-PMMA) with water aerosols to create a nanocomposite surface that generated a negative voltage from water evapora- tion. Hence, an electrostatic bond with coronaviruses′ spike protein.[82] In the absence of the novel coronavirus to test, the study used the model beer yeast cells. The mechanism appears to be linked to electrostatic interactions between the nanosys- tems (due to their nanometric size and chemical character) were proposed to develop nanodevices useful in COVID-19 and future pandemics management.[82,100] Exploiting ROS molecules′ harmfulness to coronavirus, nanofilters based on metal-decorated (Pt, Cu, Rh, and Ru)-SWCNTs, combined with H2O2, were presented as an effective platform for future experi- ments in SARS-CoV-2 absorption.[100] The nanocomposite use for elastomeric respirators and self-cleaning surfaces could be used in hospitals during future pandemics.[100] Further experi- ments combined electrostatic composite films (coagulated GO-PMMA) with water aerosols to create a nanocomposite surface that generated a negative voltage from water evapora- tion. Hence, an electrostatic bond with coronaviruses′ spike protein.[82] In the absence of the novel coronavirus to test, the study used the model beer yeast cells. The mechanism appears to be linked to electrostatic interactions between the nanosys- tems (due to their nanometric size and chemical character) with the coronaviruses′ negatively charged spike proteins. DFT calculations showed a generous capacity of metal-decorated SWCNTs for peroxide and hydroxyl radical capture with a very long recovery time.[100] Once in contact, the functional groups of GO layers and water molecules interact, thus generating an extra electric field induced through the heterostructure forma- tion with an enhanced dipolar redistribution at the interface.[82] The potential of Cu to neutralize infectious viruses and induce viral killing mediated by ROS[52] have been hypothesized to booster immune system against COVID-19 by Cu supple- mentation[52] and motivated researchers to formulate copper to fabricate nanodevices as antiviral, self-cleaning, and reusable filter media/surfaces to prevent the COVID-19 spread.[81,92,94,95] A superhydrophobic coating based on silica/copper nano- particles dispersed in silicone or epoxy polymer (flexible, superhydrophobic, and regenerative monolith surfaces) was 2000115 (10 of 18) www.global-challenges.com www.global-challenges.com www.global-challenges.com www.advancedsciencenews.com www.advancedsciencenews.com Table 6. Recent hypotheses, computational studies, and pre-clinical/clinical research into bioactive compounds and nanomaterials against coronaviruses. Table 6. Recent hypotheses, computational studies, and pre-clinical/clinical research into bioactive compounds and nanomaterials against coronaviruses. mputational studies, and pre-clinical/clinical research into bioactive compounds and nanomaterials against Nanomaterial Size Shape Phytochemical Coronavirus Potential application Ref. Carboxyl quantum dots 20 nm Spherical Catechin gallates SARS-CoVa) Antiviral agent [74] Chitosan-gel nanospheres 60 nm Spherical Genipin HCoV-NL63b) HCoV-OC43c) MHVd) Adsorbents [61] PEG-PLGAe) nanocapsules 36 nm Spherical Diphyllin FIPVf) Antiviral drugs, immunotherapy [62] CCM-CDsg) 1.5 nm Spherical Curcumin PEDVh) Antiviral drugs [68] Chitosan nanoparticles 250–450 nm Spherical/Cuboidal Curcumin FIPVf) Antiviral drugs [63] CDsi) 11.4 nm Spherical Glycyrrhizin PEDVh) Antiviral drugs [67] Ag2S-NCsj) 5.3 nm Spherical Glutathione PEDVh) Antiviral drugs [64] ZnS-NPsk) 3.8 nm Spherical Glutathione PEDVh) Antiviral drugs [66] Collagen hydrogel 50–100 nm Fibrils Polyp nanoparticles SARS-CoV-2 Antiviral agent, Immune supplementation [91] Polymeric hydrogel – – Griffithsin SARS-CoV-2 Ophthalmic drug delivery [93] Silver nanoparticles – – Artemisinin, Artemether, Artesunate SARS-CoV-2 Antiviral drugs [80] a)Severe acute respiratory syndrome coronavirus; b)Human coronavirus NL63; c)Human coronavirus OC43; d)Murine coronavirus; e)Poly(ethylene glycol-block-poly-(lactide- coglycolide); f)Type II feline infectious peritonitis virus; g)Cationic carbon dots based on curcumin; h)Porcine epidemic diarrhea virus; i)Carbon dots; j)Silver-sulfide nano- clusters; k)Zinc-sulfide nanoparticles). Polyhydroxyethylmethacrylate. a)Severe acute respiratory syndrome coronavirus; b)Human coronavirus NL63; c)Human coronavirus OC43; d)Murine coronavirus; e)Poly(ethylene glycol-block-poly-(lactide- coglycolide); f)Type II feline infectious peritonitis virus; g)Cationic carbon dots based on curcumin; h)Porcine epidemic diarrhea virus; i)Carbon dots; j)Silver-sulfide nano- clusters; k)Zinc-sulfide nanoparticles). Polyhydroxyethylmethacrylate. a)Severe acute respiratory syndrome coronavirus; b)Human coronavirus NL63; c)Human coronavirus OC43; d)Murine coronavirus; e)Poly(ethylene glycol-block-poly-(lactide- coglycolide); f)Type II feline infectious peritonitis virus; g)Cationic carbon dots based on curcumin; h)Porcine epidemic diarrhea virus; i)Carbon dots; j)Silver-sulfide nano- clusters; k)Zinc-sulfide nanoparticles). Polyhydroxyethylmethacrylate. disorder and lung injury in an acute pneumonia mouse model by nanodecoy through in vivo assay.[90] efficiently covalently conjugate with amine groups of biomol- ecules (e.g., proteins, nucleic acids), forming an amide bond through carbodiimide-mediated coupling reactions. Herein, we reviewed a nanostructure approach against the beta-corona- virus SARS-CoV to rapidly identify natural inhibitors screening of the SARS-CoV nucleocapsid (N) protein, beyond an optical method based on carboxyl quantum dots-conjugated RNA nucleotides system, with applicability for imaging analysis on a biochip[74] (Figure 6A). The systems were used as a platform for natural inhibitors based on several polyphenols, in which (-)-catechin gallate and (-)-gallocatechin gallate, obtained from green tea (Camellia sinensis), presented the higher inhibition effect against SARS-CoV N protein (more than 40% inhibition at 0.05 µg L−1).[74] The vacuolar-ATPase (v-ATPase) dysregulation has already been discussed and linked with drug resistance in viral infections[109] and cancer therapies.[110] Hence, v-ATPase blocking could be an attractive target for antiviral approaches. A nanoformulation with diphyllin (a lignan obtained from the plant Cleistanthus collinus) encapsulated by biocompatible block copolymer nanocapsules of PEG-PLGA exhibited a potent inhibitory effect against FCoV in fcwf-4 cells[62] (Figure 6B). The strategy was studied using a nanoparticulated system with diphyllin as a novel v-ATPase blocker as an alternative for the bafilomycin A1, given its compromised clinical applicability due to its high cytotoxicity, low water solubility, and potent off-target effect.[109] Besides that, genipin from Gardenia jasminoides was found as a crosslinker agent of chitosan nano/microspheres (HTCC), that was further cationized and studied as adsorbents (in aqueous suspension medium) with high selectivity against humans and animal coronaviruses (HCoV-NL63 and MHV)[61] (Figure 6C). An advantage of this attractive water-soluble nano- particle with genipin and chitosan as an antiviral strategy is that booth are non-toxic materials. 4.4. Natural Bioactive Compounds and Nanoparticles against Coronavirus and SARS-CoV-2: A Promissory, Healthy, and Bio-Friendly Strategy for Drug Delivery www.advancedsciencenews.com The sensor target detected the SARS-CoV-2 antigen protein, cultured SARS-CoV-2 virus, and SARS-CoV-2 from clinical samples.[84] The authors proposed a high dependence between SARS-CoV-2 spike protein and specific binding with the SARS-CoV-2 antigen, and the chemically modified gra- phene surface promoted this binding affinity through a pyrene backbone with an electron-withdrawing group.[84] Due to the biocompatibility nature and size of NLCs, a study hypothesized the pulmonary delivery of Salinomycin (SAL) carried by NLCs as a promising candidate to treat COVID-19 patients[88] (Figure 5D). The SAL encapsulation by NLCs 2000115 (11 of 18) Global Challenges 2021, 2000115 Global Challenges 2021, 2000115 © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 4.4. Natural Bioactive Compounds and Nanoparticles against Coronavirus and SARS-CoV-2: A Promissory, Healthy, and Bio-Friendly Strategy for Drug Delivery Eleven of the 45 articles retrieved highlighted strategies based on nanomaterials as a secondary approach to enhance natural compounds′ use, improving their bioavailability, solubility, and antiviral activity. Phenolic compounds, glycosides, terpenes, saponins, peptides, and proteins can play an essential role against viruses from the Coronaviridae family. Of these, we identified genipin, diphyllin, curcumin, glutathione, glycyr- rhizic acids, polyP nanoparticles, and griffithsin.[61,62,64,66–68] The expert opinion reported that morphology characteris- tics (size and shape) are a fundamental aspect of nanoparticle design, once it is directly connected with pharmacokinetics and cell uptake for drug delivery purposes, especially for those with minimal side effects.[106] In the case of drug carriers in blood for lymphatics channels, the geometry and aspect ratio of shape plays a crucial role in how nanoparticles will be trans- ported. From Table 6, we note that most of all nanoparticles studied are spherical and presented an impressive average size varying from 11.4 to 1.5 nm in the last 3 years reported atte mpts.[63,64,66–68,91,93] Figure 6 shows a schematic representation of some strategies reviewed on active phytochemicals (a broad-spectrum “host-tar- geted” antiviral)/nanoparticles systems as promising candidates against coronaviruses as virus adsorbents, antiviral agents. and immunomodulatory drugs. The amide coupling reaction is often used in medicinal chemistry[107] to generate novel com- pounds for antiviral drug discovery.[19,108] Carboxyl quantum dots, QD650, has carboxylic acid terminal groups that could Curcumin pyrolysis applied to prepare uniform and stable CDs with wealthy hydrophilic groups,[68] a glycyrrhizic 2000115 (12 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH edsciencenews.com www.global-ch www.global-challenges.com www.advancedsciencenews.com 2000115 (13 of 18) © 2021 The Authors. Global Challenges published by Wiley e 6. Schematic of preparation and mechanisms of phytochemicals/nanoparticles against coronaviruses reviewed: A) antiviral activity o bioconjugated QDs-based biochip by interactions with SARS-CoV N protein; B) diphyllin encapsulated by polymeric nanoparticles as peutic target against FCoV (V-ATPase inhibition); C) viral adsorption on the surface of biopolymeric nano/microspheres by electrost with HCoV-NL63 and MHV S protein. Challenges 2021, 2000115 2000115 (13 of 18) © 2021 The Authors. Global Challenges published by Wile ematic of preparation and mechanisms of phytochemicals/nanoparticles against coronaviruses reviewed: A) antiviral activity ugated QDs-based biochip by interactions with SARS-CoV N protein; B) diphyllin encapsulated by polymeric nanoparticles a arget against FCoV (V-ATPase inhibition); C) viral adsorption on the surface of biopolymeric nano/microspheres by electros CoV-NL63 and MHV S protein. es 2021, 2000115 Figure 6. 5. Conclusions and Outlook PolyP, a non-toxic and non-immunogenic inorganic polymer derived from marine bacteria, was added into a mucin/collagen- based hydrogel to simulate the mucus of the nasopharynx and bronchial epithelium on human alveolar basal epithelial A549 cells.[91] This strategy was suggested to stimulate an innate antiviral response by improved mucin barrier (high in antimi- crobial proteins) and as a potential antiviral agent by exerting protection against SARS-CoV-2-cell attachment.[91] Griffithsin is a small lectin derived from red algae (Griffithsia spp.) of broad- spectrum antiviral activity against coronavirus.[111,112] Thus, poly (2-hydroxyethyl methacrylate) hydrogel lenses with nanopar- ticles releasing griffithsin were hypothesized as therapeutic contact lenses to protect healthcare workers′ ocular surface as extra protection in daily practice against COVID-19 infection.[93] The synergistic potential between antimalarial drugs as Arte- misinin, Artemether, and Artesunate—natural sesquiterpene lactones from wormwood plant (Artemisia annua)—coated with antiviral AgNPs was optimized in a structure by mole- cular dynamics and suggested to improve the permeability and time retention of these drugs enhancing the therapeutic action against malaria and COVID-19.[80] In this systematic review, the papers retrieved and analyzed show silver, copper, and polymer-based nanomaterials as the primary with efficient anti-SARS-CoV-2 properties. The strong virucide potential of copper and silver nanomaterials with varied morphology and fabrication form were prospected to make reusable and self-cleaning surfaces (e.g., respirator facial masks and coating surfaces) in healthcare work environments to reduce the spread of COVID-19. Biopolymeric and biode- gradable polymers in nanoformulations have prospected drug delivery against SARS-CoV-2, as well as nanodecoy and manga- nese nanoparticle was suggested as a simple, safe, and robust technology for vaccine adjuvants or antiviral agents once it increased immune response by in vivo assays. Virucide surfaces and adsorbents to capture/inactivate SARS-CoV-2 and other beta-CoV were also proposed as adsorbents for coronavirus inactivation. Cutting-edge nano biosensors technologies, nano- structured carriers for pulmonary drug delivery, sVLPs, and polymeric hydrogels were pointed out as potential agents for antiviral drugs, therapeutic vaccines, and immuno-based ther- apies. A study with real clinic samples of COVID-19 patients demonstrated graphene use for immunodiagnostic assay with high specificity and celerity, potentially useful for serological tests and detection of infection. 5. Conclusions and Outlook Related to the mechanism of action, the green tea polyphe- nols, as epicatechin gallates, were reported as a potent anti- viral entry inhibitor capable of blocking the host’s binding of glycoprotein CD4 cell with glycoprotein gp120 of HIV-1 and hence, preventing the viral infection.[113] The antiviral activity of green tea catechins was suggested in previous studies against other enveloped viruses. It was mainly attributed to its hydroxyl, galloyl, and pyrogallol groups,[114] as well as phenolic OH groups on B-ring,[115] which can act at several stages of the viral entry:[113] i) affecting the expression of viral antigens or ii) inhibiting the genome replication. The desorption properties of HTCC was explained in terms of i) electrostatic Coloumb attraction of the genipin–chitosan derivatives nanospheres with the spike protein of HCoV-NL63, which can form a protein– polymer complex, resulting in virus neutralization; and ii) the high ionic strength promoted by chitosan cationization.[61] In a dose-dependent way, the diphyllin inhibited endosomal acidi- fication affecting the viral cellular susceptibility and inhibits the downstream coronavirus replication.[62] Furthermore, mul- tisite inhibition mechanisms were found for all approaches with GSH, curcumin, and glycyrrhizin tested against PEDV: i) inhibition of viral entry by changing the structure of the viral surface protein, prevent the viral RNA synthesis and budding; ii) suppression of the ROS generation; and iii) suppression of viral reproduction by activation of IFN-stimulating genes and the expression of pro-inflammatory cytokines. Likewise, it was hypothesized that the griffithsin’s capacity to block viral entry[93] due to its high affinity to glycoproteins sites of MERS-CoV[111] Molecular docking and dynamic simulations are powerful tools to study the relationship between receptor–ligand binding affinity in drug discovery using nanomaterials. Therefore, all the nanotechnologies studied by computational tools were predicted to interfering with SARS-CoV-2 adhesion to human host cell receptors and viral replication, thus inhibiting the viral infection. Primary and secondary metabolites of plants and microor- ganisms (e.g., phenolic, terpenes, glycosides, polysaccharides, and Polyp), well-known antimicrobial compounds, delivered by spherical nanoparticles, were a bio-friendly and potential strategy to produce antivirals therapies for coronavirus once nanomaterials enhanced their solubility, bioavailability, and antiviral activities. The phenolic phytochemicals acted as mul- tisite inhibitors at several stages of the viral entry, affecting viral antigens′ expression, or inhibiting the genome replication. Biodegradable polymeric nanoparticles are non-toxic and bio- compatible options for drug delivery. and SARS-CoV.[112] PolyP has already been suggested to block SARS-CoV-2-cell attachment by blocking the binding of RBD of spike protein of SARS-CoV-2 to ACE2 cell receptor in vitro.[76,91] Density functional theory (DFT) calculations predicted the highest affinity of antimalarial drugs to interact with AgNPs surfaces in the order Artesunate > Artemisinin > Artemether, due to the more negative charges on O6 atom of Artesunate, O5 atom of Artemisinin, and O3 atom of Artemether.[80] acid-based carbon dots,[67] glutathione-capped Ag2S nanoclus- ters,[64] and glutathione-modified zinc-sulfide nanoparticles,[66] were developed with high effectiveness against PEDV corona- virus. Curcumin is a polyphenol obtained from Curcuma longa that can play a vital role in antiviral activity due to its phenolic hydroxyl groups. Once the low bioavailability impairs its thera- peutic application, curcumin was encapsulated in chitosan nan- oparticles by ionic gelation technique with improved in vitro antiviral activity and bioavailability by oral administration in cats infected with FIPV.[63] © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH Global Challenges 2021, 2000115 4.4. Natural Bioactive Compounds and Nanoparticles against Coronavirus and SARS-CoV-2: A Promissory, Healthy, and Bio-Friendly Strategy for Drug Delivery Schematic of preparation and mechanisms of phytochemicals/nanoparticles against coronaviruses reviewed: A) antiviral activity of catechins on a bioconjugated QDs-based biochip by interactions with SARS-CoV N protein; B) diphyllin encapsulated by polymeric nanoparticles as a potential therapeutic target against FCoV (V-ATPase inhibition); C) viral adsorption on the surface of biopolymeric nano/microspheres by electrostatic interac- tions with HCoV-NL63 and MHV S protein. © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH 2000115 (13 of 18) 2000115 (13 of 18) Global Challenges 2021, 2000115 Global Challenges 2021, 2000115 www.global-challenges.com Keywords bioactive compounds, cell mechanisms, nanomedicine, polymeric nanoparticles, self-cleaning surfaces Received: November 6, 2020 Revised: February 1, 2021 Published online: [1] T. Ahmad, M. Khan, T. H. M Haroon, S. Nasir, J. Hui, D. K. Bonilla- Aldana, A. J. Rodriguez-Morales, Travel Med. Infect. Dis. 2020, 36, 101607. No study using the eco-friendly nanocellulose was retrieved. That was surprising, considering that nanocellulose is sustain- able, non-toxic, antimicrobial, biocompatible, relatively cheap, and a suitable carrier due to its nonspherical shape in the nanofibrous form attractive to the pharmaceutical/biomedical industries. Additionally, nanocellulose has hydroxyl groups that might form a hydrogen bonding with spike glycoproteins and stabilize the ligand–receptor complex. For future opportuni- ties, we believe in new attempts with the non-toxic nanomate- rials and more efforts in nanomaterials that have already been reported in non-cytotoxicity levels as antiviral agents. Thus, trends in toxicology evaluation and safety tests of strategies reviewed can help fill the main gaps in the literature and over- come nanomaterials′ main challenges to health surveillance. Encapsulated nanosystems with v-ATPase inhibition could be a promissive target therapeutic to overcome the challenge of antiviral drug resistance. Future directions can be identified in the opportunity to study the pharmacokinetics of phytochemi- cals delivered by nanomaterials cited to evaluate the effects in targeting, circulation time, and the ability to overcome biolog- ical barriers for drugs repurposing with more healthy options. Beyond that, the absence of attempts with nonspherical nano- particles (e.g., filamentous shapes) could encourage new future efforts. We hope that our study’s notes addressing the role of nanotechnology approaches with anti-coronavirus properties can help researchers with insights to overcome the challenges associated with the SARS-CoV-2 virus control, given direction to develop novel antiviral therapies, and prevent future pan- demics similar to the current COVID-19. [2] A. Rodriguez-Morales, D. Bonilla-Aldana, G. Balbin-Ramon, A. Rabaan, R. Sah, A. Paniz-Mondolfi, P. Pagliano, S. Esposito, InfezMed 2020, 28, 3. [3] E. de Wit, N. van Doremalen, D. Falzarano, V. J. Munster, Nat. Rev. Microbiol. 2016, 14, 523. [4] World Health Organization, WHO coronavirus disease (COVID- 19) dashboard, https://covid19.who.int/ (accessed: January 2021). [5] The World Bank, COVID-19 to plunge global economy into worst recession since World War II, https://www.worldbank.org/en/news/ press-release/2020/06/08/covid-19-to-plunge-global-economy-into- worst-recession-since-world-war-ii (accessed: September 2020). [6] S. Payne, Viruses, Elsevier, Amsterdam 2017, pp. 149–158 [7] B. Udugama, P. Kadhiresan, H. N. Kozlowski, A. Malekjahani, M. Osborne, V. Y. C. Li, H. Chen, S. Mubareka, J. B. Gubbay, W. C. W. Conflict of Interest Understanding the interface between nanomaterials and cor- onaviruses reviewed is fundamental to designing target antivi- rals for COVID-19 infection. Their varied morphology, chemical diversity, excellent physical–chemical properties, and the pos- sibility of binding several types of compounds in their surface with target functions, as well as the synergism between them (e.g., nanocapsules and nanocomposites), justified nanomate- rials′ as potential nanomedicine and prophylactics tools against COVID-19. Nanocarbons can act against coronaviruses mul- tivalent interactions (e.g., electrostatic interactions, hydrogen bonds, and hydrophobic interactions) with spike protein and lipid tails, destroying the membrane, blocking cell entry and viral replication. The primary mechanism to block cell viral infection inhibited SARS-CoV-2 cell entry by blocking the binding between RBD of spike protein and the human ACE2 receptor. However, sulfonate ligands on the gold nanoparticle surface can inhibit the SARS-CoV-2 cell attachment by inhib- iting the binding between spike protein and HS receptors. Many strategies can suppress ROS generation, inhibit host cell apoptosis, inhibit endosomal acidification, acting as multistep inhibitors on viral entry and replication. Photoactivated copper, silver, and TNP showed the highest potential as virucidal agents. The authors declare no conflict of interest. Keywords Chan, ACS Nano 2020, 14, 3822. [8] D. Wrapp, N. Wang, K. S. Corbett, J. A. Goldsmith, C.-L. Hsieh, O. Abiona, B. S. Graham, J. S. McLellan, Science 2020, 367, 1260. [9] A. Gupta, S. Kumar, R. Kumar, A. K. Choudhary, K. Kumari, P. Singh, V. Kumar, ChemistrySelect 2020, 5, 7521. g y [10] M. C. Sportelli, M. Izzi, E. A. Kukushkina, S. I. Hossain, R. 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During her Master’s and Ph.D. formation, she became an expert in the field of nanotechnology, from the synthesis to the characterization of nanocomposites, hybrids, and nanocarbons as coating materials to protect human health from electromagnetic interference. Her post-doctoral research focuses on inter- actions between nanomaterials/biomaterials with biological systems for food and biomedical applications. Anna Paula A. Carvalho is a scientist member of the Nanotechnology Network of Rio de Janeiro, Brazil. She has a Bachelor’s in Chemistry, Polymer Technologist, Master’s, and Ph.D. in Polymer’s Science and Engineering at the Federal University of Rio de Janeiro. During her Master’s and Ph.D. www.advancedsciencenews.com Wang, Bio- conjug. Chem. 2020, 31, 2553. [70] S. Khaiboullina, T. Uppal, N. Dhabarde, V. R. Subramanian, S. C. Verma, Viruses 2020, 13, 19. [42] N. Mittal, V. Garg, S. K. Bhadada, O. P. Katare, Curr. 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Global Challenges published by Wiley-VCH GmbH www.advancedsciencenews.com formation, she became an expert in the field of nanotechnology, from the synthesis to the characterization of nanocomposites, hybrids, and nanocarbons as coating materials to protect human health from electromagnetic interference. Her post-doctoral research focuses on inter- actions between nanomaterials/biomaterials with biological systems for food and biomedical applications. 2000115 (17 of 18) © 2021 The Authors. Global Challenges published by Wiley-VCH GmbH Global Challenges 2021, 2000115 www.global-challenges.com www.global-challenges.com Carlos A. Conte-Junior is a Professor of Biochemistry at the Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), Brazil. He has Bachelors in Veterinarian Medicine from Fluminense Federal University, Masters in Food Science and Technology at UFRJ, Ph.D. in Food Science and Technology at Universidad Complutense de Madrid with a stay in Vrije Universiteit Medisch Centrum Amsterdam, Postdoctorate in Karolinska Institute (Sweden), and University of California (USA). He has more than 300 peer-reviewed journal articles, 18 invited book chap- ters, 48 full-paper conference proceedings, and 156 refereed abstracts. He is Coordinator of Nanotechnology Network of Rio de Janeiro. Carlos A. Conte-Junior is a Professor of Biochemistry at the Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), Brazil. He has Bachelors in Veterinarian Medicine from Fluminense Federal University, Masters in Food Science and Technology at UFRJ, Ph.D. in Food Science and Technology at Universidad Complutense de Madrid with a stay in Vrije Universiteit Medisch Centrum Amsterdam, Postdoctorate in Karolinska Institute (Sweden), and University of California (USA). He has more than 300 peer-reviewed journal articles, 18 invited book chap- ters, 48 full-paper conference proceedings, and 156 refereed abstracts. He is Coordinator of Nanotechnology Network of Rio de Janeiro. 2000115 (18 of 18) Global Challenges 2021, 2000115 Global Challenges 2021, 2000115
https://openalex.org/W2727705276	https://link.springer.com/content/pdf/10.1007/s00251-017-1017-3.pdf	English	null	A genomic perspective on HLA evolution	Immunogenetics	2,017	cc-by	17,937	Diogo Meyer1 · Vitor R. C. Aguiar1 · B´arbara D. Bitarello1,3 · D´ebora Y. C. Brandt1,2 · Kelly Nunes1 Diogo Meyer1 · Vitor R. C. Aguiar1 · B´arbara D. Bitarello1,3 · D´ebora Y. C. Brandt1,2 · Kelly Nunes1 Received: 25 October 2016 / Accepted: 16 June 2017 / Published online: 7 July 2017 © The Author(s) 2017. This article is an open access publication Abstract Several decades of research have convincingly shown that classical human leukocyte antigen (HLA) loci bear signatures of natural selection. Despite this conclu- sion, many questions remain regarding the type of selective regime acting on these loci, the time frame at which selec- tion acts, and the functional connections between genetic variability and natural selection. In this review, we argue that genomic datasets, in particular those generated by next- generation sequencing (NGS) at the population scale, are transforming our understanding of HLA evolution. We show that genomewide data can be used to perform robust and powerful tests for selection, capable of identifying both pos- itive and balancing selection at HLA genes. Importantly, these tests have shown that natural selection can be identi- fied at both recent and ancient timescales. We discuss how findings from genomewide association studies impact the evolutionary study of HLA genes, and how genomic data can be used to survey adaptive change involving interaction at multiple loci. We discuss the methodological develop- ments which are necessary to correctly interpret genomic analyses involving the HLA region. These developments include adapting the NGS analysis framework so as to deal with the highly polymorphic HLA data, as well as developing tools and theory to search for signatures of selection, quan- tify differentiation, and measure admixture within the HLA region. Finally, we show that high throughput analysis of molecular phenotypes for HLA genes—namely transcrip- tion levels—is now a feasible approach and can add another dimension to the study of genetic variation. Keywords HLA (human leukocyte antigen) · MHC (major histocompatibility complex) · Evolution · Genomics · Balancing selection Immunogenetics (2018) 70:5–27 DOI 10.1007/s00251-017-1017-3 Immunogenetics (2018) 70:5–27 DOI 10.1007/s00251-017-1017-3 REVIEW 3 Present address: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany 1 Department of Genetics and Evolutionary Biology, University of S˜ao Paulo, 05508-090 S˜ao Paulo, SP, Brazil Introduction Genomic data is helping understand the rate at which we are burdened by deleterious mutations, and the importance of nega- tive selection—which removes deleterious variants from pop- ulations—in the human genome (Fu et al. 2013; Henn et al. 2015). Deleterious variants have been hypothesized to play an important role in explaining phenotypic variation, particularly that of common diseases, and population level exome and genome sequencing are being used to tackle this question (with their role remaining controversial, Hunt et al. 2013). Several studies have also searched for genes under balancing selection, which is the selective regime that maintains several variants in a population at intermediate frequencies, making the persistence time of each allele longer than that of neutral ones. Under this regime, the combination of alleles at a locus is often critical to defining fitness values, and the fitness of an allele may vary over time (reviewed in Key et al. 2014). g While it is clear that “documenting selection” at HLA genes is no longer a challenge, important questions regard- ing HLA evolution remain open, and can be addressed using genomic data. First, while it is accepted that bal- ancing selection increases the diversity of HLA genes, there are several types of selection that can produce this effect. Balancing selection is an umbrella term that encompasses heterozygote advantage (or overdominance), selection vary- ing over space or time, and negative frequency-dependent selection (see Box 3). Fleshing out which of these explains the high variability at HLA is a challenge (Spurgin and Richardson 2010), and we discuss the contributions of novel analytical methods and genomewide studies. Information is also increasingly available for molecular phenotypes, helping understand the functional basis of nat- ural selection. A particularly powerful method is RNAseq, which relies on next generation sequencing of RNA molecules to quantify gene expression. Using such informa- tion, Fraser (2013) showed that episodes of recent selection in humans are much more likely to affect gene expression than protein sequence. Much of the progress in our understanding of how natural selection acts in humans is based on genomewide studies. However, focusing on genes for which we have prior func- tional knowledge can provide important insights on how natural selection acts. In this review, we integrate knowledge on the function of classical human leukocyte antigen (HLA) genes with population genomic data. Introduction The availability of genomic data at the scale of populations is transforming our understanding of the processes shaping human genetic variation. We are now able to answer ques- tions which, little more than 15 years ago, seemed beyond our grasp. We can construct detailed portraits of how natu- ral selection has acted, and identify variants that increased in frequency as a consequence of positive selection (the pro- cess that drives advantageous variants to high frequencies) (reviewed in Fu and Akey 2013). In some cases, it is possible to provide mechanistic links between the favored variant and its phenotypic effect, and to estimate the timescale of selec- tion (for example, in the cases of variants involved in pig- mentation (Beleza et al. 2013), lactase persistence (Coelho et al. 2005), and adaptation to altitude (Yi et al. 2010)). 1 Department of Genetics and Evolutionary Biology, University of S˜ao Paulo, 05508-090 S˜ao Paulo, SP, Brazil There is also increasing interest in developing meth- ods for the cases in which the advantageous variant was already present in the population at the time of onset of selection (i.e., selection on standing variation) (Messer and Petrov 2013). In addition, methods are being developed to identify instances in which selection favors a combination 2 Present address: Department of Integrative Biology, University of California, Berkeley, CA, USA Immunogenetics (2018) 70:5–27 6 of genetic variants (polygenic selection), instead of a single advantageous allele (Daub et al. 2013). are firmly established regarding HLA evolution. First, it is undisputed that HLA genes bear the mark of balancing selection: there are no demographic or genetic factors that can account for the unusually high degree of polymorphism, excess of nonsynonymous variants, or linkage disequilibrium at these genes (Meyer and Thomson 2001; Garrigan and Hedrick 2003; Spurgin and Richardson 2010). Second, there are several lines of support for a role of pathogen-driven selection in shaping HLA variation: HLA genes are associ- ated with susceptibility and resistance to infectious disease (Cagliani and Sironi 2013); experimental studies show that pathogen pressure influences MHC variability (Penn et al. 2002); HLA polymorphism is correlated with pathogen diversity (Prugnolle et al. 2005); variation is highest at sites which define the peptide binding repertoire (Hedrick et al. 1991; Hughes and Nei 1988; Bitarello et al. 2016). Introduction We discuss how the genomic perspective both illuminates the study of HLA evolution, and contributes to our understanding of natural selection in the remainder of the genome. Second, the timescale of selection remains an open question. Tests of neutrality used before genomic data became available were only well-powered to detect long-term selection (Garri- gan and Hedrick 2003), whereas newer approaches—which rely on dense genetic data spanning thousands of sites—can also detect recent selection (Field et al. 2016; Albrechtsen et al. 2010; Guan 2014). We discuss the findings brought by these approaches, and argue that they indicate that selection on HLA genes can be identified at various timescales. HLA genes code for glycoproteins that bind peptides and present them to T cell receptors. If the bound peptide is non- self (i.e., possibly from a pathogen or a mutated protein), cellular and humoral responses can be mounted (see Box 1). HLA genes also interact with other molecules involved in innate and adaptive immunity. Among these are the killer cell immunoglobulin-like receptors (KIR), for which some HLA class I molecules are ligands (Trowsdale et al. 2001; Parham 2004). When cells are infected or neoplastic, the expression of classical class I loci may decrease, reducing the availability of ligand for KIR molecules. This activates cell lysis by natural killer cells (Yawata et al. 2008). Third, the increasing understanding of HLA function shows that interactions of HLA genes with other loci— and not just their immediate role in peptide binding—must also be considered in evolutionary studies (Trowsdale and Knight 2013). Further, phenotypic information, including expression levels of the HLA genes, has rarely been incor- porated into evolutionary analyses. We discuss the chal- lenges associated to bringing these functional perspectives to the study of HLA evolution. Research over the last three decades has successfully brought together knowledge on HLA function with advances in theoreti- cal population genetics, allowing evolutionary hypotheses to be tested (in particular through the implementation of neutra- lity tests, Box 2). There are now several key ideas which HLA variation in the age of genome sequencing Overall, it appears that a more accu- rate assessment of HLA variability will come from both the development of new bioinformatic tools, as well as the generation of new data (in particular with long sequencing reads). However, an understanding of the role of selection in shaping HLA variation also requires placing it in a genomewide context, so that selective and demographic fac- tors can be disentangled, and genomewide significance test- ing can be performed. In practice, this requires extracting information on HLA variation from datasets with sequence information for the entire genome. Such data are increas- ingly generated by exome or whole-genome sequencing, as well as high density SNPs arrays (e.g., The 1000 Genomes Project Consortium 2010; Fu and Akey 2013). Another development is the imputation of HLA alle- les based on dense SNP data. Imputation involves using a training set—for which both MHC region SNPs and HLA allele calls are available—to infer the HLA alleles carried by an individual with unknown HLA genotype, but for which SNP data is available (Dilthey et al. 2011; Zheng et al. 2013; Zhou et al. 2016a; Leslie et al. 2008). Zhou et al. (2016a) showed that the concordance rate between imputed HLA alleles and sequencing-based calls can reach 0.93 when using a large reference panel. Imputation is proving to be important in the context of association studies, since it allows an individual’s HLA genotype to be included as a variable (Sanchez-Mazas and Meyer 2014), or even to infer specific amino-acids and amino-acid motifs, and quantify their contribution to overall associations (Jia et al. 2013). Many genomewide studies, such as Phase I of the 1000 genomes project (The 1000 Genomes Project Consortium 2010), have analyzed HLA polymorphism using standard sequencing pipelines. Given the importance of the 1000 genomes project data to evolutionary research, we previ- ously assessed the reliability of SNP calls which they pro- vide (Brandt et al. 2015). We found that although frequency estimates for HLA SNPs are relatively robust (absolute fre- quency difference less than 0.1 for 75% of the SNPs), the SNP genotype calls within the HLA loci have alarmingly high error rates (18.6% of calls are incorrect) and are biased toward over-representing the alleles present in the reference genome. However, imputation-based estimates will be uninforma- tive with respect to novel variants, or those at very low frequencies. When interest is in identifying novel vari- ants (Klitz et al. HLA variation in the age of genome sequencing Several generations of methods have been used to identify the alleles carried by an individual: PCR-RFLP, SSOP, 7 Immunogenetics (2018) 70:5–27 An increasingly used strategy to address these chal- lenges is to map short reads, generated by NGS, to multi- ple MHC/HLA references (e.g., IPD-IMGT/HLA database (http://www.ebi.ac.uk/ipd/imgt/hla/)), as opposed to a sin- gle reference genome. Recent methods have implemented this idea to efficiently provide more reliable alignments (Castelli et al. 2015, 2017; Lima et al. 2016), HLA allele calls (see Hosomichi et al. 2015 for a review and Bauer et al. (2016) for an evaluation of 12 computational meth- ods), HLA expression estimates (Boegel et al. 2012), or to assemble individual genomes for the MHC region (Dilthey et al. 2015). Encouragingly, Phase III of the 1000 genomes project (The 1000 Genomes Project Consortium 2015) has used this strategy, allowing reads to align to 500 known HLA sequences, in addition to the human reference genome. A more general solution is to perform genomic align- ment using indices which account for the variation across the whole genome, including the MHC. These indices can be built in the form of genome graphs (Novak et al. 2017), an efficient strategy to summarize population-level variation in a graph structure, appropriate for subsequent short-read mapping. Application of such graph indices improves SNP calling in the MHC (Dilthey et al. 2015; Novak et al. 2017), and will likely supplant the use of a single linear reference index in the future. Overall, it appears that a more accu- rate assessment of HLA variability will come from both the development of new bioinformatic tools, as well as the generation of new data (in particular with long sequencing reads). An increasingly used strategy to address these chal- lenges is to map short reads, generated by NGS, to multi- ple MHC/HLA references (e.g., IPD-IMGT/HLA database (http://www.ebi.ac.uk/ipd/imgt/hla/)), as opposed to a sin- gle reference genome. Recent methods have implemented this idea to efficiently provide more reliable alignments (Castelli et al. 2015, 2017; Lima et al. 2016), HLA allele calls (see Hosomichi et al. 2015 for a review and Bauer et al. (2016) for an evaluation of 12 computational meth- ods), HLA expression estimates (Boegel et al. 2012), or to assemble individual genomes for the MHC region (Dilthey et al. 2015). HLA variation in the age of genome sequencing Encouragingly, Phase III of the 1000 genomes project (The 1000 Genomes Project Consortium 2015) has used this strategy, allowing reads to align to 500 known HLA sequences, in addition to the human reference genome. immobilized probes, PCR-SSP, and Sanger sequencing (reviewed in Erlich 2012; Carapito et al. 2016). The move to next-generation sequencing (NGS) is actively taking place, and in recent years many protocols have been described for HLA typing and SNP calling (Erlich et al. 2011; Lank et al. 2012; Wang et al. 2012; Danzer et al. 2013; Cao et al. 2013; Major et al. 2013; Langer et al. 2014; Monos and Maiers 2015; Norman et al. 2016; Zhou et al. 2016a). immobilized probes, PCR-SSP, and Sanger sequencing (reviewed in Erlich 2012; Carapito et al. 2016). The move to next-generation sequencing (NGS) is actively taking place, and in recent years many protocols have been described for HLA typing and SNP calling (Erlich et al. 2011; Lank et al. 2012; Wang et al. 2012; Danzer et al. 2013; Cao et al. 2013; Major et al. 2013; Langer et al. 2014; Monos and Maiers 2015; Norman et al. 2016; Zhou et al. 2016a). When deep-sequencing data are available, which is usu- ally the case for HLA-targeted protocols, the tiling of over- lapped reads can provide phase information and thus HLA allele sequences (Hosomichi et al. 2013). However, when polymorphisms are on different and non-overlapping reads, statistical approaches to phasing must be used (Castelli et al. 2015, 2017; Lima et al. 2016). Mayor et al. (2015) presented a solution to both the genotype ambiguity and phasing issues by using the PacBio single molecule real time (SMRT) sequencing technology, which generates long reads spanning the entire sequence of individual HLA Class I genes. The method provided accurate and unambiguous HLA genotype calls, representing a promising prospect. q g A more general solution is to perform genomic align- ment using indices which account for the variation across the whole genome, including the MHC. These indices can be built in the form of genome graphs (Novak et al. 2017), an efficient strategy to summarize population-level variation in a graph structure, appropriate for subsequent short-read mapping. Application of such graph indices improves SNP calling in the MHC (Dilthey et al. 2015; Novak et al. 2017), and will likely supplant the use of a single linear reference index in the future. HLA variation in the age of genome sequencing 2012), deep sequencing associated with mapping methods that account for variation will be required. In addition, imputation accuracy depends on the availabil- ity of reference panels with shared ancestry to the target population, representing an important challenge for studies of highly admixed populations with ancestral components This bias occurs because HLA genes are highly polymor- phic, and standard methods align short reads (50 to 250 bp) to a single reference genome. Thus, individuals which are heterozygous at a site, but have one allele which is closer to the reference genome, are likely to only map that variant, with the other one failing to align (Fig. 1). The fact that HLA genes are members of a multi-gene family further compli- cates the sequencing, since reads from one locus can be incorrectly mapped to another. 8 Immunogenetics (2018) 70:5–27 is different from the reference sequence at more positions than the mismatch threshold (in this simple example, only one mismatch is allowed). Reads from this haplotype will not align to the reference sequence and the genotype will be incorrectly called as homozygous at the SNP of interest. Modified from the Genes to Genomes blog, http:// genestogenomes.org/the-trouble-with-hla-diversity/ Fig. 1 How genotyping errors arise from the mapping of reads to a single reference genome. The left panel represents a case where sequence reads come from an individual who is heterozygous at a SNP, but the rest of the gene is similar to the reference for both haplotypes. The reads from both haplotypes can be aligned to the reference, and the SNP genotype is called correctly (i.e., determined by the analysis soft- ware). The panel on the right shows a case where one of the haplotypes is different from the reference sequence at more positions than the mismatch threshold (in this simple example, only one mismatch is allowed). Reads from this haplotype will not align to the reference sequence and the genotype will be incorrectly called as homozygous at the SNP of interest. Modified from the Genes to Genomes blog, http:// genestogenomes.org/the-trouble-with-hla-diversity/ Most genomewide scans for selection search for genes that underwent positive selection. HLA variation in the age of genome sequencing The main signatures of this mode of selection are: low variability coupled with extended linkage disequilibrium, caused by the increase in frequency of a favored variant; high population differentia- tion, due to selection favoring locally adaptive alleles; and an abundance of low frequency variants, due to mutations introducing novel variants into a region recently homoge- nized by selection (reviewed in Fu and Akey 2013) (see Box 2). Because many of these signatures can also result from non-selective events such as population expansions and bot- tlenecks, it has become standard for tests of selection to explicitly control for demographic history (e.g., by simu- lating null distributions under realistic scenarios) (Nielsen et al. 2005). These simulations are parametrized by esti- mates of the demographic history based on the genomewide data itself. In this way, sets of genes under positive selection have been identified in a robust manner (Akey 2009). which are relatively poorly studied (Levin et al. 2014; Nunes et al. 2016). In conclusion, we now have accesstoa wide array of options for uncovering HLA variation. Whereas genomewide sequencing based on alignment to a reference genome generates biased allele frequency estimates, pipelines that accountfor known HLA diversity can generate accurate infor- mation (Dilthey et al. 2015). Importantly, whole genome sequencing places HLA data in a genomewide context, an ideal scenario for separating demographic and selective contributions to variation, as we discuss in the next section. Genome scans for balancing selection Appropriate tests include search- ing the genome for regions with ancient shared polymor- phisms (e.g., Leffler et al. 2013; Teixeira et al. 2015), extreme patterns of polymorphism relative to divergence (e.g., DeGiorgio et al. 2014; Andr´es et al. 2009; Bitarello et al. 2017), an excess of intermediate frequency variants (DeGiorgio et al. 2014; Andr´es et al. 2009; Bitarello et al. 2017; Hedrick and Thomson 1983), an excess of identity by descent (IBD) (Albrechtsen et al. 2010), or unusually low differentiation between populations (Hofer et al. 2012; Sanchez-Mazas 2007) (Box 2). Finally, a recent study sequenced genomes of an extant population from the Northwest coast of North America, along with ancient genomes of individuals presumably from the same group, but from before contact with Europeans (Lindo et al. 2016). The study found that at HLA-DQA1 there was a shift from past positive to recent negative selection, bringing about marked allele frequency changes. The authors conjecture that this may have resulted from environmental or social changes. Tests using these approaches have been implemented, and the findings for HLA genes are summarized in Table 1. All studies show hits in the MHC region, with HLA-B appearing in five out of the six scans (Andr´es et al. 2009; DeGiorgio et al. 2014; Leffler et al. 2013; Teixeira et al. 2015; Hofer et al. 2012; Bitarello et al. 2017). In addition, HLA genes show the most extreme evidence of balancing selection in tests based on ancient shared polymorphisms (Klein et al. 1993; Teixeira et al. 2015; Leffler et al. 2013), and are highly enriched for extreme p-values in tests based on polymorphism and divergence (e.g., DeGiorgio et al. 2014; Andr´es et al. 2009; Bitarello et al. 2017). This not only confirms that HLA genes have been under long-term balancing selection but also shows that they are extreme in their patterns of diversity, compared to non-HLA loci. In summary, genomic scans for selection have revealed two important patterns. First, when tests designed to iden- tify balancing selection are used, evidence for selection at HLA genes is strong and extreme with respect to the remain- der of the genome, confirming what was known based on candidate gene approaches. Second, two studies have iden- tified selection within the MHC region that is consistent with regimes other than heterozygote advantage, and involv- ing very recent time frames (Albrechtsen et al. 2010; Field et al. 2016). Genome scans for balancing selection The early work on selection at HLA loci was carried out in the “candidate gene” framework, wherein specific HLA loci were tested for selection (see Box 2) (e.g., Hedrick and Thomson 1983, 1986; Hughes and Nei 1988). With genomewide data, on the other hand, it is no longer neces- sary to a priori define which loci will be queried for selec- tion, allowing us to investigate how extreme the evidence for selection at HLA loci is with respect to the remainder of the genome. Although there was strong support for positive selection on genes related to immunity (e.g., Nielsen et al. 2005; Tang et al. 2007b; Carlson et al. 2005), few genomic scans found evidence for it in the extended MHC region. Exceptions are the studies of de Bakker et al. (2006) and Sabeti et al. (2006), which identified long range haplotypes in the MHC Immunogenetics (2018) 70:5–27 9 than expected under drift alone. Within the MHC region of Europeans there are at least seven independent signals for selective changes (consistent with both balancing selection or the occurrence of multiple sweeps). New findings also came from the study of Field et al. (2016) which used the theoretical prediction that recently selected variants should be associated with a less diverse genetic neighborhood than the non-selected variants. Leveraged by very large samples of sequence data, they identified genomic regions where selection has driven advantageous alleles to high frequen- cies in a time frame as recent as 2 000 years, and found that at least three independent SNPs within the extended MHC region were among the most significant targets (Field et al. 2016). This test is designed to detect recent positive selec- tion, implying that balancing selection should not be seen as the only regime relevant to HLA evolution. region. The weak support for selection on HLA genes across several genomewide studies (Akey 2009) is largely a con- sequence of the fact that they used tests designed to detect positive—and not balancing—selection (Box 2). region. The weak support for selection on HLA genes across several genomewide studies (Akey 2009) is largely a con- sequence of the fact that they used tests designed to detect positive—and not balancing—selection (Box 2). In order to detect balancing selection, it is necessary to develop statistics sensitive to deviations expected under this selective regime. Genome scans for balancing selection According to these studies, and also a recent ancient-DNA study of Lindo et al. (2016), selection drove recent changes in allele frequencies (e.g., via frequency- dependent selection, or selection in a fluctuating selective environment). This supports the view that several selective regimes account for the patterns of variation of HLA genes. The MHC region is also the most extreme in a test based on identity-by-descent (IBD), which identifies genomic regions with extensive identity among individuals, consis- tent with the hypothesis that they descend from an advan- tageous ancestral variant (Albrechtsen et al. 2010). This signature supports very recent selection (<500 generations, or 10,000 years), which can be positive or balancing. Inter- estingly, Albrechtsen et al. (2010) showed that the increase in IBD is not expected under heterozygote advantage, lead- ing them to argue that selection at HLA loci may be frequency-dependent, or to fluctuate over time, possibly tracking changes in the evolving pool of pathogens that individuals are exposed to. Disease associations Identifying HLA variants that contribute to resistance to infectious diseases has important evolutionary implications. Simply put, alleles conferring disease resistance are com- pelling evidence for past and ongoing selection. Important developments in our understanding of HLA evolution have also come from two recent technological breakthroughs: the ability to sequence ancient samples and the genomic analysis of extremely large samples. Using over 200 ancient genomes, Mathieson et al. (2015) found several loci in modern Europeans which experienced greater changes in allele frequencies (with respect to their pre- sumed ancestors, as inferred using the ancient samples), A standard approach for identifying genetic variants that contribute to disease phenotypes is to carry out association studies. These compare the frequencies of genetic variants in groups that differ in a phenotype of interest, such as the occurrence of a specific disease. Thus, for example, if a variant Immunogenetics (2018) 70:5–27 10 Table 1 Findings for HLA genes in genome scans for balancing selection Reference Method Selection timescaled Selection at HLA Andr´es et al. (2009) SFS and polymorphism/divergence ratio Ancient HLA-Ba Albrechtsen et al. (2010) Excess IBD regionsb Recent Entire MHC region Leffler et al. (2013) Long-term shared polymorphism Ancient HLA-Bc, HLA-DQA1, HLA-DQB1, HLA-DPB1 DeGiorgio et al. (2014) Composite likelihood Long-term HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPB1 Teixeira et al. (2015) Long-term shared polymorphism Ancient HLA-C, HLA-DQA1, HLA-DPB1 Bitarello et al. (2017) SFS and polymorphism/divergence ratio Long-term HLA-B, HLA-C, HLA-DPA1, HLA-DQA1, HLA-DPB1, HLA-DRB1, HLA-DRB5, HLA-DQB2, HLA-DQB1, HLA-G IBD identity-by-descent, SFS site-frequency spectrum aOut of five HLA genes analyzed bA signature compatible with both positive and balancing selection cThe shared polymorphism falling in this gene is a CpG site (has higher mutation rate and could reflect recurrent mutation) dLong-term: more than 1 million years ago; ancient: greater than species-divergence time (6 million years, for humans and chimps) Table 1 Findings for HLA genes in genome scans for balancing selection (Fellay et al. 2007), leprosy (Zhang et al. 2009), hepatitis (Kamatani et al. 2009), and tuberculosis (Sveinbjornsson et al. 2016). is significantly less common among those with the disease than those without it, it is said to be associated with protection from the disease (provided that case and control groups are carefully controlled for possible confounding variables). Through much of the 1980s and 1990s, HLA variants were tested for association with resistance or susceptibility to infectious diseases. Disease associations These studies revealed a large number of associations with infectious diseases, some of the most studied being leprosy, malaria, chronic viral hepatitis, and further into the 90s, HIV/AIDS (see Blackwell et al. 2009, for a thorough review). However, these early studies carried important limitations: samples sizes were modest, typically on the order of hundreds, and a priori selected candidate genes were investigated, making it difficult to differenti- ate between associations which were causal or driven by linkage disequilibrium. Second, diseases which until recently were impractical to study in a GWAS setting can now be investigated. A remark- able example is the analysis led by the personal genomics company 23andMe, which performed an association study for infectious diseases in a sample of 200,000 customers which had volunteered information on various medical con- ditions (Tian et al. 2016). The study found that variation at HLA genes or within the MHC region is associated with viral (chickenpox, shingles, cold sores, mononucleo- sis, mumps, warts caused by papillomavirus, strep throat, scarlet fever, pneumonia) and bacterial (tonsil infections, ear infections) diseases. Third, because GWAS query SNPs throughout the entire MHC region, it is possible to fine-map associations, i.e., identify associations within a narrower region of the genome. This has shown that several associations involve sites with regulatory function. For example, AIDS pro- gression is associated with a 5’ UTR regulatory variant of HLA-C (Kulkarni et al. 2011) and hepatitis B recovery is associated with variation at a 3’ UTR site which modulates -DPB1 expression (Thomas et al. 2012). From an evolution- ary perspective, this indicates that selection on HLA genes is not restricted to the structural domains involved in peptide binding, but also involves regulatory variants. The explosion of data that has occurred in the last decade has brought about important changes. Millions of genetic markers are now queried in extremely large samples, allow- ing genomewide association studies (GWAS) to identify genes or genomic regions associated with diseases, with- out having to define beforehand the candidate loci to be queried. These association studies are bringing important contributions to our understanding of how genetic variation at HLA genes is related to response to pathogens. Below, we highlight four insights. First, the recent generation of GWAS have confirmed that variation at HLA genes is directly associated with the out- come of many infectious diseases. Disease associations Among these are HIV Fourth, dense SNP data allows HLA alleles to be imputed (see Section 3) and thus the amino acid sequence coded 11 Immunogenetics (2018) 70:5–27 by HLA genes to be inferred. In this way, it is possible to study associations at the molecular level, identifying specific changes in a protein that are associated with disease resistance or susceptibility (Nishida et al. 2016; Tian et al. 2016). involving HLA genes to be polygenic, which we discuss below. by HLA genes to be inferred. In this way, it is possible to study associations at the molecular level, identifying specific changes in a protein that are associated with disease resistance or susceptibility (Nishida et al. 2016; Tian et al. 2016). There is support for epistatic interactions between vari- ants at distinct HLA loci, driving advantageous haplotypes to higher frequencies than expected by chance, and thus explaining the high linkage disequilibrium in the MHC. One reason why a haplotype may be favored is that it car- ries a combination of alleles that presents a broader range of pathogenic peptides than expected for a random pair of alleles. This hypothesis was recently supported by a theo- retical model, as well as data analyses showing that alleles in linkage disequilibrium on average have a lower overlap in the peptide binding repertoire than expected by chance (Penman et al. 2013). Using a simulation-based approach, van Oosterhout (2009) also illustrated that epistasis among HLA loci can play an important role in shaping extant patterns of diversity. Finally, GWAS for HLA loci found multi-locus effects, as is the case of the association of the DR2 haplotype (DRB11501 and DRB50101) with multiple sclerosis (Gregersen et al. 2006). Even more activity has taken place in the study of genetic associations with autoimmune diseases. Samples of tens of thousands have routinely been assembled, and copious associations with the MHC region or specific HLA genes have been firmly established, including diabetes, arthritis, celiac disease, lupus, ankylosing spondylitis, multiple scle- rosis, psoriasis, and Crohn’s disease (reviewed in Trowsdale and Knight 2013). From an evolutionary perspective, the existence of autoimmune conditions associated with rela- tively common HLA alleles poses an important question: if the disease reduces an individual’s chances of survival and reproduction, why have the underlying alleles not been driven to low frequencies? Disease associations To answer this question, an influential working hypoth- esis that the same alleles which conferred resistance to infectious diseases and rose in frequency are also asso- ciated with autoimmune conditions (Corona et al. 2010; Sams and Hawks 2014; Abadie et al. 2011). This suggests a trade-off occurs, where the benefits brought by disease resistance outweigh the fitness costs of autoimmunity. A formal test involves asking whether alleles that are associ- ated with autoimmune disease risk have increased evidence of having experienced selection. In the context of non-HLA variants, Fumagalli et al. (2011) found a correlation between the abundance of autoimmune disease predisposing vari- ants and pathogen abundance, an indirect support for the trade-off hypothesis. Specifically for HLA, Abadie et al. (2011) examined whether the HLA-DQA1 variant which predisposes to celiac disease showed evidence of past selec- tion, but found no support. Corona et al. (2010) surveyed GWAS for complex diseases, and found that for type 1 dia- betes strongly predisposing SNPs are also those with strong evidence for positive selection. Second, multi-locus interactions have also been docu- mented between HLA genes and those outside the extended MHC (see Box 1). For example, Kirino et al. (2013) found a strong epistatic interaction between HLA-B51 and the ERAP1 locus, with one specific genotype greatly increas- ing the susceptibility to Behc¸et’s disease. ERAP1 codes for the protein responsible for trimming the pathogens to be loaded and presented by HLA class I molecules, making interactions between it and HLA genes functionally plausible. Another case of epistasis involves the interaction between HLA and KIR. KIR molecules can recognize HLA class I molecules carrying HLA-A3, -A11, -Bw4, -B27, -C1, or -C2 epitopes, as well as HLA-F and possibly HLA-G (reviewed in Parham et al. 2012). In a study of 30 human populations, Single et al. (2007) found a strong negative correlation between the frequency of HLA-B alleles of the Bw4 group, which carry an isoleucine at position 80, and the presence of KIR3DS1 gene. Because Bw4 alleles are lig- ands for KIR3DS1, which is an “activator” (a gene whose protein product initiates a cytotoxic response), the combina- tion of high frequencies of ligand and receptors would result in an abundance of excessively activating genotypes, which are prone to autoimmunity. At the other extreme, combi- nations of low frequencies of ligand and KIR3DS1 would result in an excessively weak KIR response, increasing the susceptibility to infection. Disease associations Selection against genotypes at these extremes could account for the observed correlations seen in Single et al. (2007). Using a similar approach, Hollenbach et al. (2013) found strong (r > 0.79) and signif- icant correlations between the frequencies of KIR2DL3 and HLA-C1 in 45 populations. Although this approach has not yet delivered a clear pic- ture, the strong evidence of pathogen-driven selection at HLA genes, coupled with the extreme abundance of HLA involvement in autoimmunity, call for further development of evolutionary approaches investigating the possibility that there is a causal connection between evolutionary response to infectious diseases and autoimmunity. Multilocus effects: epistasis and hitchhiking In the context of the MHC region, a natural hypothe- sis is that genes close to the classical HLA loci will show an enrichment of deleterious variants, with respect to the expectations based on genomewide controls. Mendes (2013) investigated this hypothesis, and in an analysis of the 1000 Genomes data (The 1000 Genomes Project Consortium 2010) found that genes that hitchhike with HLA loci have an increased proportion of putatively deleterious variants (Fig. 2). This hypothesis was also tested by Lenz et al. (2016), who used a larger exome-based dataset to show an excess of intermediate frequency deleterious polymorphism within the MHC. Further, these authors used simulations to show that strong balancing selection—comparable in strength to that seen at HLA genes—makes deleterious vari- ants more common than would be expected without the hitchhiking effect. These findings are particularly important given the large number of disease associations in the MHC region (includ- ing the flanking non-HLA loci), suggesting that balancing selection in HLA genes may drive the accumulation of deleterious variants in their neighborhood, contributing to the associations with disease phenotypes. The epistatic interactions between KIR and HLA also influence reproduction. For example, mothers homozygous for the KIR haplotype from group A (defined by the presence of four framework genes—KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1—and KIR2DL1, KIR2DL3, KIR2DS4, and KIR3DL1) have an increased rate of miscar- riage, pre-eclampsia, and weight restriction at birth when they also carry an HLA-C1 allele and the fetus has an HLA- C2 allele. This results from a less effective remodeling of blood vessels, necessary for placentation (Penman et al. 2016; Hiby et al. 2014; Hiby et al. 2004). On the other hand, individuals with group A KIR haplotypes and HLA- C1 alleles respond to viral infections more efficiently than individuals with group B haplotypes (which carry genes encoding KIRs with decreased or no binding to HLA class I molecules, such as KIR2DS2, KIR2DS3, and KIR2DS5) in combination with HLA-C2 alleles (e.g., hepatitis C and HIV clearance). This tradeoff may result in alternating episodes of reproductive and pathogen-driven selection, explaining the maintenance of polymorphism for KIR haplotypes and for the HLA-C1 and -C2 group alleles in many human populations. This scenario was supported by computer sim- ulations (Penman et al. Multilocus effects: epistasis and hitchhiking There is increasing awareness that many adaptive traits are polygenic, and that searching for allele frequency changes at multiple loci is an important improvement over “sin- gle locus” approaches (Daub et al. 2013; Berg and Coop 2014). There are several reasons why we expect adaptation Support for these interactions also comes from the study of specific populations. In the African KhoeSan, the C2 12 Immunogenetics (2018) 70:5–27 allotype occurs at an unusually high frequency (63%), whereas in the Yucpa of South America it is the C1 allotype that is common (83%) (Hilton et al. 2015; Gendzekhadze et al. 2009). Strikingly, in both populations, the receptors for these common allotypes show evidence of having been recently selected and driven to high frequencies, with the mutant forms having reduced or complete lack of function. In both cases, these population-specific variants may have been favored due to their ability to restore a balance between C1, C2, and the KIR inhibitory allotypes, providing the ben- efits of reducing the chances of originating preeclampsia predisposing genotypes (see below). Functional studies pro- vide further support for epistasis, showing that homozygotes for HLA-C1 respond more intensely to a viral infection than those carrying HLA-C2 alleles (Ahlenstiel et al. 2008, see also Augusto et al. 2015, for an example involving the autoimmune disease pemphigus). allotype occurs at an unusually high frequency (63%), whereas in the Yucpa of South America it is the C1 allotype that is common (83%) (Hilton et al. 2015; Gendzekhadze et al. 2009). Strikingly, in both populations, the receptors for these common allotypes show evidence of having been recently selected and driven to high frequencies, with the mutant forms having reduced or complete lack of function. In both cases, these population-specific variants may have been favored due to their ability to restore a balance between C1, C2, and the KIR inhibitory allotypes, providing the ben- efits of reducing the chances of originating preeclampsia predisposing genotypes (see below). Functional studies pro- vide further support for epistasis, showing that homozygotes for HLA-C1 respond more intensely to a viral infection than those carrying HLA-C2 alleles (Ahlenstiel et al. 2008, see also Augusto et al. 2015, for an example involving the autoimmune disease pemphigus). Multilocus effects: epistasis and hitchhiking 2016) and is consistent with patterns of HLA and KIR polymorphism in many human popula- tions (see details in Trowsdale and Moffett 2008; Parham and Moffett 2013; Augusto and Petzl-Erler 2016). To conclude, we emphasize that ongoing research recom- mends that variation at HLA genes be studied with reference to both the genes they interact with, as well as considering how physical linkage leads to changes in polymorphism at neighboring sites. Placing HLA variation in a genomewide context will be essential in order to achieve these goals. Fig. 2 The value of ψ, a statistic that measures the proportion of deleterious variants, in three sets of SNPs. The statistic is defined by ψ = LS.PN LN .(PS+1), where P represents the number of polymorphic sites, L represents the number of potentially mutable sites, and S and N subscripts refer to synonymous and nonsynonymous sites. Higher val- ues of ψ indicate a greater proportion of deleterious (or functional, in the case of the SNPs from the classical HLA genes) variants. Values are shown for exons of classical HLA genes, genes in the immediate neighborhood of the HLA genes (“peri-HLA”), and genes outside the MHC region. Values were computed for sites with a minor allele fre- quency (MAF) greater than 0.05, to avoid the effect of rare deleterious variants, which are overrepresented in the control set. The peri-HLA genes have higher load (ψ) than the controls Fig. 2 The value of ψ, a statistic that measures the proportion of deleterious variants, in three sets of SNPs. The statistic is defined by ψ = LS.PN LN .(PS+1), where P represents the number of polymorphic sites, L represents the number of potentially mutable sites, and S and N subscripts refer to synonymous and nonsynonymous sites. Higher val- ues of ψ indicate a greater proportion of deleterious (or functional, in the case of the SNPs from the classical HLA genes) variants. Values are shown for exons of classical HLA genes, genes in the immediate neighborhood of the HLA genes (“peri-HLA”), and genes outside the MHC region. Values were computed for sites with a minor allele fre- quency (MAF) greater than 0.05, to avoid the effect of rare deleterious variants, which are overrepresented in the control set. The peri-HLA genes have higher load (ψ) than the controls Fig. Population differentiation various demographic models, among others (Table 2). Third, the power to detect balancing selection may vary depending on the timescale of separation of populations, and features of their demographic histories (reduced HLA differentiation being harder to detect in admixed popula- tions, for which genomewide FST is lower). Fourth, SNPs with low heterozygosities are constrained to low FST , implying that HLA and non-HLA SNPs must be compared in a way that accounts for this effect (Bhatia et al. 2013). If distinct populations are under a regime of selection favor- ing HLA heterozygotes, population differentiation, mea- sured by FST , is expected to be lower at HLA than at neutral loci (Schierup et al. 2000). This is because balancing selec- tion maintains alleles segregating in populations for longer than expected under neutrality, reducing FST (Box 2). An alternative scenario is that selection favors different alleles in distinct populations, driving locally adaptive HLA alleles to higher frequencies, increasing population differen- tiation.This expectation is consistent with pathogen-driven selection at HLA, for which there is theoretical (Borghans et al. 2004; Hedrick 2002) and empirical support (e.g., Prugnolle et al. 2005; Hedrick 2006). Given the premise that pathogen populations differ between regions, pathogen-driven selection could drive locally adaptive HLA alleles to higher frequencies, and thus cause an increase in population differentiation. In order to overcome these issues, we analyzed HLA dif- ferentiation among major continental groups, accounting for these effects (Brandt 2015) (Fig. 3). Marker-type effects are accounted for by only analyzing SNP data. The non-HLA SNPs provide expectations due to demographic processes, allowing a statistical assessment of how extreme the dif- ferentiation is for SNPs within HLA loci. FST values for SNPs in the HLA and non-HLA groups are averaged using an approach that controls for the differing heterozygosity distributions in those groups (Reynolds et al. 1983; Bhatia et al. 2013). With these methodological controls in place, the results in Fig. 3 show that SNPs within HLA genes have lower FST than genomewide SNPs when we com- pare highly diverged populations (i.e., those from different continents). Population pairs from the same continent have higher differentiation for SNPs in the HLA genes compared to other genomic regions. Surprisingly, support for both of these markedly different expectations has been found (Table 2), with some studies showing HLA to be unusually highly differentiated, and oth- ers reporting unusually low differentiation at HLA. Multilocus effects: epistasis and hitchhiking 2 The value of ψ, a statistic that measures the proportion of deleterious variants, in three sets of SNPs. The statistic is defined by ψ = LS.PN LN .(PS+1), where P represents the number of polymorphic sites, L represents the number of potentially mutable sites, and S and N subscripts refer to synonymous and nonsynonymous sites. Higher val- ues of ψ indicate a greater proportion of deleterious (or functional, in the case of the SNPs from the classical HLA genes) variants. Values are shown for exons of classical HLA genes, genes in the immediate neighborhood of the HLA genes (“peri-HLA”), and genes outside the MHC region. Values were computed for sites with a minor allele fre- quency (MAF) greater than 0.05, to avoid the effect of rare deleterious variants, which are overrepresented in the control set. The peri-HLA genes have higher load (ψ) than the controls Strong selection at a locus can also influence variation at linked sites through genetic hitchhiking. Under pathogen- driven selection an advantageous variant is driven to higher frequencies at a greater speed than would be expected under drift, and can thus drag linked variants (Charlesworth 2006). This selective regime can increase the frequency of slightly de- leterious mutations near the selected gene. Accordingly, Chun and Fay (2011) showed that for regions in the neigh- borhood of sites with strong evidence for positive selection, there is an enrichment for deleterious polymorphism. 13 Immunogenetics (2018) 70:5–27 aSee Box 1 for the definition of HLA allele Population differentiation What is the cause for the inconsistency among studies? Analyses using FST are sensitive to various aspects of the methodology, all of which can influence the results, as we discuss below. First, studies which compare different markers, such as HLA alleles and microsatellites, are sensitive to the effects of the mutational mechanism and mean heterozygosity on FST , making direct contrasts between HLA and non-HLA markers unreliable (a challenge for the studies of Meyer et al. 2006; Sanchez-Mazas 2007). Second, the statistical tests used to define extreme FST differ among studies, including outlier approaches, tree-based tests, simulation under an How do these findings compare to those of previous studies? Low differentiation among HLA SNPs is consistent with the findings of Hofer et al. (2012), which detected a similar pattern in a dataset including highly divergent human populations. The increased differentiation seen by Bhatia et al. (2011) among African populations is also consistent with this result, since that study analyzed closely related populations. Table 2 Population differentiation at HLA genes relative to neutral markers Reference Neutral marker HLA marker Method FST in HLA Akey et al. (2002) SNP SNP (genomewide Empirical outlier Not an outlier scan) Meyer et al. (2006) Microsatellites HLA allelea Empirical outlier Not an outlier Sanchez-Mazas (2007) Microsatellites HLA allelea Empirical outlier Lower in HLA and RFLPs Bhatia et al. (2011) SNP SNP (genomewide Tree-based test Higher in HLA scan) Nunes (2011) Microsatellites Microsatellites Simulation Higher in HLA Hofer et al. (2012) SNP SNP (genomewide Simulation Lower around HLA-C scan) Colonna et al. (2014) SNP SNP (genomewide Empirical outlier + clustering Not an outlier scan) Brandt (2015) SNP SNP and HLA alleles Empirical outlier Lower for HLA SNPs; HLA alleles are not outliers aSee Box 1 for the definition of HLA allele Table 2 Population differentiation at HLA genes relative to neutral markers 14 Immunogenetics (2018) 70:5–27 FST at HLA SNPs than genomewide SNPs for pairs of populations in the same continent (Fig. 3). Also, one of the SNPs driving the high differentiation reported in Bhatia et al. (2011) was linked to HLA-DPA1, a locus we excluded because it did not show strong evi- dence of balancing selection in previous studies (Solberg et al. 2008; Begovich et al. 2001), and showed instances of directional selection (Hollenbach et al. 2001). Selection and admixture Individuals in admixed populations have genomes which are a mosaic of different ancestries (Winkler et al. 2010). The size and ancestry of segments is determined by fac- tors which are demographic (e.g., proportion of ancestors from each ancestry, timing of admixture) and genetic (e.g., recombination rates). If genetic variants from one of the First, the signal of local adaptation (high differentiation) may only be detectable when comparing closely related populations, such as the ones in the same continent. Indeed, previous studies have detected high differentiation in HLA alleles between populations within the same continent (Cao et al. 2004; Qian et al. 2013), and we have detected higher Fig. 3 FST among pairs of populations. Each point depicts the mean FST for non-HLA (x-axis) and HLA (y-axis) SNPs between pairs of populations in each continent (AFR: Africa; EAS: East Asia; EUR: Europe; SAS: Southeast Asia). Pairs of populations from the same continent are represented by white-filled points, and pairs of popula- tions from different continents, by solid black points. SNP data was acquired from the 1000 Genomes data phase III (The 1000 Genomes Project Consortium 2015), and HLA SNPs were filtered according to Brandt et al. (2015) to avoid errors due to mapping bias. FST values were weighted by allele frequency, so that the excess of rare variants in the non-HLA SNPs does not cause a reduction of mean FST in that class. Notice that HLA differentiation is higher than genomewide for population pairs from the same continent, and lower than genomewide when populations from different continents are compared Fig. 3 FST among pairs of populations. Each point depicts the mean FST for non-HLA (x-axis) and HLA (y-axis) SNPs between pairs of populations in each continent (AFR: Africa; EAS: East Asia; EUR: Europe; SAS: Southeast Asia). Pairs of populations from the same continent are represented by white-filled points, and pairs of popula- tions from different continents, by solid black points. SNP data was acquired from the 1000 Genomes data phase III (The 1000 Genomes Project Consortium 2015), and HLA SNPs were filtered according to Brandt et al. (2015) to avoid errors due to mapping bias. FST values were weighted by allele frequency, so that the excess of rare variants in the non-HLA SNPs does not cause a reduction of mean FST in that class. Population differentiation Indeed, for HLA-DPA1 population differentiation was higher than genomewide in our data as well, consistent with local pos- itive selection. Interestingly, HLA-DPA1 has one of the strongest signatures of long term balancing selection in Bitarello et al. (2017). A plausible scenario is that HLA- DPA1 is under a selective regime that varies through time, leaving a signature of past balancing selection and more recent local positive selection. Second, low differentiation at SNPs and high differenti- ation at HLA alleles may be expected if we consider that HLA alleles are defined by multiple SNPs, and that most SNPs are shared between two or more alleles. The impor- tant role that intragenic recombination and gene conversion play in generating HLA allele diversity also contributes to the sharing of SNPs among different HLA alleles (Parham and Ohta 1996). Thus, a plausible scenario is that individual SNPs have low FST , but the haplotypes which they define may show high divergence. Biologically this amounts to considering that balancing selection favors the maintenance of polymorphism at specific sites, key to defining peptide binding specificities (Bitarello et al. 2016). However, the specific combinations of variants (i.e., the HLA alleles) that become more frequent differ among populations as a function of the pathogens driving the selection. Given the overall result that natural selection on HLA genes, over long periods of time, results in decreased population differentiation (Fig. 3, y-axis), it is natural to consider how to reconcile this with the expectation that pathogens would drive local adaptation, making populations more different from one another at HLA genes. There are two possible ways in which low differentiation at HLA SNPs can be reconciled with a model of local adaptation of HLA alleles. Selection and admixture Notice that HLA differentiation is higher than genomewide for population pairs from the same continent, and lower than genomewide when populations from different continents are compared 15 Immunogenetics (2018) 70:5–27 Fig. 4 Deviation from average genomewide ancestry in four admixed populations along chromosome 6. The degree to which local ances- try deviates from genomewide averages is shown for African ancestry (black lines). The region encompassing the MHC region is indicated by gray shading. Ancestral and admixed populations are from the 1000 genomes project (African and European; ftp://ftp.1000genomes. ebi.ac.uk/vol1/ftp/release/20110521/), except for the ancestral Native American sample, which is from the HGDP-CEPH (http://www.cephb. fr/hgdp/index.php). Local ancestries were estimated using RFMIX (Maples et al. 2013). The ancestry deviation measure is the differ- ence between ancestry at a given genomic position with respect to the genomewide average, normalized by the standard deviation of the ancestry estimate (thus providing a measure of the number of standard deviations each ancestry departs from its genomewide average) Fig. 4 Deviation from average genomewide ancestry in four admixed populations along chromosome 6. The degree to which local ances- try deviates from genomewide averages is shown for African ancestry (black lines). The region encompassing the MHC region is indicated by gray shading. Ancestral and admixed populations are from the 1000 genomes project (African and European; ftp://ftp.1000genomes. ebi.ac.uk/vol1/ftp/release/20110521/), except for the ancestral Native American sample, which is from the HGDP-CEPH (http://www.cephb. fr/hgdp/index.php). Local ancestries were estimated using RFMIX (Maples et al. 2013). The ancestry deviation measure is the differ- ence between ancestry at a given genomic position with respect to the genomewide average, normalized by the standard deviation of the ancestry estimate (thus providing a measure of the number of standard deviations each ancestry departs from its genomewide average) parental populations are advantageous to individuals in the admixed population, they will rise in frequency and thus cause an over-representation of a specific parental ances- try in the genomic region under selection. Thus, regions of the genome exhibiting ancestry proportions that deviate from the genomewide average provide evidence for recent selection. chromosome 6, we find that two of the populations (Colom- bian and Mexican) have an excess of African ancestry in the MHC region (the threshold of significance set at 4.4 standard deviations, following Seldin et al. 2011) (Fig. 4). bMexican, Guatemalan, Costa Rican, Colombian, Chilean, Argentinean, Brazilian aDominican Republic, Colombian, Puerto Rican, Ecuadorian Selection and admixture in local ancestry for the MHC region is strong and recurrent, prompting us to consider both its possible biological basis as well as the likelihood of methodological artifacts. A basic concern is whether local ancestry methods are biased by features of the MHC region (other than a true shift in ancestry proportions). For example, Price et al. (2008) pointed out that most deviations in ancestry reported by Tang et al. (2007a) (both within and outside the MHC region) were associated with regions of high linkage dis- equilibrium (LD). However, new methods for detecting local ancestry control for LD, but still detect an excess of African ancestry in the MHC (Guan 2014; Brisbin et al. 2012) (Table 3). An additional concern is that some ances- try inference methods require phased data, something that is challenging for the MHC, given the high polymorphism. However, ancestry results are consistent across methods that do (e.g., Brisbin et al. 2012) and do not (Guan 2014) require phased data, suggesting this is not the factor driving the findings. h bl f l l i i i d b Yasukochi and Ohashi (2016) argue that this evidence is circumstancial, noting that B73 was not found in any archaic genome and that strong long-term balanc- ing selection could maintain the alleles independently in both species. Also, if Denisova introgression into modern humans occurred in Southeast Asia, that is where HLA- B73 should have higher frequency. Further problems for local ancestry estimation were raised by Pasaniuc et al. (2013), who found that loci with increased devi- ation in local ancestry show high polymorphism and increased rates of mendelian inconsistency. These authors also showed that inappropriate parental reference panels (e.g., distantly related from the true parental populations) can introduce errors in the analysis. This fact is of extreme relevance since samples from the true parental populations are not always available. On the other hand, Abi-Rached et al. (2011) found even more compelling evidence for adaptive introgression coming from the HLA-A11 allele, which occurs at high frequencies in Papua New Guinea and China (but is absent from Sub-Saharan African) and is found in long haplotypes with HLA-C15 and HLA-C12, both of which exhibit higher diversity in Asia than in Africa. A likely explanation is that all HLA-A11 found in modern humans came from Denisovan introgression, followed by a rise in frequency in Asia. Selection and admixture To explore this pattern further, we reviewed the findings of eight studies that investigated the distribution of local ancestries and recorded how often the MHC showed unusual ancestry proportions with respect to genomewide averages. In total, six out of eight studies report an excess of African ancestry in the MHC region for at least one admixed population (Table 3). Interestingly, this effect is seen in populations with different admixture histories, distinct African parental populations and proportion of contributions, and using different methods to estimate local ancestry. Overall, the support for deviation To illustrate the power of this approach in understanding selection at HLA genes, we calculated local ancestries (i.e., the ancestry of a specific position of the genome) for indi- viduals from four admixed populations (The 1000 Genomes Project Consortium 2010). For each position in the genome, we quantified how much the ancestry proportions differed from the genomewide average, within each population. For Table 3 Ancestry proportions in the MHC region vs genomewide Reference Admixed population Method Observation Tang et al. (2007a) Puerto Rican Frape Excess African Johnson et al. (2011) Mexicans SABER+ Excess European Brisbin et al. (2012) Four Latino populationsa PCAdmix Excess African in Colombian, Puerto Rican and Ecuadorian Bhatia et al. (2014) African Americans RFmix non-significant increase in African Guan (2014) Mexican ELAI Excess African Rishishwar et al. (2015) Colombian SUPORTMix Excess African Zhou et al. (2016b) Mexican ELAI Excess African Deng et al. (2016) Seven Latino populationsb Structure and Z-test Excess African aDominican Republic, Colombian, Puerto Rican, Ecuadorian bMexican, Guatemalan, Costa Rican, Colombian, Chilean, Argentinean, Brazilian Table 3 Ancestry proportions in the MHC region vs genomewide Immunogenetics (2018) 70:5–27 16 groups (Denisova and Neanderthal) into modern humans (reviewed in Racimo et al. 2015), including in the MHC region. Abi-Rached et al. (2011) suggested that a highly divergent allele, HLA-vB73, entered the modern human gene pool through introgression from archaic hominins. In mod- ern populations, HLA-B73 is practically absent everywhere except West Asia, and almost all haplotypes carrying HLA- B73 also carry HLA-C15:05, which only reaches appreciable frequencies in Asia (Abi-Rached et al. 2011). Simulations showed that introgression from archaic hominins provides a bet- ter fit to the datathan a model in which the allele arose in Africa before the Out-of-Africa event (Abi-Rached et al. 2011). Selection and admixture In brief, it may well be that when humans left Africa, they encountered new selective pressures to which archaic hominins were better adapted on a local scale, and strong selection favored those adaptive variants acquired through introgression. However, current evidence for adaptive introgression of HLA alleles should be inter- preted with caution because of the technical difficulties in assessing variability of HLA genes, and small sample sizes of archaic species. Also, apparent introgression might result from incomplete lineage sorting, which is particularly likely in the MHC region, where long-term balancing selection results in trans-specific polymorphisms (Klein et al. 1993; Teixeira et al. 2015; Leffler et al. 2013). Further studies will be needed so as to evaluate whether technical artifacts underlie the shifts in ancestry propor- tions in the MHC region. In this sense, a promising result was reported by Deng et al. (2016), who used simulations under a human demographic model to show that the ances- try deviation in the MHC of Latin American populations is not expected in the absence of selection. In addition, Tang et al. (2007a) showed that an unusual African ancestry pro- portion in the MHC region of Puerto Rican individuals is found using local ancestry analysis based on SNPs, as well as more traditional admixture estimates using classical HLA markers and microsatellites, providing additional evidence that the shifts in ancestry are not a feature observed with one type of marker or inference method. Ancestry deviations place the MHC as a striking example of a genomic region under strong recent selection. Neverthe- less, even if this general picture is confirmed in new studies, several questions remain to be addressed. First, how many and which HLA alleles are favored by selection, causing the deviation in local ancestry? Second, is the recurrent finding of excess African ancestry explained by higher genomewide diversity in Africans (which indirectly could lead to the har- boring of more advantageous variants)? Clearly, a biological understanding of these patterns is still lacking. From genome to transcriptome In the event that the surveyed individual is highly divergent from the sequences in the index (as is often the case due to the high polymorphism of HLA genes), it is likely that many reads will be discarded due to large numbers of mismatches, failing to document expression, and biasing the estimates toward the overexpression of variants which are more simi- lar to the one in the index. This results in inaccurate and/or biased gene expression estimates and can cause spurious eQTLs to be identified (Panousis et al. 2014). This problem is similar to that of read mapping for HLA genes in NGS, discussed in Section 3 (Brandt et al. 2015). among tumor types, ranging from loss/downregulation to high expression (Boegel et al. 2014). Such opposing effects of expression levels may account for the selective maintenance of differential expression across HLA alleles or haplotypes. Despite the potential importance of HLA expression to evolutionary and medical studies, few datasets with this information have been generated. To a large degree, this results from the the difficulty in quantifying expression for genes which show an unusually high polymorphism and are members of a multi-gene family. For highly polymorphic genes, array-based expression requires probes that avoid polymorphic regions, which if not accounted for can cause differential binding due to genetic variation, biasing expres- sion estimates. The same difficulty applies to quantitative PCR, which needs primers that can bind the entire range of alleles of a specific locus, posing an important challenge when developing the experimental design. To overcome these difficulties, customized arrays (Vandiedonck et al. 2011) and qPCR primer sets (Ramsuran et al. 2015) have been developed. These account for polymor- phism and can provide locus-level expression estimates. However, these studies are limited in the number of samples and population diversity surveyed, and the requirement of custom arrays or primer sets makes repetition of surveys on additional populations and extension to other HLA loci challenging. Further, the expression of each allele can- not be directly estimated, and is instead imputed from the locus-level expression of homozygotes (Ramsuran et al. 2015). This places the quantification of HLA expression as an enterprise still in its infancy, although the studies carried out to date show that HLA expression varies between alle- les, loci, and tissues (Boegel et al. 2012; Boegel et al. 2014; Ramsuran et al. 2015; Mel´e et al. 2015). From genome to transcriptome While most studies on selection at HLA genes focus on pep- tide binding properties, expression levels are also important in determining phenotypes related to disease progression, both for infection and cancer (Blais et al. 2012; Thomas et al. 2012; Apps et al. 2013; Boegel et al. 2014). For exam- ple, high expression of HLA-C enhances an individual’s ability to respond to HIV infection, whereas low expression confers protection against Crohn’s disease (Blais et al. 2012; Apps et al. 2013). Additionally, expression varies broadly Selection favoring alleles of a specific ancestry can also be seen through the analysis of archaic genomes. These studies found evidence for adaptive introgression from archaic 17 Immunogenetics (2018) 70:5–27 Fig. 5 Fold change in expression estimates obtained by kallisto (Bray et al. 2016) using a supplemented index relative to a standard refer- ence index (y = 0). Results are presented for genotypes with different degrees of similarity to the reference genome (bar colors). We used 48 CEU individuals for which RNAseq data are available from the Geu- vadis consortium (Lappalainen et al. 2013) and HLA genotypes were determined by Sanger sequencing (Gourraud et al. 2014). Genotypes at each locus were divided according to quartiles of differences from the ref- erence allele at that locus. “Most similar” and “Most different” correspond to the first and fourth quartiles respectively (12 individuals each). Fig. 5 Fold change in expression estimates obtained by kallisto (Bray et al. 2016) using a supplemented index relative to a standard refer- ence index (y = 0). Results are presented for genotypes with different degrees of similarity to the reference genome (bar colors). We used 48 CEU individuals for which RNAseq data are available from the Geu- vadis consortium (Lappalainen et al. 2013) and HLA genotypes were determined by Sanger sequencing (Gourraud et al. 2014). Genotypes at each locus were divided according to quartiles of differences from the ref- erence allele at that locus. “Most similar” and “Most different” correspond to the first and fourth quartiles respectively (12 individuals each). The RNAseq technology, which quantifies expression using NGS, is increasingly being used in genomewide stud- ies and has the potential to provide large-scale information on HLA expression, but also has challenges. The technology relies onthe mapping of short reads (generated by sequencing the transcriptome) to an index, so as to quantify the abun- dance of mRNA originating from each gene or exon. From genome to transcriptome As a consequence, large studies which surveyed the whole- transcriptome in many individuals (e.g., Lappalainen et al. 2013; Battle et al. 2014) using high-throughput technologies do not provide reliable estimates for the expression of HLA genes. An alternative is the development of bioinformatic tools that use whole-transcriptome RNAseq data to accurately esti- mate HLA expression. This has the benefit of placing the HLA expression data within the context of genomewide expression levels, and allows the use of RNAseq datasets that are already available (Lappalainen et al. 2013; Battle et al. 2014; Mel´e et al. 2015). A promising approach is to use of an index with thousands of HLA sequences reported in databases such as IPD-IMGT/HLA, instead of relying on a single reference genome. For example, seq2HLA is a pipeline proposed by Boegel et al. (2012) Immunogenetics (2018) 70:5–27 18 reased differentiation (e.g., Hofer et al. 2012), as well as of increased differentiation (Bhatia et al. 2011). Such studies will help investigate which HLA variants represent adapta- tions to local selective pressures, and which are shared exten- sively at global scale, as an outcome of long-term balancing selection. We are now also able to investigatepatterns of admix- ture in HLA genes (Tang et al. 2007a; Guan 2014), providing insights into the time frame and mode of selection that occurs when populations of different ancestries meet and interbreed. which uses a form of in silico genotyping to both infer the geno- types at HLA genes as well as estimate the expression of each HLA allele at a locus. Such allele-specific estimates are not obtained when RNAseq data is processed by standard pipelines, which provide expression estimates at the level of genomic features such as annotated genes, exons or isoforms. The work by Boegel et al. (2012) showed that the use of an appropriate index (i.e., the set of reference sequences to which the short reads generated by the NGS will be aligned) is the key element for the improvement in the estimates. The benefits of this approach are shown in Fig. 5: expres- sion estimates increase when using indices supplemented with many HLA sequences, relative to expression estimated using the single reference genome. This effect is more pro- nounced for individuals carrying alleles which are most different from the reference. This is expected, since these are the cases where the use of the reference genome leads to the greatest underestimation of expression. From genome to transcriptome We can increasingly test co-evolutionary hypotheses, such as the relation between KIR and HLA polymorphism (e.g., Single et al. 2007), and test hypotheses of epistatic interactions. Genomic data also allows us to test the effect of strong selection on HLA upon linked variants, a pro- cess which may be driving the accumulation of deleterious mutations near HLA genes (e.g., Lenz et al. 2016). A whole new layer of information, namely expression levels, canbegeneratedon alarge scale, andintegratedwithinformation on genetic variation. This will contribute to association stud- ies, by incorporating a key cellular phenotype—expression level—as a covariate. Such approaches will also help bring functional information to the investigation of HLA evolu- tion (for example, in the form of allelic lineages (Bitarello et al. 2016) or supertype grouping (Francisco et al. 2015)). This result suggests that bioinformatic methods tailored to deal with HLA diversity can bring important changes to expression estimates and thus to eQTLs mapped, providing new hypotheses for functional elements which drive HLA expression variation. Promising candidates will include UTR sites, promoter/enhancer polymorphism, transcription factor binding sites, etc, all of which have been documented as enriched categories of eQTLs in standard genomewide studies (e.g., Lappalainen et al. 2013). Our perspective is that, increasingly, we will see the immunogenetics community working closely with researchers in genomics. Placing HLA within the genomic context is key to understanding HLA genes; complementarily, immu- nogenetics expertise will be key to interpreting genomewide studies, within which HLA genes are frequent and striking findings (be it in GWAS, selection, admixture or expression studies). In addition, lessons and challenges associated with studying a highly polymorphic region under intense balancing selection, as is the case for the MHC, can be carried over to the study of other genes or genomic regions under bal- ancing selection (Leffler et al. 2013; Teixeira et al. 2015; DeGiorgio et al. 2014; Bitarello et al. 2017). It will also be possible to further explore initial findings regarding expression differences among genes and alleles (revealed by qPCR studies). In particular, the pattern of rel- atively even expression among HLA-B alleles (Ramsuran et al. 2017), and variable expression levels among lineages at HLA-A (Ramsuran et al. 2015) and HLA-C (Apps et al. 2013) will be amenable to investigation on a wider scale. BOX 1. HLA terminology The vocabulary within the immunogenetic literature is often quite challenging to non-specialists. Here is a brief summary of key terms used in this review. MHC region. The MHC (Major Histocompatibility Complex) genomic region, lo- cated on the short arm of chromosome 6 (6p21.3), which contains the HLA (Human Leukocyte Antigen) genes, as well as at least 200 other genes (approximately 40% of which are involved in some aspect of the immune response), spanning around 4 Mb (reviewed in Shiina et al, 2009). It is the most gene-dense region of the genome (with on average one gene every 16 kb) and, due to the presence of HLA loci, shows unusually high levels of polymorphism. It is also referred to as the HLA region, in humans. Extended MHC region. The extended MHC comprises around 8 Mb of chromosome 6, containing over 250 protein-coding genes. This region expands the MHC borders to segments with high linkage disequilibrium with the MHC and contains additional genes involved in the immune response (Horton et al, 2004; Shiina et al, 2009). HLA genes (or loci). Classical HLA genes are the highly polymorphic loci that code for the proteins that present peptides to the T-cell receptors (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRA1), and distinguish themselves from the Non-Classical genes, which have reduced polymorphism and do not have a role in peptide presentation. HLA genes (or loci). Classical HLA genes are the highly polymorphic loci that code for the proteins that present peptides to the T-cell receptors (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRA1), and distinguish themselves from the Non-Classical genes, which have reduced polymorphism and do not have a role in peptide presentation. HLA allele. This refers to a speciﬁc DNA sequence at an HLA gene, and can be thought of as a haplotype of SNP variants. There are extraordinarily large numbers of HLA alleles, with more than 12,000 for class I alleles and more than 4,000 for class II. (see details in http://www.ebi.ac.uk/ipd/imgt/hla/stats.html). HLA haplotype. The combination of alleles at several HLA genes on a single chro- mosome of a given individual. HLA function. HLA class I molecules, expressed in most cells, bind peptides of intracellular origin and present them on the cell surface to the T-cell receptors of CD8+ T-cells, initiating a cytotoxic response if they are recognized as foreign. Conclusions Our current knowledge of HLA evolution differs with respect to that of a decade ago in many ways. To a large degree, this results from our ability to place HLA variation within the context of the entire genome. Genomewide studies have contributed to our understanding of selection by increasing the power of tests (thanks to the large number of samples and genetic markers) and by allowing variation from the entire genome to be used as a control for complicating factors, including population history.We nowhave evidence that selection on classical HLA genes extends beyond the het- erozygote advantage model and has operated from ancient to very recent timescales (Albrechtsen et al. 2010; Field et al. 2016; Tang et al. 2007a; Mathieson et al. 2015). Acknowledgements We thank three anonymous reviewers for their helpful comments. We are grateful to Maria Luiza Petzl-Erler, Glenys Thomson, Danillo Augusto, Erick Castelli, Richard Single, and Cibele Masotti for their thoughtful reading and useful sug- gestions and criticisms. Scholarships provided by the S˜ao Paulo Research Foundation (FAPESP):# 2014/12123-2 (VRCA), #12/22796- 9 (DYCB), #11/12500-2 (BDB), #12/09950-9, and Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES) #1645581 (KN). FAPESP research grant 12/18010-0 (DM) and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) grant 305888/2015-3. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. By comparing genetic differentiation at HLA genes to that of the remainder of the genome, we have found instances of dec- 19 Immunogenetics (2018) 70:5–27 What are "neutrality tests"? Since the proposition of the neutral theory of molecular evolution, a myriad of tests have been proposed for the null hypothesis of neutrality (i.e., that evolutionary change results exclusively from mutation and genetic drift). When the neutral model is rejected, plausible alternative hypotheses include positive or balancing selection. Because the null hypothesis is based on the neutral model of evolution, these tests are called ’neutrality tests’. Tests based on differentiation among populations. If the intensity of positive selec- tion at a locus varies among populations, an allele can become common exclusively in the population where it is advantageous. As a consequence, differentiation at this selected locus will be greater than for the rest of the genome (Lewontin and Krakauer, 1973). However, for loci under balancing selection, differentiation is ex- pected to be reduced because variants will be kept at intermediate frequencies in all populations. Tests based on population differentiation typically use FST to compare differentiation at focal loci to simulated expectations or genomic controls (i.e., all other genes). These tests can detect relatively recent selection. Tests based on linkage disequilibrium and diversity. Variants under strong positive selection rise in frequency rapidly, reducing diversity at neutral loci in their vicinity. For balancing selection, because two or more variants are maintained, local genetic variation increases in the region. These tests are powerful to detect recent and strong positive selection, and less powerful for long-term balancing selection. Tests based on patterns of allelic frequency distribution (or "Site Frequency Spec- trum", SFS). These tests compare the distribution of observed allelic frequencies to those expected under neutrality. These include Tajima’s D, Fu and Li’s D, Fu and Li’s F, Fay and Wu’s H tests, the T2 test (DeGiorgio et al, 2014) and the NCD statistics (Bitarello et al, 2017). Positive selection is expected to increase the num- ber of low frequency variants, whereas balancing selection increases the number of intermediate frequency variants around the selected site. These tests can detect selection at comparatively deeper timescales than those based on LD or FST and are inﬂuenced by demographic parameters. For example, a bottleneck will prefer- entially remove low frequency variants, thus shifting the SFS to more intermediate frequencies even in the absence of balancing selection (Gattepaille et al, 2013). BOX 1. HLA terminology HLA class II molecules, expressed in antigen presenting cells, typically bind peptides of extracellular origin and present them to the TCRs of CD4+ cells, triggering a signaling process that leads to the multiplication of T-helper cells, leading to the stimulation of B-cells, which produce antibodies to the antigen that triggered the response. 20 Immunogenetics (2018) 70:5–27 What are "neutrality tests"? A crucial point of SFS-based tests is that the expectations should be evaluated for a neutral scenario that includes demographic parameters, since various demographic trajectories can mimic the effects of selection (e.g., population structure results in many of the signatures expected under balancing selection). 21 Immunogenetics (2018) 70:5–27 Tests contrasting intra and inter-speciﬁc variation. These include the dN/dS (non- synonymous to synonymous substitution rates ratio), HKA and MK tests, as well as the T1 and T2 tests (DeGiorgio et al, 2014) and the NCD2 statistic (Bitarello et al, 2017). While both positive and balancing selection are compatible with ele- vated dN/dS levels, they produce opposing signatures for HKA and MK tests (both of which compare polymorphism and divergence levels): positive selection causes lower polymorphism levels compared to divergence within the target species, bal- ancing selection causes higher polymorphism relative to divergence. These tests are most powerful to detect long-term (> 1 million years) selection in humans. Shared polymorphisms between species. If species divergence is not extremely re- cent and population sizes are not extremely large, we expect lineages to share their most recent common ancestor with other lineages from the same species. In the case of humans, the probability that two lineages do not coalesce before the split between humans and chimpanzees from their common ancestor, in the absence of selection, is on the order of 10−4 (Leffler et al, 2013). Considering the size of the human genome, one would still expect to ﬁnd 100 of such sites. However, by also requiring the same to occur for chimpanzees, this sharing is extremely unlikely for neutrally evolving sites, thus providing the basis for a test of neutrality (Klein et al, 1993). If a polymorphism is maintained by balancing selection for a sufficiently long time, it may be found in two sister species, such as human and chimpanzee. This test is appropriate to detect ancient balancing selection, operating at a deeper timescale than the divergence between humans and the sister species (e.g. 6 myr for humans and chimpanzees). BOX 3. Mechanisms of balancing selection BOX 3. Mechanisms of balancing selection Balancing selection is a term that encompasses a broad range of selective regimes, all of which generate high levels of adaptive genetic variation. An important challenge in the study of HLA genes is teasing apart which of these forms of selection is operating. Heterozygote advantage (or overdominance). Occurs whenever the ﬁtness of the heterozygous genotype is higher than that of both homozygous genotypes. Under this scenario, two or more alleles can be maintained indeﬁnitely in a population, eventually reaching a frequency equilibrium. Assuming genotype ﬁtnesses are constant through time and that marginal frequencies are the same, a polymorphic equilibrium can be achieved, with both alleles being kept in a population. This has been proposed as one (non-exclusive) biologically plausible mechanism through which variants are maintained in the HLA class I and class II classical loci because heterozygote individuals would be able to present a vaster repertoire of antigens than homozygote individuals (Doherty and Zinkernagel, 1975). Variable selection (over time and space). If selection coefficients (and hence the ﬁtnesses of the genotypes) vary over time or space, a population may maintain levels of polymorphism which are greater than those expected under neutrality. The observed levels of diversity may be similar to those expected under heterozygote advantage, even if the heterozygote is not constantly the ﬁttest genotype. Instead, the time or space-averaged ﬁtness of heterozygotes must be higher than that of homozygotes (Gillespie, 2004). Negative frequency-dependent selection. Under this selective regime the ﬁtness of a genotype is inversely proportional to its frequency in the population. This model is biologically plausible, since pathogens are likely to evolve escape mutations to the most common HLA alleles. This reduces these HLA alleles’ ﬁtness, and thus their frequency. Relaxation of selection upon the pathogen may make this variant once again capable of conferring resistance, causing it to rise in frequency, thus driving a cycle which results in the maintenance of a polymorphic state (Spurgin and Richardson, 2010). "Outlier" vs "model-based" approaches Commonly, summary statistics are calculated for genomic windows or bio- logically deﬁned entities (such as genes). There are two main strategies that the statistic can be used to assess if this genomic region conforms to neutral expectations. Simulation-based tests. Using simulations that assume neutral evolution, a null dis- tribution is generated for the test statistic. Empirical data that are extreme with respect to this distribution are considered to reject the null hypothesis of neutrality. 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https://openalex.org/W3011270104	https://researchonline.lshtm.ac.uk/id/eprint/4656293/1/A%20qualitative%20study%20of%20attitudes%20towards%2C%20typologies%2C%20and%20drivers%20of%20concurrent%20partnerships%20among%20people%20of%20black%20Caribbean.pdf	English	null	A qualitative study of attitudes towards, typologies, and drivers of concurrent partnerships among people of black Caribbean ethnicity in England and their implications for STI prevention	BMC public health	2,020	cc-by	10,987	A qualitative study of attitudes towards, typologies, and drivers of concurrent partnerships among people of black Caribbean ethnicity in England and their implications for STI prevention Sonali Wayal1,2,3* , Makeda Gerressu1,3, Peter Weatherburn3,4, Victoria Gilbart3, Gwenda Hughes1,2,3† and Catherine H. Mercer1,3† Sonali Wayal1,2,3* , Makeda Gerressu1,3, Peter Weatherburn3,4, Victoria Gilbart3, Gwenda Hughes1,2,3† and Catherine H. Mercer1,3† Abstract Background: Partner concurrency, (having sexual partnerships overlapping in time), especially when condoms are not used, can facilitate sexually transmitted infections (STI) transmission. In Britain, STI diagnoses rates and the reporting of concurrency are higher among black Caribbeans than other ethnic groups. We explored attitudes towards, drivers, characteristics, and contexts of concurrent partnerships, and their implications for STI risk among black Caribbeans in England. Methods: Purposive sampling, by sex and age-groups, was used to recruit participants (overall n = 59) from five sexual health clinics and community settings in London and Birmingham, England. Audio-recorded four focus group discussions (n = 28 participants), and in-depth interviews (n = 31) were conducted (June 2014–December 2015). Transcribed data were thematically analysed using Framework Analysis. Results: ‘Main plus’ and ‘non-main’ concurrency were identified in this population. Main plus concurrency involves an individual having a main partner with whom s/he has a “relationship” with, and the individual and/or their partner secretly or explicitly have other non-main partners. In contrast, non-main concurrency entails having multiple, non-committed partners overlapping in time, where concurrency is usually taken as a given, making disclosure to partners irrelevant. While main partnerships were usually long-term, non-main partnerships ranged in duration from a single event through to encounters lasting several months/years. Condomless sex was common with ex/long-term/married/cohabiting partners; whereas condoms were typically used with non-main partners. However, condom use declined with partnership duration and familiarity with partners. Awareness of partners’ concurrency facilitated condom use, STI-testing, and partner notification. While unresolved feelings, or sharing children with ex-partners, usually facilitated main plus concurrency; non-main concurrency was common among young, and single people. Gender norms, notions of masculinity, and sexual desires influenced concurrency. Black Caribbean popular music, social media, peer pressure, and relationship norms among black Caribbeans were also perceived to encourage concurrency, especially among men and young people. (Continued on next page) * Correspondence: sonali.wayal@developmentmedia.net †Gwenda Hughes and Catherine H. Mercer are joint senior authors. 1Centre for Population Research in Sexual Health and HIV, Institute for Global Health, University College London (UCL), London WC1E 6JB, UK 2HIV & STI Department, Public Health England, Centre for Infectious Disease Surveillance and Control (CIDSC), Public Health England, London NW9 5EQ, UK Full list of author information is available at the end of the article A qualitative study of attitudes towards, typologies, and drivers of concurrent partnerships among people of black Caribbean ethnicity in England and their implications for STI prevention Wayal et al. BMC Public Health (2020) 20:188 https://doi.org/10.1186/s12889-020-8168-0 Wayal et al. BMC Public Health (2020) 20:188 https://doi.org/10.1186/s12889-020-8168-0 Methods We conducted focus group discussions to explore atti- tudes towards and factors influencing concurrency among people of black Caribbean ethnicity, followed by one-to-one in-depth interviews to explore their personal experiences and their implications for sexual health choices and STI risk. People aged ≥15 years, who could read and speak English, and who identified as having a black Caribbean heritage were eligible to participate. The lower age restriction of 15 years was chosen because of the high prevalence of bacterial STIs among young black Caribbeans [19]. Due to variations in STI diagno- ses rates by sex and age, participants were purposively sampled by sex and age groups (15–24, 25–34, ≥35 years). Participants aged < 16 years were only recruited from sexual health clinics because of the greater likeli- hood of them being sexually active, minimising ethical concerns related to discussing sexual behaviour. Research on HIV in the USA has shown that variations in the prevalence of concurrency and sexual mixing pat- terns by race exacerbated racial disparity in the epidemic potential [10]. Variation in the duration of concurrent part- nerships also influences HIV transmission [11]. For ex- ample: long-term concurrent partnerships were associated with reduced HIV risk potentially due to the closed nature of the sexual networks [12]. Additionally, condom use in concurrent partnerships can influence STI transmission dy- namics. Studies have shown that partnership type (i.e. steady or non-steady) can influence condom use [13, 14]. A high proportion of steady male partners of women diag- nosed with Trichomonias vaginalis had concurrent partners and engaged in condomless sex with the women [15]. These data highlight the significance of understanding the nature and context of concurrent partnerships to better comprehend their implications for STI transmission [16]. Convenience sampling was used to recruit participants from five sexual health clinics and community settings such as colleges; with the help of community-based or- ganisations working with black Caribbean communities on a range of issues such as depression, unemployment, teenage pregnancy, youth groups, and condom promo- tion. Based on analysis of England’s STI surveillance data, four sexual health clinics in London and one in Birmingham were chosen because a high proportion of attendees at these clinics are of black Caribbean ethni- city. Background In Britain, since 2000 sexually transmitted infection (STI) surveillance and national probability survey data have shown that rates of bacterial STI diagnoses, for example: gonorrhoea, chlamydia, among people of black Caribbean ethnicity have remained higher than in people of other eth- nicities [1–4]. Socio-economic deprivation and individual- level behavioural risk factors do not fully explain these eth- nic inequalities in STIs [1, 4] suggesting the need to under- stand the role of sexual networks [4–6] in influencing STI risk. Sexual networks can influence whether STIs remain endemic within a population and explain inequalities in STI rates [5–7]. Specifically, patterns of partner concur- rency, defined as overlapping sexual partnerships in which sexual intercourse with one partner occurs between two acts of intercourse with another partner [8], can influence not only the size but also the speed of spread of STIs in a population [9]. (Continued from previous page) (Continued from previous page) Conclusions: Concurrency among black Caribbeans is shaped by a complex interaction between emotional/psychological, interpersonal, sociocultural, and structural factors. Concurrency type, its duration, and awareness influence sexual health choices, and thus STI risk in this population. Collecting these data during clinic consultations could facilitate offering partner notification methods tailored to concurrency type. Gender- and age-specific, culturally-sensitive interventions addressing STI risks associated with concurrency are needed. Keywords: Concurrency, Ethnicity, Sexually transmitted infection, Qualitative, Sexual behaviour in sexual health as part of England’s National Institute for Health Research’s Health Protection Research Unit in Blood Borne and Sexually Transmitted Infections [17, 18]. © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 14 Wayal et al. BMC Public Health (2020) 20:188 Results Four focus group discussions and 31 interviews were conducted from June 2014–December 2015 (Table 1). Altogether fifty-nine participants (n = 24 men) aged 15– 70 years participated in our study. All the in-depth inter- view participants identified as black Caribbean whereas some focus group discussion participants identified as being from a black British or mixed ethnic background. None of the study participants identified as being gay or bisexual. Typology and characteristics of concurrent partnerships Overall, two partnership types ‘main/regular’ and ‘non- main/casual’ were identified from the focus group discus- sions and the personal experiences shared by the interview participants. A main/regular partner is typically someone with whom a person has “a relationship”, an “emotional connection” to. These feelings are mutual, and as such, there is an expectation of exclusivity of sexual relationship. In contrast, a non-main/casual partnership entails being with a person primarily for “having sex”, “fun”, without any commitment or expectation of exclusivity. Two key typologies of concurrency identified were ‘main plus’ and ‘non-main’ concurrent partnerships: Piloted topic guides, informed by a systematic review [22], were used for the focus group discussions and in- terviews conducted during June 2014–December 2015. Female researchers with substantial qualitative research experience facilitated focus group discussions and con- ducted in-depth interviews. Each focus group discussion and in-depth interview lasted for approximately 60–90 and 50–60 min respectively and were audio-recorded. Participants were reimbursed for travel costs and re- ceived £20 as a token of appreciation. Audio recordings were transcribed verbatim and the- matically analysed using qualitative data analysis soft- ware NVivo 11 for Windows (QSR International Pty Ltd., Australia). We used Framework Analysis to induct- ively and thematically analyse data [23]. Firstly, two re- searchers familiarised themselves with a sub-set of transcripts and open-coded data using broad themes for key areas explored in the topic guides and developed sub-themes. These were revised based on discussion be- tween the researchers and an index of themes was cre- ated which was applied to the entire dataset. Subsequently, all the data coded for a topic of interest, for example, “personal experience of concurrency” were retrieved and coded to refine sub-themes that sum- marised participants’ common and divergent views and experiences related to concurrency. Anonymised quota- tions are used to illustrate the analysis. Methods Initial recruitment from community settings showed that most participants had no personal experi- ences of concurrency so subsequently in-depth interview participants were recruited only from sexual health clinics because clinic attendees are more likely to report STI risk behaviour, including concurrent partnerships than individuals in the general population [20]. In Britain, the prevalence of concurrency has been shown to be higher among black Caribbean than white British people [4]. Due to the dearth of data on concur- rency in the context of STIs other than HIV generally and specifically among people of black Caribbean ethnicity, we conducted a qualitative study to explore the nature of con- current partnerships among black Caribbeans in England. This study was undertaken as part of a larger study which sought to understand factors contributing to inequalities Posters with contact details of the researchers were used to advertise the study in the sexual health clinics, commu- nity-based organisations and on Twitter so potential par- ticipants could directly contact the researcher. Black Caribbean community oriented commercial enterprises Page 3 of 14 Wayal et al. BMC Public Health (2020) 20:188 Page 3 of 14 such as supermarkets, restaurants/take-away shops etc. were approached directly by researchers or targeted via Twitter (if they had a Twitter presence). Additionally, sex- ual health clinic and community-based organisation staff approached potential participants about the study, and passed their contact details to the research team. Subse- quently a member of the research team contacted them to determine eligibility. Eligible participants were invited to participate in an in-depth interview or a focus group dis- cussion in the city of their residence (no-one was recruited to participate in both), in a quiet place at either the par- ticipating sexual health clinic or the community-based or- ganisation office. All participants were given a Study Information Sheet. Written informed consent was ob- tained. Parental consent of participants aged < 16 years was not sought due of the confidential nature of sexual health clinic services; however, participants < 18 years old were informed of our duties and legal limitations to confi- dentiality [21]. purport that health inequalities arise from the inter- action between individuals’ characteristics and their soci- etal and ecological contexts which influence their behaviours [24, 25]. UCL Research Ethics Committee (Project ID: 6887/001) and National Research Ethics Ser- vice Committee of South Central-Oxford C (reference: 15/SC/0223) approved the study. Results We present quotes in the manuscript from a range of participants; however, sometimes quotes from the same participant are used more than once if their quote articulates the theme more clearly. We have also used terms and phrases quoted by individual participants where these express the sentiments of several participants for brevity so as not to disrupt the narrative’s flow. Our analysis draws on the ecosocial theoretical frameworks which Main plus concurrent partnerships BMC Public Health (2020) 20:188 Page 4 of 14 Participant 1: I suppose it does happen (having Focus group 1, young women aged 15-24 years, East Table 1 Characteristics of study participants Focus group discussion participants characteristics Number of focus group discussions Men Women Total Age group Ethnicity Area Employment/occupation status 1 0 5 5 15–24 3 Black British and 2 mixed ethnicity (black Caribbean & English; black Caribbean & Turkish) East London All students 2 3 8 11 15–24 All, except 2 participants who identified as black Caribbean/black British, others identified as black Caribbean North London All students 3 2 3 5 35–48 3 participants identified as black British and 2 as black Caribbean West London 2 were unemployed, 2 employed, 1 other (HIV+ and receiving benefits) 4 4 3 7 > 48 Except one participant who identified as black British, others identified as black Caribbean South London People living with mental health problems and were largely unemployed/voluntarily retired Total 9 19 28 – – – – In-depth interview participants characteristics Men Women All Age group 15–24 5 6 11 25–35 5 5 10 > 35 5 5 10 Recruitment site Sexual health clinics 10 12 22 Community settings 5 4 9 Location London 9 14 23 Birmingham 6 2 8 Total 15 16 31 Table 1 Characteristics of study participants Focus group discussion participants characteristics Number of focus group discussions Men Women Total Age group Ethnicity Area Employment/occupation status 1 0 5 5 15–24 3 Black British and 2 mixed ethnicity (black Caribbean & English; black Caribbean & Turkish) East London All students 2 3 8 11 15–24 All, except 2 participants who identified as black Caribbean/black British, others identified as black Caribbean North London All students 3 2 3 5 35–48 3 participants identified as black British and 2 as black Caribbean West London 2 were unemployed, 2 employed, 1 other (HIV+ and receiving benefits) 4 4 3 7 > 48 Except one participant who identified as black British, others identified as black Caribbean South London People living with mental health problems and were largely unemployed/voluntarily retired Total 9 19 28 – – – – In-depth interview participants characteristics Table 1 Characteristics of study participants Focus group discussion participants characteristics In-depth interview participants characteristics In depth interview participants characteristics Men Men Men Women All Age group 15–24 5 6 11 25–35 5 5 10 > 35 5 5 10 Recruitment site Sexual health clinics 10 12 22 Community settings 5 4 9 Location London 9 14 23 Birmingham 6 2 8 Total 15 16 31 Focus group 1, young women aged 15-24 years, East London Participant 1: I suppose it does happen (having different sexual partners at the same time), because there’s the thing that they’ve got a side chick and all that; like if they’re in a relationship then they’ll have another girl and another girl and another girl. Facilitator: Sorry, what did you mean by a side chick? Facilitator: Sorry, what did you mean by a side chick? Participant 1: You have like a main chick, side chick, mistress … Participant 2: yeah, you’ve got loads of faces. So if you’re in a relationship, you’ve got obviously the main girl that you’ve obviously got an emotional connection with, but then you’ve got something on the side, and then you’ve got something else on the side! Main plus concurrent partnerships However, the majority of participants described main plus concurrency to be “kind of wrong”. Some women felt that if they were to find out about their main male partner’s concurrency it would “make (you) think (you) are not good enough”, “make (you) question (your) womanhood”. Similarly, some men felt that awareness of their main female partner’s concur- rency would make them feel inadequate: “(you’d) start feeling like a wimp or (you’re) not manly enough”. Some men also felt that it could lead to violence with her concurrent partner, and potentially, relationship break-up. Facilitator: Sorry, what did you mean by a side chick? Main plus concurrent partnerships These were characterised by an individual having a main/regular sexual partner, and they and/or their part- ner having non-main/casual partner(s) concurrently. These partnerships could be: i) ‘closed from one-end’ where only one partner in the ‘main’ partnership has other non-main sex partner(s), which the main partners are either aware or unaware of, ii) ‘open-ended’ where both partners in a main partnership implicitly or expli- citly agree to have sex with non-main partners, either to- gether (i.e. threesomes/group sex) or individually. These partnerships were perceived to be more common among black Caribbean men than women. Women were more likely to be in ‘closed on their-end’ concurrent partner- ships. Main plus partnerships were usually of longer dur- ation or recurrent. Participants used terms such as “side chick”, “thot”, “jez” to refer to men’s non-main concur- rent female partners, and “side boy”, “side dick” to refer to women’s non-main concurrent partners which high- lights an awareness of existence of concurrency in this population: Wayal et al. Impact of concurrent partnership typologies on sexual health related behaviours Concurrency typology and its characteristics influenced sexual health choices and STI risk in several ways. Participant: In the summer time, I had one or two other partners. Concurrency typology and its characteristics influenced sexual health choices and STI risk in several ways. Interviewer: OK. And how old were they? Participant: One was 23/24 and one was 18/19. Interviewer: And you were seeing both around the same time? Participant: Yeah. Interviewer: And they were both women? Participant: Yeah. Interviewer: And what kind of partnership did you have with them? Participant: Sexual. Interviewer: So you didn’t have any emotional relationship with them in a way? Participant: Well, in a way, but not serious. Interviewer: And how long did your relationship with them last? Interviewer: OK. And how old were they? Participant: One was 23/24 and one was 18/19. Interviewer: And you were seeing both around the same time? Participant: Yeah. Interviewer: And they were both women? Participant: Yeah. Interviewer: And what kind of partnership did you have with them? Participant: Sexual. Interviewer: So you didn’t have any emotional relationship with them in a way? Participant: Well, in a way, but not serious. Interviewer: And how long did your relationship with them last? Interviewer: OK. And how old were they? Interviewer: OK. And how old were they? Interview 1, female aged >35 years, Birmingham Interview 1, female aged >35 years, Birmingham Some participants felt that in main plus partnerships, the awareness of a partner’s concurrency could lead to women - usually as the non-main partner - trying to get pregnant to “trap” their partner, thus leading to not using contraception nor condoms. In non-main partnerships, pregnancy prevention was usu- ally not discussed; however, some participants felt that in non-main concurrent partnerships, the “no strings attached” nature of these partnerships facilitated condom use: Interviewer: And did you use condoms with him? Participant: No because I was in love and I was stupid. Participant: No because I was in love and I was stupid. Non-main concurrent partnerships These were characterised as having multiple non-main partners overlapping in time. Several focus group and interview participants perceived such partnerships to be common among young people and single people regard- less of their age. However the importance of being hon- est about “if people/they (are) in an open relationship” Participant 3: It’s not just with guys as well, it could be girls. This is another thing, you can’t say it’s just like a side girl, there are side boys, you know? Participant 2: You get side men. Page 5 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 Participant: No, he wasn’t married, he was co- habiting. Participant: No, he wasn’t married, he was co- habiting. with sexual partners and to give the other person the “choice” to decide if they want to be in this situation was emphasised, although they also recognised that this may not necessarily happen. In contrast, some interview participants felt that disclosure of their concurrent partnerships to non-main partners was irrelevant be- cause they were “not in a relationship” with them nor answerable to them. Interviewer: Did any of your ex-partners’ (participant had concurrent partnerships of similar nature at dif- ferent points in her life with different partners) other partner find out that they were seeing you? Participant: Not to my knowledge. Participant: Not to my knowledge. Sometimes participants assumed that their non-main partners would know that their sexual partnership was not exclusive. Some focus group and interview participants felt that if a person is not in a “serious relationship” then having partners concurrently would be acceptable because they would “not be hurting anyone”. However, mismatch of ex- pectations about the nature of relationship between non- main partners, where one person may want to explore the potential of having a committed “emotional relationship”, whereas the other does not, was also highlighted. In terms of duration, such types of partnerships were long-term, involving regular or occasional meetings for sex, with participants describing these partners as “fuck buddies” or “friends with benefits”. In contrast, some were spontaneous, brief partnerships, involving one-off sex through to several weeks. Interviewer: And did you use condoms with him? Impact of concurrent partnership typologies on sexual health related behaviours Impact of concurrent partnership typologies on sexual health related behaviours Concurrency typology and its characteristics influenced sexual health choices and STI risk in several ways. Condom and contraception use Some participants in main plus concurrent partnerships did not usually use condoms with their long-term main part- ner, despite being aware of their partner’s concurrency: Interviewer: What type of partnership did you have with him? Participant: Always closed on my side. He was already with somebody else when I got with him, because when you’re young, you don’t care about stuff like that. So, yeah, he was obviously sleeping with somebody else and me at the same time. He was my first love. I really fell hard for him. I can honestly say I wasn’t thinking about it from a sexual point of view with regards to infection and diseases. Interviewer: How did you know that he had another partner? Participant: Because he did, that’s how I met him, he was a family friend, like he knew my dad. Interviewer: So was he married or was he seeing somebody? Wayal et al. BMC Public Health (2020) 20:188 Page 6 of 14 Participant: About two months or three months; I’m not too sure to be honest. Interviewer: And did you use condoms with these partners? Participant: Yes. Interviewer: Why did you use condoms with these partners? Participant: Because they weren’t my girlfriend. Or I didn’t make them my girlfriend. But that’s what I normally do when I don’t have a girlfriend, so … yeah. Interview 10, male age range 15-24 years, East London Participant: About two months or three months; I’m not too sure to be honest. Interviewer: And did you use condoms with these partners? Participant: Yes. Interviewer: Why did you use condoms with these partners? Participant: Because they weren’t my girlfriend. Or I didn’t make them my girlfriend. But that’s what I normally do when I don’t have a girlfriend, so … yeah. Interview 10, male age range 15-24 years, East London previously been diagnosed with an STI due to a part- ner “cheating” on them: previously been diagnosed with an STI due to a part- ner “cheating” on them: Interviewer: So why did you come to the clinic this time? Participant: This time I came because my partner stepped out and he says that he used protection, but I don’t believe that he did, because I know him! And obviously we’ve been having sex unprotected (because partner does not like using condoms). Interview 11, female age range 25-35 years, East London Facilitator: What do others feel about what she just said? Whereas participants who were/had been in non- main concurrent partnerships said that they tested regularly, usually at the end of a sexual relationship and/or at the beginning of a new partnership, some- times along with their new partner. Male participant 1: I’d do the same thing. If I was just sleeping with a person, then I would be more protective, but if I was in a relationship and then not With regards to notifying the main partner follow- ing STI diagnosis, fear of losing them if they were unaware of an individual’s concurrent partners was a barrier. However, some participants had done so due to concerns of re-infection. Partner notification some- times led to partners blaming each other for having concurrent partners. It also often led to them breaking-up, especially if the main partner was rela- tively new and there had been no prior awareness of the partner’s concurrency: Female participant 3: I think because as well you don’t know if they’re sharing sex with other people (murmurs of agreement) because from the get-go you only know it’s not a thing like that … Condom and contraception use So I’m just taking my precautions and going and checking it out just to make sure that everything is OK down there. Participant: Because they weren’t my girlfriend. Or I didn’t make them my girlfriend. But that’s what I normally do when I don’t have a girlfriend, so … yeah. Interviewer: So how did you know he’d stepped out? Interviewer: So how did you know he’d stepped out? Interview 10, male age range 15-24 years, East London Participant: The girl messaged me and told me about it, so that’s how I found out that they saw each other and that they had sex. So it’s been a bit of a tough one but, again, you’ve got to put your feelings aside to a degree and just focus on getting things sorted out. So that’s where I’m at right now. Female participant 1: I think a relationship is like more you don’t use the condom or whatever, but friends with benefit, because you’re not actually going out with the person, you’re just sleeping with the person or whatever, you use it more or whatever. Interview 11, female age range 25-35 years, East London Focus group 2, mixed sex participants’ age range 15-24 years, North London Some participants however also shared that the like- lihood of condom use with non-main partners de- clined with increasing partnership duration and familiarity between partners. Condoms were usually used with one-off partners, but inconsistently in the event of alcohol consumption, and dependant on the degree of sexual attraction towards the person. Interviewer: Have you ever asked anybody to go to a clinic when you’ve found out that you’ve had an infection? Participant: Yeah. Like every time I’ve ever had an infection, I’ve, like, just contact them, phone them up and say, “You know what, I’ve got something to tell you, basically I’ve got an infection and you need to go to the clinic.” Like before when it happened to me, like when that girl gave me something, I had a relationship and I split up through that. Like, she was like my ex- girlfriend and then obviously I slept with this regular girlfriend and then I had to tell my regular girlfriend STI testing and partner notification In main plus concurrent partnerships, awareness of partners’ concurrency - regardless of whether it was their main or non-main partner - usually led an indi- vidual to test for STIs, especially if they had Page 7 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 that I caught something off that one, and then that split up my relationship. Interview 7, male aged 25-35 years, Birmingham Participant: Oh, it’s different, innit, that’s why, because obviously you meet an individual partner and if you have sex with them, that sex is different to the other person that you’re having sex with or whatever. I suppose you get bored of one thing. Like say I was wearing this coat all the time, all the time, and I thought to myself, you know what, I want to feel different or I want a different design, just get a different design. Like, I’m getting bored of this now. Or like you’re driving a car, you have a car for a few years and then you’re like, oh, I feel like a change, buy a new car, like that. In some situations, developing symptoms had prompted individuals to test for STIs, and confront their partner about sexual concurrency in the event of STI diagnosis. A few participants also described being diagnosed with an STI after they had had sex with a main partner who lives abroad and/or new non-main partners they met whilst abroad, mainly in the Caribbean. They had usually in- formed the main partner of the STI diagnosis; however notifying non-main partners was challenging due to lim- ited, if any, contact details. Unwillingness to see the part- ner again and perceptions that they were the source of infection also hindered notification of non-main partners. But, long-term non-main partners were sometimes noti- fied to avoid the risk of re-infection. Interview 7, male age range 25-35 years, Birmingham Interview 7, male age range 25-35 years, Birmingham Where participants had notified their partner, some had told their partner to “go to the clinic” as opposed to informing them about their STI diagnosis, which some- times led to re-infection if their partner had then not done so: Male participant 1: You know, sometimes a man might have a woman for a long time and she’s not giving him what he wants, so he goes astray, he goes and looks for somebody outside. And then he’s still with you, he still loves you and he comes back with this disease! Participant: I said, “I’ve been to the clinic today and I’m not sure what it is yet, but they gave me tablets and I think you probably should go and have a check-up.” And that was it. But I think it’s because, I don’t know if she did go and got tested or not from the last time, and because I’ve been to Sweden and came back, and she is the only person that I have had intercourse with since I’ve been back, and I’ve got it again, so I think that it might be from the last time. And a couple of days before I got tested, I had had sex with her. Male participant 2: Yes, but it’s not only the men, women do it as well! I think women get fed up of men more quicker than a man getting fed up of a woman. Focus group 4, mixed sex participants’ aged >45 years, South London Focus group 4, mixed sex participants’ aged >45 years, South London Some female participants who reported having concurrent partnerships in the past due to lust for sex and variety had changed their behaviour after entering a committed relation- ship, especially after having children. Interview 31, male age range 25-35 years, West London “Biological thing” Lack of fulfilment of sexual needs by one sexual partner, need for “variety”, “lust” and it being a “biological thing” were often cited as drivers of concurrency, especially among men. This was further fuelled by perceived no- tions of “West Indian” mens’ masculinity: Interviewer: How long had you been together in this regular relationship? Participant: Not long, you know, about eight months. Participant: Not long, you know, about eight months. Participant: Not long, you know, about eight months. Interviewer: What makes you seek out others (partners) when you’ve got a regular partner? Interview 7, male aged 25-35 years, Birmingham Interviewer: How long had you been together in this regular relationship? Participant: Not long, you know, about eight months. Past relationships Both men and women who were/had been in main plus con- current partnerships felt that meeting an ex-partner for shared childcare responsibilities was one of the most com- mon contexts in which such partnerships occur. A complex interaction between the individual-level, rela- tionship/interpersonal, community-level and societal factors (Fig. 1) influenced attitudes towards, and experi- ences of, concurrency among black Caribbeans. Focus group and interview data reflected that most factors in- fluence both types of concurrency, although some fac- tors facilitated specific type(s) of concurrency. Participant: Just going back round there, chilling and then gathering our feelings again, innit, and then stay Wayal et al. BMC Public Health (2020) 20:188 Page 8 of 14 Fig. 1 Ecosocial model of factors influencing concurrent partnerships among black Caribbeans in England Fig. 1 Ecosocial model of factors influencing concurrent partnerships among black Caribbeans in England there and then next we just have sex. It became more casual kind of thing. particularly happy about, but you go through things and you learn about why it’s not good for you. At the end of the day, if you’re not with someone you might as well just wait and just work on you, rather than to be giving yourself and tiring out yourself for other people. Because sex is not just sex, it’s a connection. Interview 7, male age range 25-35 years, Birmingham Participants, especially women, felt that lack of self- esteem and self-confidence about finding another partner left them feeling lonely and vulnerable; like “spoiled goods”, which led them to continue to have sex with their ex- partner with whom they share a child. “Unresolved feelings” for an ex-partner (with whom a person does not necessarily share a child) was another reason cited for being in a main plus concurrent partnership. Some female participants, who were usually the non-main partner and aware of their partner’s main sex partner, cited “being young”, “being in love” as reasons for being in main plus concurrency. Interview 11, female age range 25-35 years, East London Interview 11, female age range 25-35 years, East London Interview 11, female age range 25-35 years, East London Mass media Parental/observed norms of intimate relationships Some participants felt that some people engage in concur- rency because it is a “learnt behaviour”. They “think that’s the norm” because they have seen their parent(s) or other family members do so and consciously or subconsciously they engage in similar relationships. Teenage pregnancies, single-parent households, and unmarried, non-cohabiting parents with both or either parent having other partner(s) were perceived to be common and enhance the practice of concurrency. Several participants had grown up in a single- parent family (usually women-led) with minimal to regular presence of a father and some with “an absent father”, i.e., he was never around when they were growing up. Participants felt that this influences young people’s attitudes towards sex, relationships, notions of family because they think that if their parent could do it, so could they: Several participants felt that mass media plays a piv- otal role in shaping, maintaining and facilitating cul- tural notions of masculinity, gendered norms of sex and sexuality which in turn influence attitudes to- wards concurrency. Some participants, especially younger ones, felt that glorification of partner concur- rency via music that is popular among the black com- munity, and via celebrity culture plays a role in normalising concurrency and even celebrating it. Some participants also felt that social media platforms such as Twitter are used, especially by men to ap- plaud and encourage other men and sometimes to name and shame women perceived to have multiple partners. The changing norms and attitudes towards sex, facilitated by the ease of meeting sex partners online were also perceived to promote concurrency. Participant: My mum’s partner lived with us. My dad was a fly-by-night type of guy; dad always lived by himself. He always had his own house and then who- ever he was co-habiting with at the time, he’d be there for that period of time, but then if that went to pot, he would have somewhere to go back to. Participant: I just think it’s the culture difference. So even, like, just down to the music that comes out of the Caribbean, it’s all about … well, not all, but a lot of it is about being sexually promiscuous! Peer pressure Both men and women talked about peer pressure to initi- ate sexual activity when young. Men also mentioned peer pressure to have multiple sex partners as a factor influen- cing their concurrent partnerships. This was influenced by cultural notions of black Caribbean men’s masculinity and media as described below. However, among young people and single people, non-main concurrency was common due to their pursuit for an appropriate long-term partner. Although not commonly reported, previous failed in- timate relationships facilitated non-main concurrent partnerships: Participant: I think it was peer pressure because if I was to turn back the hands of time, I don’t think I would have, but I was in a situation where it was just me and that other person, and they were like, “Oh, come on, come on!” So I think it was peer pressure, but I can’t put the blame on them, I played a part in it as well. Participant: There was one time in my life and that was around the time that I had broken up with my daughter’s dad, that I had my casual partners. I was heartbroken so I was just like, something’s got to fill the void, which it didn’t and obviously that’s a life lesson. And it’s something that I’m not very Page 9 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 Interview 9, female age range 15-24 years, East London any grudges with him because, at the end of the day, people like to blame their papas, but for me that’s not really the case. Relationships grow, they work and they fail, and all you can do is thank them for trying their best, if they try their best, and try to understand the situation. Erm. I don’t know, I’ve just never been interested in being promiscuous like that, just having lots of different girlfriends at one time, it doesn’t appeal to me. Participant: It’s almost encouraged amongst young Black men by other young Black men, and older Black men, to be promiscuous, so I think that might be a reason. Interview 3, male age range 25-35 years, Birmingham Mass media And I think even from a base level, things like that can subconsciously affect the minds of some people and they’ll think, OK, they’re doing it or celebrities are doing it, that’s what I can do as well, and it’s something I should be doing. Interviewer: And how did you feel about your parents’ relationships? Participant: It was what it was, I didn’t know any different. So you’re learning from them without even knowing that you’re learning and they’re teaching you without them knowing that they’re teaching you. Interview 3, male age range 25-35 years, Birmingham Interview 25, male aged >35 years, South London Interview 3, male age range 25-35 years, Birmingham Participant 2: I think it’s (concurrent partnerships) always been there. Participant 2: I think it’s (concurrent partnerships) always been there. Interview 1, female aged >35 years, Birmingham But not all participants who had experienced similar situations growing up felt the same: Participant 3: It’s always been there but I think now it’s almost glorified … … it’s like it’s not just social media, now it’s music as well. Like nearly every new kind of R&B or rap song that comes out, they are always talking about, OK, I’ve got my side bitch and I’ve this one and I’ve got that one (murmur of agreement) and, oh, my god, I’m so good, look at all Participant: Growing up with my mum, my mum was there; she said that my dad was there, my biological dad was there, but I don’t remember, I’m too young to remember. I met him again at 15; I don’t really hold Page 10 of 14 Page 10 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 my chains! And boys look at that and think I need to be that; that’s what girls want! But we don’t! Interviewer: And where does that come from do you think? Interviewer: And where does that come from do you think? Interview 25, male aged >60 years, South London Female participant 1: They’d look at her as a slut and say, “My god, how can you do that!?” Concurrency was perceived to be a common practice among men; however it was felt that increasingly women too are engaging in concurrent partnerships. Partici- pants, especially females, highlighted the “double stan- dards” in attitudes towards men and women who have concurrent (and multiple) sexual partners. Unlike men, women were at a greater risk of stigma which was reflected in terms such as “ho” and “slag” being com- monly used to describe women who engage in concur- rency. Participants felt that concurrency is “not acceptable, but men get away with it” due to the “trad- itional” roles that are associated with being a woman or a man. Male participant 4: Yeah, they’d put on her and call her a slut, all of these things, you know? All the loose words, you know? A woman get put down because of what she’s done, you know? (murmurs of agreement) Interview 7, male aged 25-35 years, Birmingham Participant: The West Indian men that I know, a lot of them have got a very strong sexual drive and it pushes them to go that way, because sometimes their wife or girlfriend alone doesn’t sexually satisfy them. So they go somewhere else. Because I used to do that. It’s lust too. You see a nice girl walking down the street and you go, oh! Yes, I won’t lie about it. I used to have a lot of different girlfriends at one stage. Female participant 3: We are called all names under the sun. Yeah, it’s not acceptable for a woman to carry herself like that, for some unknown reason; I have no idea why. Female participant 2: I think people don’t think much of women who have multiple men. Focus group 1, young women aged 15-24 years, East London Participant: Well, I think it’s died out now, because men get called dogs or whatever, but where do I think it’s come from? Don’t know, it’s come through traditional whatever. I couldn’t tell you really where it’s come from … Can’t think of a word, but that’s what women are perceived as, with more than one partner; for a man, it’s probably not looking that bad, but for a woman it is, for some reason. Notions of masculinity and gender Notions of masculinity and gender Most men and some women felt that the perceived no- tions of “West Indian” men as “promiscuous” facilitates concurrency. Some men also felt that women, particu- larly from non-black ethnicities prefer black men as sex- ual partners due to perceptions of their greater “sexual prowess”. Discussions about “notches on bedposts” among peers made some men feel that having multiple partners is “something to be proud of” and encouraged them to do so. Interviewer: And do you think that as well, generally what people are thinking? Participant: Yeah, I kind of do, yeah, in a way. I don’t know. A little bit. Interview 7, male aged 25-35 years, Birmingham Interview 7, male aged 25-35 years, Birmingham Discussion Our study conducted among black Caribbeans in Eng- land highlights that two key concurrency typologies exist among black Caribbeans, main plus and non-main, and their characteristics, especially awareness of partner con- currency and duration, influence sexual health choices and thus STI risk. Our results also highlight the range of emotional/psychological, interpersonal, sociocultural, and structural factors that can interact and shape the context in which concurrency occurs. In the following sections, we discuss the similarities and differences of our research findings in comparison to other research studies, and the implications of our study findings for clinical practice, research and policy. Interviewer: And what about women having more than one partner at the same time? Participant: For women, I don’t know if it’s a bit different or not, but the stigma of it these days is calling them slags, innit, like if a woman has more than one, it makes them look bad a bit; I don’t know why. Interviewer: Who calls them that? Participant: Men. Or women. Interviewer: Who calls them that? Participant: Men. Or women. Participant: Men. Or women. Page 11 of 14 Page 11 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 in our study in the context of the need to satisfy sexual de- sires, and as a result of men’s biological needs [33, 34]. Moreover young people, especially men, were more likely to report peer pressure to have multiple sexual partners due to norms of black Caribbean men’s masculinity, per- petuated by social media; whereas psychosocial aspects usually influenced women’s decisions to be in concurrent partnerships. Perceptions of greater sexual prowess of black Caribbean men, particularly among women of non- black Caribbean ethnicity was mentioned by men in our interviews. These concur with the high prevalence of ethnic-mixing among black Caribbean men reported in a parallel quantitative study, although ethnic-mixing did not explain the high prevalence of STIs among these men compared to white British men [35]. The gendered double-standards towards concurrency reflected in the perceived greater tolerance of black Caribbean men - ra- ther than women - having concurrent partnerships, tallies with greater prevalence of concurrency among black Caribbean men [4]. Modelling partnership patterns ob- served in one’s family/community [27, 34] appears to be one of the key factors influencing engaging in main plus concurrency among black Caribbeans. Discussion Sociological re- search is needed to understand the implications of familial structures on the sexual behaviour and thus sexual health of black Caribbeans. Our findings also highlight that fu- ture epidemiological studies should examine the preva- lence of different types and characteristics of concurrency as they present varying levels of STI risk [13] and should assess the strength of their association with STI risk. Implications for policy f Current UK safer sex guidelines [36] recommend retest- ing for asymptomatic STIs to all individuals with a prior STI diagnosis including HIV. Given the complex range of factors that influence and sustain concurrent partner- ships, enhancing STI risk, anyone in a concurrent part- nership who is diagnosed with STI(s) should be retested. These guidelines also recommend screening for asymp- tomatic STIs at least annually (and in some cases as fre- quently as every 3 months) to all individuals at risk of acquisition or transmission of HIV. Although the risk of HIV is low among people of black Caribbean ethnicity, given the high STI burden among them, STI testing at least annually should be encouraged, especially among young black Caribbeans reporting concurrent partnerships. Young participants were more likely than the older participants to report experiencing non-main concurrent partnerships. Younger people of all ethnicities generally are more likely to report other STI risk behaviours such as larger partner numbers than older people [32]. Add- itionally, young black Caribbeans are more likely than other ethnic groups to report early sexual debut [4]. This in part may explain the disproportionate STI burden ex- perienced by young black Carribeans in England [2, 19]. Therefore promoting frequent STI testing among young people, especially young black Caribbeans is vital. Our data also suggest that gender-sensitive and age- specific, multi-faceted [37] interventions among black Ca- ribbeans should be developed to address STI risks associ- ated with different concurrency types, and psychosocial vulnerabilities that lead to maintenance of some concurrent partnerships. Given that STI diagnoses rates among black Caribbeans attending sexual health clinics in England are Implications for clinical practice p p Similar to another study we found that one or both part- ners in a sexual relationship may have concurrent part- ners [26], which has implications for the spread of STIs if either partner is infected [9]. However, black Carib- bean women commonly-described being in ‘closed- from-one-end’ main plus concurrent partnerships, im- plying that they are in a ‘passive’ concurrency, thus their STI risk is determined by their partner’s concurrent partnerships [27]. STI risk is likely to be especially heightened in the context of long-term and recurrent main plus concurrency due to an increased likelihood of condomless sex. Additionally often the lack of awareness of partner concurrency could hinder partner notification, which potentially explains the high rates of repeat STI diagnoses among black Caribbeans [28]. Conversely, ir- respective of typology, perceived or actual awareness of partners’ concurrency facilitated condom use [14], STI testing, and partner notification. However, our data also suggest that condom use could change with changes in partnership status or increasing familiarity over time [29] and a mismatch of partnership expectations could also influence condom use and thus STI risk [27]. Because people may or may not be aware of their part- ner’s concurrency, which has been shown to be an inde- pendent predictor of STI risk [30, 31], regular STI testing and partner notification among black Caribbeans in concurrent partnerships should be promoted to pre- vent re-infections, and onward STI transmission. Col- lecting information from index patients in concurrent partnerships about the nature of the partnership(s) and their perceptions about their sex partners’ awareness of their concurrency could facilitate the process of offering partner notification by using methods that take account of concurrency type to facilitate case-finding. For ex- ample, using provider-led rather than patient-led partner notification if the index patient wants to remain an- onymous in the event that their partner(s) is unaware of their concurrency. Funding g The research and SW, VG, EG, LS are funded by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Blood Borne and Sexually Transmitted Infections at UCL in partnership with Public Health England (PHE) and in collaboration with the London School of Hygiene and Tropical Medicine. The study design, data collection and analysis, interpretation of the study data and views expressed in this publication are those of the author(s) and not necessarily those of the National Institute for Health Research, the Department of Health, or Public Health England. Finally, our interview data are not at a partnership- level that is, we did not interview all partners in a sexual relationship. We are therefore unable to confirm partici- pants’ reports of their partners’ characteristics and be- haviours. Perceptions of partners’ concurrency or lack of Implications for research Similar to studies conducted among African Americans in the USA, concurrency was often justified by participants Page 12 of 14 Wayal et al. BMC Public Health (2020) 20:188 Wayal et al. BMC Public Health (2020) 20:188 Page 12 of 14 it, and its implications for sexual health choices there- fore should be interpreted with caution. high [2], they provide a setting to offer interventions target- ing black Caribbeans at STI risk. Interactive digital inter- ventions are an effective means for promoting sexual health knowledge [38] and so could be used to offer STI risk- reduction interventions tailored to black Caribbeans report- ing self or partner concurrency. Conclusions Overall, our findings suggest that concurrency type, its duration, and awareness influence sexual health choices, and thus STI risk among black Caribbeans. Collecting these data during clinic consultations could facilitate of- fering appropriate tailored interventions to black Carib- beans in concurrent partnerships. However, such interventions should factor in challenges posed by its role in enhancing sexual pleasure, and its social and structural determinants. Our findings should be interpreted in the context of the following limitations. Unlike some USA studies, neither economic dependence nor the sex ratio [13, 39] were men- tioned in our study as driving factors for concurrency, highlighting the significance of qualitative studies in under- standing context-specific factors that influence behaviours. Nonetheless this could be an anomaly of our sampling strategy, and we recognise that the concurrency typologies described in our study may not be generalisable to the black Caribbean population across England. Nevertheless, we re- cruited our sample from areas in England with high con- centrations of black Caribbeans and included men and women of different ages. Furthermore, by sampling partici- pants from sexual health clinics and community-based or- ganisations, we achieved a diverse group of black Caribbeans. We conducted all, except one, focus group dis- cussions with mixed-sex groups, to facilitate an open dis- cussion on social norms and practices related to concurrency among this ethnic group and to understand its gendered patterns. These were followed by one-to-one in- terviews, separately with men and women. Abbreviations PR l h P HPRU: Health Protection Research Unit; STI: Sexually transmitted infections; UCL: University College London Acknowledgements W h k ll h d We thank all the study participants and staff of the sexual health clinics and community-based organisations involved in this study. We acknowledge the members of the NIHR HPRU in Blood Borne and Sexually Transmitted Infections Steering Committee: Caroline Sabin (Director), Anthony Nardone (PHE Lead), Catherine Mercer, Gwenda Hughes, Greta Rait, Jackie Cassell, William Rosenberg, Tim Rhodes, Kholoud Porter, Sema Mandal, and Samreen Ijaz and the members of Theme-A of the NIHR HPRU in Blood Borne and Sexually Transmitted Infections Steering Committee: Catherine Mercer, Gwenda Hughes, Hamish Mohammed, Jackie Cassell, Fiona Burns, Makeda Gerressu, Jonathan Elford, David Phillips, Gary Brook, Nicola Low, Anthony Nardone and Paul Crook. We would also like to thank Dr. Emma Garnett for her help with conducting the focus group discussions and initial coding of the focus group discussions data, and Ms. Lorna Sutcliffe for her help with coding the data. The gendered attitudes towards concurrency may have influenced reporting of concurrent partnerships. More- over, the interviewers were mindful of the impact of stigma associated with concurrency and participants’ con- cerns about its discussion in the context of their ethnicity and STIs (especially as both interviewers were of non- black Caribbean ethnicity: one was white and one from another minority ethnic background). In an attempt to minimise the impact of their characteristics on partici- pants’ reporting of sexual behaviours, prior to recruiting participants, interviewers explained the epidemiological evidence of high STI prevalence among black Caribbeans in England and the scientific importance of the informa- tion disclosed during interviews/discussions by partici- pants for gaining a greater understanding of the factors influencing these STI trends. In this respect, it is worth noting the similarities in some of the themes identified by our study with previous studies on concurrency con- ducted in the UK which had ethnicity-matched inter- viewers [27] and from the USA where sex- and ethnicity- matched interviewers were used [34] suggesting that the information elicited from our study participants is reliable. Authors’ contributions SW d i d h d SW designed the study and secured ethics, and R&D permissions in NHS study sites with support from GH and CM. CM and GH contributed to the conception and design of the study. SW conducted all the focus group discussions. SW and VG conducted in-depth interviews. SW coded and the- matically analysed the data, conceived the paper, and wrote the manuscript. All co-authors contributed to the drafting of the manuscript, and read and approved the manuscript, including authors MG and PW. CM and GH se- cured funding from the National Institute for Health Research for the Health Protection Research Unit (NIHR HPRU) in Blood Borne and Sexually Transmit- ted Infections at University College London in partnership with Public Health England (PHE), in collaboration with London School of Hygiene & Tropical Medicine (LSHTM). All authors read and approved the final manuscript. References F Garnett GP, Hughes JP, Anderson RM, Stoner BP, Aral SO, Whittington WL, Handsfield HH, Holmes KK. Sexual mixing patterns of patients attending sexually transmitted diseases clinics. Sex Transm Dis. 1996;23(3):248–57. 29. 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https://openalex.org/W2947128067	https://www.research-collection.ethz.ch/bitstream/20.500.11850/313361/2/PhysRevD.98.112005.pdf	English	null	Measurement of the differential cross sections for <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:mi>W</mml:mi></mml:mrow></mml:math> -boson production in association with jets in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:mi>p</mml:mi><mml:mover accent="true"><mml:mrow><mml:mi>p</mml:mi></mml:mrow><mml:mrow><mml:mo accent="true" stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:mrow></mml:math> …	Physical review. D/Physical review. D.	2,018	cc-by	19,920	ETH Library Measurement of the differential cross sections for W-boson production in association with jets in pp¯ collisions at √s=1.96 TeV Author(s): CDF Collaboration; Aaltonen, Timo A.; Casal, Bruno; Wallny, Rainer ; et al. Originally published in: Physical Review D 98(11), https://doi.org/10.1103/PhysRevD.98.112005 Originally published in: This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. PHYSICAL REVIEW D 98, 112005 (2018) Measurement of the differential cross sections for W-boson production in association with jets in p¯p collisions at ﬃﬃs p = 1.96 TeV Takemasa,49 Y. Takeuchi,49 J. Tang,11 M. Tecchio,31 P. K. Teng,1 J. Thom,15,f E. Thomson,40 V. Thukral,47 D. Toback,47 S. Tokar,12 K. Tollefson,32 T. Tomura,49 D. Tonelli,15,e S. Torre,17 D. Torretta,15 P. Totaro,39a M. Trovato,41d F. Ukegawa,49 S. Uozumi,25 F Vázquez 16,l G Velev 15 C Vellidis 15 C Vernieri 41d M Vidal 43 R Vilar 9 J Vizán 9,dd M Vogel 34 G Volpi 17 P Wagner 40 y g S. Wilbur,7 H. H. Williams,40 J. S. Wilson,31 P. Wilson,15 B. L. Winer,35 P. Wittich,15,f S. Wolbers,15 H. Wolfmeister,35 T. Wright,31 X. Wu,18 Z. Wu,5 K. Yamamoto,37 D. Yamato,37 T. Yang,15 U. K. Yang,25 Y. C. Yang,25 W.-M. Yao,26 G. P. Yeh,15 K. Yi,15,m J. Yoh,15 K. Yorita,51 T. Yoshida,37,k G. B. Yu,25 I. Yu,25 A. M. Zanetti,48a 14 h 14 d h lli6b (CDF Collaboration) 112005-1 2470-0010=2018=98(11)=112005(22) Published by the American Physical Society T. Aaltonen,21 S. Amerio,39b D. Amidei,31 A. Anastassov,15,w A. Annovi,17 J. Antos,12 G. Apollinari,15 J. A. Appel,15 T. Arisawa,51 A. Artikov,13 J. Asaadi,47 W. Ashmanskas,15 B. Auerbach,2 A. Aurisano,47 F. Azfar,38 W. Badgett,15 T. Bae,25 A. Barbaro-Galtieri,26 V. E. Barnes,43 B. A. Barnett,23 P. Barria,41c P. Bartos,12 M. Bauce,39b F. Bedeschi,41a S. Behari,15 G. Bellettini,41b J. Bellinger,53 D. Benjamin,14 A. Beretvas,15 A. Bhatti,45 K. R. Bland,5 B. Blumenfeld,23 A. Bocci,14 A. Bodek,44 D. Bortoletto,43 J. Boudreau,42 A. Boveia,11 L. Brigliadori,6b C. Bromberg,32 E. Brucken,21 J. Budagov,13 H. S. Budd,44 K. Burkett,15 G. Busetto,39b P. Bussey,19 P. Butti,41b A. Buzatu,19 A. Calamba,10 S. Camarda,4 M. Campanelli,28 F. Canelli,11,ee B. Carls,22 D. Carlsmith,53 R. Carosi,41a S. Carrillo,16,l B. Casal,9,j M. Casarsa,48a A. Castro,6b P. Catastini,20 D. Cauz,48b,48c V. Cavaliere,22 A. Cerri,26,e L. Cerrito,28,r Y. C. Chen,1 M. Chertok,7 G. Chiarelli,41a G. Chlachidze,15 K. Cho,25 D. Chokheli,13 A. Clark,18 C. Clarke,52 M. E. Convery,15 J. Conway,7 M. Corbo,15,z M. Cordelli,17 C. A. Cox,7 D. J. Cox,7 M. Cremonesi,41a D. Cruz,47 J. Cuevas,9,y R. Culbertson,15 N. d’Ascenzo,15,v M. Datta,15,hh P. de Barbaro,44 L. Demortier,45 M. Deninno,6a M. D’Errico,39b F. Devoto,21 A. Di Canto,41b B. Di Ruzza,15,p J. R. Dittmann,5 S. Donati,41b M. D’Onofrio,27 M. Dorigo,48d A. Driutti,48b,48c K. Ebina,51 R. Edgar,31 A. Elagin,11 R. Erbacher,7 S. Errede,22 B. Esham,22 S. Farrington,38 J. P. Fernández Ramos,29 R. Field,16 G. Flanagan,15,t R. Forrest,7 M. Franklin,20 J. C. Freeman,15 H. Frisch,11 Y. Funakoshi,51 C. Galloni,41b A. F. Garfinkel,43 P. Garosi,41c H. Gerberich,22 E. Gerchtein,15 S. Giagu,46a V. Giakoumopoulou,3 K. Gibson,42 C. M. Ginsburg,15 N. Giokaris,3,* P. Giromini,17 V. Glagolev,13 D. Glenzinski,15 M. Gold,34 D. Goldin,47 A. Golossanov,15 G. Gomez,9 G. Gomez-Ceballos,30 M. Goncharov,30 O. González López,29 I. Gorelov,34 A. T. Goshaw,14 K. Goulianos,45 E. Gramellini,6a C. Grosso-Pilcher,11 J. Guimaraes da Costa,20 S. R. Hahn,15 J. Y. Han,44 F. Happacher,17 K. Hara,49 M. Hare,50 R. F. Harr,52 T. Harrington-Taber,15,m K. Hatakeyama,5 C. Hays,38 J. Heinrich,40 M. Herndon,53 A. Hocker,15 Z. Hong,47,w W. Hopkins,15,f S. Hou,1 R. E. Hughes,35 U. Husemann,54 M. Hussein,32,cc J. Huston,32 G. Introzzi,41e,41f M. Iori,46b A. Ivanov,7,o E. James,15 D. Jang,10 B. Jayatilaka,15 E. J. Jeon,25 S. Jindariani,15 M. Jones,43 K. K. Joo,25 S. Y. Jun,10 T. R. Junk,15 M. Kambeitz,24 T. Kamon,25,47 P. E. Karchin,52 A. Kasmi,5 Y. Kato,37,n W. Ketchum,11,ii J. Keung,40 B. Kilminster,15,ee D. H. Kim,25 H. S. Kim,15,bb J. E. Kim,25 M. J. Kim,17 S. H. Kim,49 S. B. Kim,25 Y. J. Kim,25 Y. K. Kim,11 51 15 51 * 25 16 14 24 40 14 E. Gramellini, C. Grosso Pilcher, J. Guimaraes da Costa, S. R. Hahn, J. Y. Han, F. Happacher, K. Hara, M. Hare, R. F. Harr,52 T. Harrington-Taber,15,m K. Hatakeyama,5 C. Hays,38 J. Heinrich,40 M. Herndon,53 A. Hocker,15 Z. Hong,47,w W. Hopkins,15,f S. Hou,1 R. E. Hughes,35 U. Husemann,54 M. Hussein,32,cc J. Huston,32 G. Introzzi,41e,41f M. Iori,46b A. Ivanov,7,o E. James,15 D. Jang,10 B. Jayatilaka,15 E. J. Jeon,25 S. Jindariani,15 M. Jones,43 K. K. Joo,25 S. Y. Jun,10 T. R. Junk,15 M. Kambeitz,24 T. Kamon,25,47 P. E. Karchin,52 A. Kasmi,5 Y. Kato,37,n W. Ketchum,11,ii J. Keung,40 15 ee 25 15 bb 25 17 49 25 25 11 Published by the American Physical Society Measurement of the differential cross sections for W-boson production in association with jets in p¯p collisions at ﬃﬃs p = 1.96 TeV pp R. F. Harr,52 T. Harrington-Taber,15,m K. Hatakeyama,5 C. Hays,38 J. Heinrich,40 M. Herndon,53 A. Hocker,15 Z. Hong,47,w W. Hopkins,15,f S. Hou,1 R. E. Hughes,35 U. Husemann,54 M. Hussein,32,cc J. Huston,32 G. Introzzi,41e,41f M. Iori,46b A. Ivanov,7,o E. James,15 D. Jang,10 B. Jayatilaka,15 E. J. Jeon,25 S. Jindariani,15 M. Jones,43 K. K. Joo,25 S. Y. Jun,10 T. R. Junk,15 M. Kambeitz,24 T. Kamon,25,47 P. E. Karchin,52 A. Kasmi,5 Y. Kato,37,n W. Ketchum,11,ii J. Keung,40 15 25 15 bb 25 17 49 25 25 11 N. Kimura,51 M. Kirby,15 K. Kondo,51,* D. J. Kong,25 J. Konigsberg,16 A. V. Kotwal,14 M. Kreps,24 J. Kroll,40 M. Kruse,14 24 49 43 15 28 35 41 25 25 S. Leo,22 S. Leone,41a J. D. Lewis,15 A. Limosani,14,s E. Lipeles,40 A. Lister,18,a Q. Liu,43 T. Liu,15 S. Lockwitz,54 A. Loginov,54 D. Lucchesi,39b A. Luc`a,17,15 J. Lueck,24 P. Lujan,26 P. Lukens,15 G. Lungu,45 J. Lys,26,* R. Lysak,12,d R. Madrak,15 P. Maestro,41c S. Malik,45 G. Manca,27,b A. Manousakis-Katsikakis,3 L. Marchese,6a,jj F. Margaroli,46a P. Marino,41d , , , , , g , , K. Matera,22 M. E. Mattson,52 A. Mazzacane,15 P. Mazzanti,6a R. McNulty,27,i A. Mehta,27 P. Mehtala,21 C. Mesropian,45 T Miao 15 E Michielin 39b D Mietlicki 31 A Mitra 1 H Miyake 49 S Moed 15 N Moggi 6a C S Moon 25 R Moore 15,ff,gg M. J. Morello,41d A. Mukherjee,15 Th. Muller,24 P. Murat,15 M. Mussini,6b J. Nachtman,15,m Y. Nagai,49 J. Naganoma,51 I. Nakano,36 A. Napier,50 J. Nett,47 T. Nigmanov,42 L. Nodulman,2 S. Y. Noh,25 O. Norniella,22 L. Oakes,38 S. H. Oh,14 , p , , g , , , , , , Y. D. Oh,25 T. Okusawa,37 R. Orava,21 L. Ortolan,4 C. Pagliarone,48a E. Palencia,9,e P. Palni,34 V. Papadimitriou,15 W. Parker,53 48b 48 10 30 14 15 40 22 8 G. Pauletta, M. Paulini, C. Paus, T. J. Phillips, G. Piacentino, E. Pianori, J. Pilot, K. Pitts, C. Plager, L. Pondrom,53 S. Poprocki,15,f K. Potamianos,26 A. Pranko,26 F. Prokoshin,13,aa F. Ptohos,17,g G. Punzi,41b I. Redondo Fernández,29 P. Renton,38 M. Rescigno,46a F. Rimondi,6a,* L. Ristori,41a,15 A. Robson,19 T. Rodriguez,40 S. Rolli,50,h y F. Scuri,41a S. Seidel,34 Y. Seiya,37 A. Semenov,13 F. Sforza,41b S. Z. Shalhout,7 T. Shears,27 P. F. Shepard,42 M. Shimojima,49,u M. Shochet,11 I. Shreyber-Tecker,33 A. Simonenko,13 K. Sliwa,50 J. R. Smith,7 F. D. Snider,15 H. Song,42 V. Sorin,4 R. St. Denis,19,* M. Stancari,15 D. Stentz,15,w J. Strologas,34 Y. Sudo,49 A. Sukhanov,15 I. Suslov,13 K. FIN-00014, Helsinki, Finland; Helsinki Institute of Physics, FIN-00014, Helsinki, Finland 22University of Illinois, Urbana, Illinois 61801, USA 3 23The Johns Hopkins University, Baltimore, Maryland 21218, USA Experimentelle Kernphysik, Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany enter for High Energy Physics: Kyungpook National University, Daegu 702-701, Korea; Seoul National University, Seoul 151-742, Korea; Sungkyunkwan University, Suwon 440-746, Korea; Korea Institute of Science and Technology Information, Daejeon 305-806, Korea; nd Technology Information, Daejeon 305-806, Kore Chonnam National University, Gwangju 500-757, Korea; Chonbuk National University, Jeonju 561-756, Korea; Ewha Womans University, Seoul, 120-750, Korea ando Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA 27 27University of Liverpool, Liverpool L69 7ZE, United Kingdom f Liverpool, Liverpool L69 7ZE, United Kingdom 28University College London, London WC1E 6BT, United Kingdom lege London, London WC1E 6BT, United Kingdom Investigaciones Energeticas Medioambientales y Tecnologicas, E-28040 Madrid, Spain Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 31 31University of Michigan, Ann Arbor, Michigan 48109, USA 32 32Michigan State University, East Lansing, Michigan 48824, USA ate University, East Lansing, Michigan 48824, US 34University of New Mexico, Albuquerque, New Mexico 87131, USA 35 w Mexico, Albuquerque, New Mexico 87131, US 35The Ohio State University, Columbus, Ohio 43210, USA 36 6Okayama University, Okayama 700-8530, Japan 37Osaka City University, Osaka 558-8585, Japan 38University of Oxford, Oxford OX1 3RH, United Kingdom Istituto Nazionale di Fisica Nucleare, Sezione di Padova, I-35131 Padova, Italy 39b 41bUniversity of Pisa, I-56127 Pisa, Italy 41 41cUniversity of Siena, I-56127 Pisa, Italy 040 01 Kosice, Slovakia 13Joint Institute for Nuclear Research, RU-141980 Dubna, Russia 14 14Duke University, Durham, North Carolina 27708, USA 15 15Fermi National Accelerator Laboratory, Batavia, Illinois 60510, USA 16 18University of Geneva, CH-1211 Geneva 4, Switzerland 19 19Glasgow University, Glasgow G12 8QQ, United Kingdom 20 20Harvard University, Cambridge, Massachusetts 02138, USA n of High Energy Physics, Department of Physics, University of Helsinki, 112005-1 Published by the American Physical Society T. AALTONEN et al. PHYS. REV. 112005-1 D 98, 112005 (2018) 1Institute of Physics, Academia Sinica, Taipei, Taiwan 11529, Republic of China 2Argonne National Laboratory, Argonne, Illinois 60439, USA 3University of Athens, 157 71 Athens, Greece 4Institut de Fisica d’Altes Energies, ICREA, Universitat Autonoma de Barcelona, E-08193, Bellaterra (Barcelona), Spain 5Baylor University, Waco, Texas 76798, USA 6aIstituto Nazionale di Fisica Nucleare Bologna, I-40127 Bologna, Italy 6bUniversity of Bologna, I-40127 Bologna, Italy 7University of California, Davis, Davis, California 95616, USA 8University of California, Los Angeles, Los Angeles, California 90024, USA 9Instituto de Fisica de Cantabria, CSIC-University of Cantabria, 39005 Santander, Spain 10Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA 11Enrico Fermi Institute, University of Chicago, Chicago, Illinois 60637, USA 12Comenius University, 842 48 Bratislava, Slovakia; Institute of Experimental Physics, 040 01 Kosice, Slovakia 13Joint Institute for Nuclear Research, RU-141980 Dubna, Russia 14Duke University, Durham, North Carolina 27708, USA 15Fermi National Accelerator Laboratory, Batavia, Illinois 60510, USA 16University of Florida, Gainesville, Florida 32611, USA 17Laboratori Nazionali di Frascati, Istituto Nazionale di Fisica Nucleare, I-00044 Frascati, Italy 18University of Geneva, CH-1211 Geneva 4, Switzerland 19Glasgow University, Glasgow G12 8QQ, United Kingdom 20Harvard University, Cambridge, Massachusetts 02138, USA 21Division of High Energy Physics, Department of Physics, University of Helsinki, FIN-00014, Helsinki, Finland; Helsinki Institute of Physics, FIN-00014, Helsinki, Finland 22University of Illinois, Urbana, Illinois 61801, USA 23The Johns Hopkins University, Baltimore, Maryland 21218, USA 24Institut für Experimentelle Kernphysik, Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany 25Center for High Energy Physics: Kyungpook National University, Daegu 702-701, Korea; Seoul National University, Seoul 151-742, Korea; Sungkyunkwan University, Suwon 440-746, Korea; Korea Institute of Science and Technology Information, Daejeon 305-806, Korea; Chonnam National University, Gwangju 500-757, Korea; Chonbuk National University, Jeonju 561-756, Korea; Ewha Womans University, Seoul, 120-750, Korea 26Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA 27University of Liverpool, Liverpool L69 7ZE, United Kingdom 28University College London, London WC1E 6BT, United Kingdom 29Centro de Investigaciones Energeticas Medioambientales y Tecnologicas, E-28040 Madrid, Spain 30Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 31University of Michigan, Ann Arbor, Michigan 48109, USA 32Michigan State University, East Lansing, Michigan 48824, USA 33Institution for Theoretical and Experimental Physics, ITEP, Moscow 117259, Russia 34University of New Mexico, Albuquerque, New Mexico 87131, USA 35The Ohio State University, Columbus, Ohio 43210, USA 36Okayama University, Okayama 700-8530, Japan 37Osaka City University, Osaka 558-8585, Japan 38University of Oxford, Oxford OX1 3RH, United Kingdom 39aIstituto Nazionale di Fisica Nucleare, Sezione di Padova, I-35131 Padova, Italy 39bUniversity of Padova, I-35131 Padova, Italy 40University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 41aIstituto Nazionale di Fisica Nucleare Pisa, I-56127 Pisa, Italy 41bUniversity of Pisa, I-56127 Pisa, Italy 41cUniversity of Siena, I-56127 Pisa, Italy 41dScuola Normale Superiore, I-56127 Pisa, Italy 41eINFN Pavia, I-27100 Pavia, Italy 41fUniversity of Pavia, I-27100 Pavia, Italy 42 y g 3University of Athens, 157 71 Athens, Greece MEASUREMENT OF THE DIFFERENTIAL CROSS … PHYS. REV. D 98, 112005 (2018) 44University of Rochester, Rochester, New York 14627, USA 45 44University of Rochester, Rochester, New York 14627, USA 45 The Rockefeller University, New York, New York 10065, USA to Nazionale di Fisica Nucleare, Sezione di Roma 1, I-00185 Roma, Italy 46b nale di Fisica Nucleare, Sezione di Roma 1, I-0018 b 46bSapienza Universit`a di Roma, I-00185 Roma, Italy p y 47Mitchell Institute for Fundamental Physics and Astronomy, Texas A&M University, College Station, Texas 77843, USA 48aIstituto Nazionale di Fisica Nucleare Trieste, I-33100 Udine, Italy 48b ituto Nazionale di Fisica Nucleare Trieste, I-33100 Gruppo Collegato di Udine, I-33100 Udine, Italy 48 48cUniversity of Udine, I-33100 Udine, Italy 48d 48dUniversity of Trieste, I-34127 Trieste, Italy 49University of Tsukuba, Tsukuba, Ibaraki 305, Japan 50 50Tufts University, Medford, Massachusetts 02155, USA 51Waseda University, Tokyo 169, Japan 52Wayne State University, Detroit, Michigan 48201, US 3 53University of Wisconsin–Madison, Madison, Wisconsin 53706, USA 54 of Wisconsin–Madison, Madison, Wisconsin 53706 54Yale University, New Haven, Connecticut 06520, USA (Received 8 August 2018; published 13 December 2018) This paper presents a study of the production of a single W boson in association with one or more jets in proton-antiproton collisions at ﬃﬃﬃs p ¼ 1.96 TeV, using the entire data set collected in 2001–2011 by the aVisitor from University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada. b bVisitor from Istituto Nazionale di Fisica Nucleare, Sezione di Cagliari, 09042 Monserrato (Cagliari), Ital y , , , sitor from Institute of Physics, Academy of Sciences of the Czech Republic, 182 21, Czech Republic. eVisitor from CERN, CH-1211 Geneva, Switzerland. f eVisitor from CERN, CH-1211 Geneva, Switzerland. f fVisitor from Cornell University, Ithaca, New York 14853, USA. fVisitor from Cornell University, Ithaca, New York 14853, USA g y om University of Cyprus, Nicosia CY-1678, Cyprus gVisitor from University of Cyprus, Nicosia CY-1678, Cyprus. h y yp yp hVisitor from Office of Science, U.S. Department of Energy, Washington, DC 20585, USA. i hVisitor from Office of Science, U.S. Department of Energy, Wa i iVisitor from University College Dublin, Dublin 4, Ireland. j iVisitor from University College Dublin, Dublin 4, Ireland. j Visitor from University College Dublin, Dublin y g jVisitor from ETH, 8092 Zürich, Switzerland. k y g jVisitor from ETH, 8092 Zürich, Switzerland. k y , y, , p lVisitor from Universidad Iberoamericana, Lomas de Santa Fe, M´exico, C.P. 01219, Distrito Federal. m y y lVisitor from Universidad Iberoamericana, Lomas de Santa Fe, M´e mVisitor from University of Iowa, Iowa City, Iowa 52242, USA. mVisitor from University of Iowa, Iowa City, Iowa 52242, USA. y y nVisitor from Kinki University, Higashi-Osaka City, Japan 577-8502. o nVisitor from Kinki University, Higashi-Osaka City, Japan 577-8502. oVisitor from Kansas State University, Manhattan, Kansas 66506, USA. p Visitor from Kansas State University, Manhattan, Kansas 66506, USA. pVisitor from Brookhaven National Laboratory, Upton, New York 11973, USA. y, , , pVisitor from Brookhaven National Laboratory, Upton, New York 11973, USA. qVisitor from Istituto Nazionale di Fisica Nucleare, Sezione di Lecce, Via Arnesano, I-73100 Lecc r qVisitor from Istituto Nazionale di Fisica Nucleare, Sezione di Lecce, Via Arnesan rVisitor from Queen Mary, University of London, London, E1 4NS, United Kingdom. s rVisitor from Queen Mary, University of London, London, E1 4NS, United Kingdom s sVisitor from University of Melbourne, Victoria 3010, Australi t itor from Muons, Inc., Batavia, Illinois 60510, USA tVisitor from Muons, Inc., Batavia, Illinois 60510, USA. (Received 8 August 2018; published 13 December 2018) u uVisitor from Nagasaki Institute of Applied Science, Nagasaki 851-0193, Japan. v uVisitor from Nagasaki Institute of Applied Science, Nagasaki 851-0193, Japan. itor from Nagasaki Institute of Applied Science, Na g pp g p vVisitor from National Research Nuclear University, Moscow 115409, Russia. vVisitor from National Research Nuclear University, Moscow 115409, Russia. y wVisitor from Northwestern University, Evanston, Illinois 60208, USA. wVisitor from Northwestern University, Evanston, Illinois 60208, USA. itor from University of Notre Dame, Notre Dame, I y yVisitor from Universidad de Oviedo, E-33007 Oviedo, Spain. yVisitor from Universidad de Oviedo, E-33007 Oviedo, Spain. zVisitor from CNRS-IN2P3, Paris, F-75205 France. aaVisitor from Universidad Tecnica Federico Santa Maria, 110v Valparaiso, Chile. bb aaVisitor from Universidad Tecnica Federico Santa Maria, 110v Valparaiso, Chile. bb bbVisitor from Sejong University, Seoul 143-747, Korea. cc r from Sejong University, Seoul 143-747, Korea. j g y ccVisitor from The University of Jordan, Amman 11942, Jordan. dd ccVisitor from The University of Jordan, Amman 11942, Jordan. dd y ddVisitor from Universite catholique de Louvain, 1348 Louvain-La-Neuve, Belgium. ee ddVisitor from Universite catholique de Louvain, 1348 Louvain-La-Neuve, Belgium. ee ddVisitor from Universite catholique de Louvain, 1348 Louvain-L eeVisitor from University of Zürich, 8006 Zürich, Switzerland. ff eeVisitor from University of Zürich, 8006 Zürich, Switzerland. ff p ggVisitor from Harvard Medical School, Boston, Massachusetts 02114 USA. hh ggVisitor from Harvard Medical School, Boston, Massachusetts 02114 USA. hh hhVisitor from Hampton University, Hampton, Virginia 23668, USA. ii iiVisitor from Los Alamos National Laboratory, Los Alamos, New Mexico 87544, USA. jj jjVisitor from Universit`a degli Studi di Napoli Federico II, I-80138 Napoli, Italy. jjVisitor from Universit`a degli Studi di Napoli Federico II, I-80138 Napoli, Italy. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. I. INTRODUCTION sections that considered only electron final states in a sample corresponding to 320 pb−1 of integrated luminosity [3]. The current measurement improves upon previous CDF studies in that it uses the entire Run II data set and it includes the investigation of the muon channel, resulting in more data and a partially complementary set of system- atic uncertainties. Recent studies of the W þ jets process in p¯p collisions have been reported by the D0 Collaboration [4] and in pp collisions by the ATLAS [5], CMS [6], and LHCb [7] Collaborations. The production of W bosons in association with jets in proton-antiproton (p¯p) collisions requires high momentum transfer between the interacting constituents of the incom- ing hadrons. It follows that this process is suitable for testing perturbative quantum chromodynamics (pQCD). Moreover, it is an important background for standard- model (SM) processes such as Higgs-boson and t¯t production at hadron colliders. This paper reports mea- surements of the inclusive production cross sections σN ¼ σðWð→lνÞþ ≥N jets), where l is either an electron or a muon, for each of the jet multiplicities N ¼ 1, 2, 3, or 4 in p¯p collisions. In addition to these inclusive cross sections, differential cross sections (dσ1=dEjet T ) as functions of the leading-jet energy transverse to the beam direction (Ejet T ) are presented. Moreover, it is an important background for standard- model (SM) processes such as Higgs-boson and t¯t production at hadron colliders. This paper reports mea- surements of the inclusive production cross sections σN ¼ σðWð→lνÞþ ≥N jets), where l is either an electron or a muon, for each of the jet multiplicities N ¼ 1, 2, 3, or 4 in p¯p collisions. In addition to these inclusive cross sections, differential cross sections (dσ1=dEjet T ) as functions of the leading-jet energy transverse to the beam direction (Ejet T ) are presented. This paper is structured as follows. The CDF II detector is described in Sec. II. In Sec. III the details of the W þ jets event selection are presented. Section IV describes how the background is estimated and subtracted. The procedure used to unfold the data is presented in Sec. V, and the systematic uncertainties are discussed in Sec. VI. Section VII describes the combination of electron and muon results. Comparisons of theoretical predictions with the data are discussed in Sec. VIII and are presented with the results in Sec. PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. Collider Detector at Fermilab at the Tevatron, which corresponds to an integrated luminosity of 9.0 fb−1. The W boson is identified through its leptonic decays into electron and muon. The production cross sections are measured for each leptonic decay mode and combined after testing that the ratio of the Wð→μνÞ þ jets cross section to the Wð→eνÞ þ jets cross section agrees with the hypothesis of e-μ lepton universality. The combination of measured cross sections, differential in the inclusive jet multiplicity (Wþ ≥N jets with N ¼ 1, 2, 3, or 4) and in the transverse energy of the leading jet, are compared with theoretical predictions. DOI: 10.1103/PhysRevD.98.112005 I. INTRODUCTION IX. Finally, the results are summarized and conclusions are drawn in Sec. X. The Appendices A and B detail the background validation and the unfolding procedure, respectively. T p These measurements are performed by selecting W-boson decays with one electron or one muon detected in the central region of the Collider Detector at Fermilab (CDF) and by requiring the presence of at least one hadronic jet. The transverse energies (momenta) [1] of electrons (muons) are required to exceed 25 GeV (25 GeV=c) as are the transverse energies of jets. Jets are defined using a cone-based jet clustering algorithm. Although the presence of one high-transverse-momentum lepton is a distinctive signature for identifying the W boson, background contamination remains significant. One of the most challenging tasks is the subtraction from the selected sample of the multijet background made of jets that have experimental signatures similar to those of the leptons and are therefore reconstructed as electrons or as muons. The techniques used to model this background are optimized to obtain a better identification of the signal sample and to reduce the systematic uncertainties of the results. The measured cross sections are then corrected for detector effects using an unfolding procedure for a straightforward comparison with theoretical predictions at the particle level. 112005-3 112005-3 PHYS. REV. D 98, 112005 (2018) DOI: 10.1103/PhysRevD.98.112005 III. EVENT RECONSTRUCTION AND SELECTION Events enriched in decays of a W boson into an electron or muon, and a neutrino, are selected using an inclusive- lepton online event selection system (trigger) [8]. In the offline data reduction, electron and muon candi- dates are identified using standard requirements [8]. Electron candidates are identified as charged particles whose trajectories geometrically match significant energy deposits in a few adjacent calorimeter cells, while muon candidates are tracks that match signals in the muon detectors and deposit no significant energy in the calorim- eters. Electron and muon candidates are required to satisfy requirements on the minimum number of COT hits and the primary-vertex position [23]. Requirements are also applied to the fraction of particle energy, inferred from the momentum measurement, deposited in the calorimeter. At least 95% of the energy is required in the electro- magnetic calorimeter for the electrons, and little energy in both calorimeters for the muons. Selection requirements for the electron candidates include conditions, referred to as identification (ID) criteria [2], that are used to reduce the probability that a jet is misidentified as an electron. These ID criteria include requirements on the ratio between the energy deposited in the electromagnetic and hadronic calorimeters and on the shape and position of the shower produced by the electron candidate. Finally, both electron and muon candidates are required to be isolated [24]. Only the leptons in the central part of the detector (jηj < 1.0), where the track reconstruction efficiency is optimal and the calorimeter is well instrumented, are considered. pT T The sampling calorimeter system was located outside the solenoid. It included inner electromagnetic and outer hadronic calorimeters, both comprising central and forward (end-plug) sections. The central section, which included the cylindrical central electromagnetic [13] and central hadronic calorimeters, followed by the hadronic end-wall calorimeter [14] covered the range of pseudorapidy jηj < 1.1. The end-plug electromagnetic and hadronic calorimeters extended the coverage up to jηj < 3.64 [15]. All calorimeter sections were subdivided into projective modules (towers) pointing to the nominal interaction point. Each projective tower in the central region covered 0.1 in η and 15° in ϕ. The size of the projective towers in the plug calorimeters changed progressively from 0.1 in η and 7.5° in ϕ at jηj ¼ 1.1 to 0.5 in η and 15° in ϕ at jηj ¼ 3.64 [8]. III. EVENT RECONSTRUCTION AND SELECTION Sampling of the energy deposited in all calorimeters was obtained by an interleaving active scintillator with passive metal layers (lead in the electromagnetic and steel in the hadronic sections). Shower profiles were measured by strip detectors located near the shower maxima (at ∼6 radiation lengths) in the electromagnetic calorimeters: the central electromagnetic strip chambers and the plug electromagnetic strip detector [16] with 2 cm and 1.5 cm resolution, respectively. The unresolved gamma back- ground is reduced by scintillator-tile preshower counters located near the front faces of all electromagnetic calorimeters [17,18]. Candidate W þ jets events are selected from this inclu- sive lepton data set by requiring the presence of exactly one central electron or muon candidate with ET > 25 GeV or pT > 25 GeV=c, respectively, and at least one jet. Jets are reconstructed using the JETCLU cone algorithm [25] with jet-radius parameter R¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΔϕÞ2 þðΔηÞ2 p ¼0.4. Only jets having ET > 25 GeV, jηj < 2, electromagnetic fraction (i.e., the fraction of the total calorimetric energy of the jet deposited in the electromagnetic calorimeter) lower than 0.9, and that are well separated from the lepton candidate [ΔRðl; jetsÞ > 0.4] are considered. The energy of each jet is corrected using the jet-energy scale (JES) correction detailed in Ref. [26]. The muon detector [19,20] included four independent detectors located behind the hadronic calorimeter. Coverage for pseudorapidities jηj < 0.6 was provided by a central muon detector, located behind the central hadronic calorim- eter and followed by a central muon upgrade detector after an additional layer of shielding steel. The pseudorapidity region 0.6 < jηj < 1.0 was covered by a central muon extension detector. These three muon detectors comprised wire drift chambers operating in proportional mode inter- leaved with scintillator planes. Finally, coverage was extended to the region 1.0 < jηj < 1.5 by the barrel muon upgrade detector [21]. A threshold is imposed on the transverse mass of the W candidate [27], mW T > 40 GeV=c2. MEASUREMENT OF THE DIFFERENTIAL CROSS … PHYS. REV. D 98, 112005 (2018) measuring the average rate of inelastic p¯p collisions in each bunch crossing [22]. measuring the average rate of inelastic p¯p collisions in each bunch crossing [22]. Charged particle trajectories (tracks) were reconstructed by a silicon microstrip system [9,10] located just outside the interaction region, surrounded by the central outer tracker (COT) [11,12]. Together they provided high- resolution tracking information for pseudorapidities jηj < 1. The silicon microstrip system consisted of a central part (SVXII) which covered jηj ≤1 and an intermediate part (ISL) which extended coverage (with degraded resolution) to jηj ¼ 2. The SVXII comprised a layer of single-sided silicon microstrip detectors at 1.6 cm from the beam and a five-layer double-sided silicon microstrip detector at radii ranging from 2.5 to 11 cm. The ISL was located between the radii of 19 and 29 cm at higher jηj. The transverse momentum resolution was σpT=p2 T ¼0.0017 ðGeV=cÞ−1. II. THE CDF II DETECTOR The CDF II detector was a general-purpose apparatus that collected p¯p collision data from the Tevatron between 2001 and 2011 [8]. The detector consisted of a tracking system contained in a 1.4 T solenoidal magnetic field, surrounded by sampling calorimeters and muon detectors. The CDF II detector was cylindrically symmetric around the beam axis. The coordinate system has its origin in the center of the detector and consists of the radius r, the azimuthal angle ϕ, and the polar angle θ measured from the z axis, which is oriented along the incoming proton beam. The pseudorapidity is defined as η ¼ −ln ðtan θ 2Þ; the transverse energy as ET ¼ E sin θ, E being the energy detected by the calorimeters; and the transverse momentum as pT ¼ p sin θ, p being the mag- nitude of the momentum reconstructed by the tracking system. The angular distance between two reconstructed particles or clusters of particles P1 and P2 is defined as ΔRðP1; P2Þ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðϕP1 −ϕP2Þ2 þ ðηP1 −ηP2Þ2 q . The measurements are obtained using the entire p ¯p collision data set collected with the CDF II detector in Run II (2001–2011) at the Tevatron collider, corresponding to 9.0 fb−1 of integrated luminosity. They follow previous studies of jet pairs produced in association with a W boson [2] and a measurement of W þ jets production cross 112005-4 A. Simulated background processes Electroweak (EW) processes consist of decays into electrons or muons of gauge bosons produced in Wð→τνÞ þ jets, Zð→lþl−Þ þ jets, and WW, WZ, and ZZ processes. Top-quark processes involve the production and decay of top quarks, singly or in pairs. The two classes of processes are modeled using Monte Carlo (MC) sam- ples. Samples of Z þ jets and Wð→τνÞ þ jets events are generated using ALPGEN V1.3 [28] interfaced with PYTHIA V6.3 [29] for parton showering and hadronization. The contribution of the underlying event [30] is included in the PYTHIA generator using the Tevatron-tuned parameters of TUNE A [2], and final jets are matched to the original partons with the MLM matching procedure described in [28]. Production of WW, WZ, and ZZ pairs and top-quark pairs is modeled with the PYTHIA event generator, also using TUNE A and assuming a top-quark mass of 172.5 GeV=c2. Production of single top quarks (both in the s channel and in the t channel) is modeled with the MADEVENT [31] generator followed by PYTHIA for parton showering and hadronization [32]. All simulated samples are generated assuming the CTEQ5L parton distribution functions (PDFs) [33]. Multijet (MJ) background events enter the signal sample if one of the jets is incorrectly identified as a lepton. This background gives a large contribution in the electron channel, but is almost negligible in the muon channel because a jet, in order to mimic a muon, must also generate a matching track in the muon detectors. The multijet background is modeled using data, following Ref. [2]. The background data samples are obtained from the same data set as that used for the analysis (and described in Sec. III) by requiring the failure of two (one) of the electron (muon) ID criteria [8]. In the electron channel, the multijet background events modeled in this way are referred to as “nonelectron” events [2]. Only the ID criteria that introduce the least bias in the kinematic distributions of nonelectrons (i.e., the fraction of energy deposited in the hadronic calorimeter by the electron candidate and the shape its shower) are inverted, so as to minimize differences with respect to the candidate electrons. The ET of nonelectron in the W þ 2jet sample is tuned following Ref. [2], and the tuning procedure is generalized to other jet multiplicities. The tuning procedure includes two steps. A. Simulated background processes First, the contamination from all processes with a real lepton (e.g., weak-boson decay) is evaluated using a MC technique and subtracted from the nonelectron event sample as a function of the variable of interest. Then, in order to model the kinematic properties of the event correctly, the ET of the nonelectron is taken to be the ET of the corresponding jet (i.e., the jet with ΔR < 0.4 with respect to the nonelectron). After this procedure, the following two corrections to the ET of the jet producing a nonelectron have been applied. ( ) The contributions expected from each process are based on theoretical cross-section predictions. The rate of diboson production (WW, WZ, and ZZ) is scaled to the cross section calculated at next-to-leading order (NLO) in pQCD [34]; the t¯t sample is normalized using a next-to-next-to- leading order plus next-to-next-to-leading logarithm (NNLO þ NNLL) pQCD cross-section calculation [35]; and the single top-quark process is normalized to approxi- mate NNLO þ NNLL calculations [36] for the s channel and approximate NNLO þ NLL calculations [37] for the t channel. The Zð→lþl−Þ þ jets and Wð→τνÞ þ jets cross sections are normalized to leading order (LO) pQCD calculations [28] and scaled by a K factor of 1.4 to account for higher-order effects [3,38]. The uncertainties of these cross sections are 3% and 11% for top-quark pair produc- tion and single-top-quark production, respectively; 20% for Zð→lþl−Þ þ jets and 40% for Wð→τνÞ þ jets; and a fully correlated 6% for WW, WZ, and ZZ. The background contributions are also affected by the uncertainties in the integrated luminosity measurement (6%) [39], the lepton acceptance (2.2%), and the jet-energy scale (see Sec. VI for details). The first correction, called the “nonelectron energy-scale correction,” accounts for the difference in the energy scale between a jet producing a nonelectron and a jet producing a misidentified electron, i.e., a jet fulfilling all the electron selection criteria. To correct the nonelectron transverse energy, the same energy-scale correction as was previously evaluated for the W þ 2jets sample [2] is used. This ET correction is tested in a multijet-enriched region [control region (CR)] and shows very good agreement between data and MC expectations. The CR is defined by reversing the W-boson transverse-mass requirement for the signal region (SR), i.e., by requiring that mW T < 40 GeV=c2. IV. BACKGROUND MODELING AND VALIDATION The resulting sample is expected to include two classes of backgrounds, (i) electroweak and top-quark processes and (ii) multijet production. Background of the first type is Cherenkov counters located at a small angle, 3.7 < jηj < 4.7, were used to determine the luminosity by 112005-5 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. modeled by using simulated samples. A reliable model of the multijet background is particularly difficult to produce using simulation, so this background is estimated using data. so that the distribution of the number of reconstructed primary vertices, due to the additional p ¯p interactions in the same bunch crossing (pileup), matches the distribution in the data. A. Simulated background processes All generated samples are processed using the CDF II detector simulation based on GEANT3 [40], and the same event reconstruction and selection procedures used for the experimental data, described in Sec. III, are applied. Moreover, the events in each simulated sample are weighted The second correction, called the “trigger-bias correc- tion,” is required to fully account for the efficiency of the trigger selection. The need for such a correction arises from the inversion of the ratio of hadronic-to-electromagnetic energy criteria. The nonelectron ET multijet distribution is 112005-6 PHYS. REV. D 98, 112005 (2018) MEASUREMENT OF THE DIFFERENTIAL CROSS … NMJjCR ¼ NjCR; ð4Þ NMJjCR ¼ NjCR; ð4Þ NMJjCR ¼ NjCR; ð4Þ NMJjCR ¼ NjCR; corrected by applying weights evaluated bin by bin in the control region. The weights (wTB) for each bin of the nonelectron ET distribution (ET bin) are evaluated as follows: σWþjets has been calculated using the equation σWþjets has been calculated using the equation σWþjets ¼ NjSR −njSR −NMJjSR LAϵ ; ð5Þ ð5Þ ð5Þ wTBðETbinÞ ¼ NðETbinÞ −nðETbinÞ NMJðETbinÞ ; ð1Þ ð1Þ where LAϵ is the product of the integrated luminosity, the acceptance in the SR, and the total efficiency; the number njSR is the estimated yield of simulated background events in the SR; and NMJjSR is evaluated by replacing NMJjCR of Eq. (4) into Eq. (2). The number NsjCR is then calculated using Eq. (3) with σWþjets. On the next iteration NMJjCR is then calculated with the measured value of NsjCR. The process is iterated until the multijet scale factor fl MJ ¼ NMJjCR=N MJjCR changes by less than 1% between subsequent iterations. where NðET binÞ is the event yield in a bin of the electron ET data distribution, nðET binÞ is the predicted event yield of electroweak and top-quark background events, and NMJðET binÞ is the estimated number of multijet events in the same bin. To account for the possible dependence of the correction on the choice of the control region, two sets of weights from two nonoverlapping subsets of the CR, defined by the events with mW T < 20 GeV=c2 and by the events with 20 < mW T < 40 GeV=c2, have been calculated. The two sets of corrections are then applied to the events populating the whole control region, and the differences with respect to the nominal correction are assigned as systematic uncertainties. C. Background model validation The modeling of the background distributions, for both electrons and muons, is validated by comparing them with data in the CR. Examples of validation histograms are shown in Fig. 1. Validation of the modeling of other important kinematic variables is discussed in Appendix A. The good agreement between the data and the predictions in the control region supports the validity of the back- ground models. The shaded areas in Fig. 1 represent the total uncertainty in the evaluation of the backgrounds previously discussed. The main systematic uncertainty in the control region is the uncertainty in the multijet model. The fractional size of this uncertainty on the control region of the muon channel is larger than that on the electron channel. The reason for this difference in uncertainties is that the identification requirements that are reversed to define the nonelectron sample have less impact on the kinematic properties of the event than the modification of the isolation requirement applied to the muon channel. However, in the SR the background of the muon channel is much smaller than that in the electron channel. After these two corrections are applied, the missing transverse energy of the event is recalculated. In the muon channel, the multijet background is modeled using muon candidates that pass all the muon requirements [8] but with isolation [24] between 0.1 and 0.2, rather than greater than 0.1, as was previously used in [8] to define nonisolated muons. In this paper these muons are referred to as “loosely isolated muons.” Events with isolation greater than 0.2 are used to evaluate a systematic uncer- tainty of the model. The multijet background yield expected in the SR (NMJjSR) is estimated using the following equation: NMJjSR ¼ NMJjCR N MJjCR · N MJjSR; ð2Þ ð2Þ where N MJjCR and N MJjSR are the multijet event yields in the control and in the signal regions, respectively, that pass the nonelectron or the loosely isolated muon selections after the subtraction of contributions from processes with real leptons. The multijet yield in the CR, NMJjCR, corresponds to the number of candidates in the control region (NjCR) minus the number of simulated “electroweak and top-quark processes" background (njCR) and signal (NsjCR) contributions, D. Estimated background The background contributions for each inclusive jet multiplicity in the SR are summarized in Table I. For N ≥1 and N ≥2 jets, multijet production and Z þ jets represent the main background contributions to the electron and the muon channels, respectively, while the t¯t back- ground contribution is the largest single contribution for the sample with N ≥4 jets in both channels. For N ≥3, the main background contributions are multijet production in the electron channel and t¯t in the muon channel. The contributions of the WW, WZ, ZZ, and the single-top backgrounds are largest for N ≥2–4 jet multiplicities but do not exceed 4% in either channel. Table I reports also the number of selected events for each inclusive number of jets. NMJjCR ¼ ðN −n −NsÞjCR: ð3Þ ð3Þ To avoid circularity, NsjCR is estimated using the measured cross section σWþjets, instead of the theoretical calculation. The process is iterative. Starting with the approximation of a control region entirely populated of multijet events, 112005-7 112005-7 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. 30 40 50 60 100 200 300 Events per bin 0 100 200 300 400 500 600 700 800 900 3 10 × hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 hdata0 Entries 1457810 Mean 40.05 RMS 16.49 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. D. Estimated background hratio0 Entries 8 Mean 104.7 RMS 69.44 (GeV) T Leading jet E 30 40 50 60 70 100 200 300 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 Data-to-prediction ratio Syst. unc. (a) 30 40 50 60 100 200 300 Events per bin 0 10 20 30 40 50 60 70 3 10 × hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. hr En Me RM (GeV) T Leading jet E 30 40 50 60 70 100 200 300 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM Data-to-prediction ratio Syst. unc. (b) FIG. 1. Leading jet ET distribution in the control region for (a) the Wð→eνÞþ ≥1jets sample and (b) the Wð→μνÞþ ≥1jets sample. The data are represented with black points, while the signal and background predictions are represented with filled stacked histograms. D. Estimated background hratio0 Entries 8 Mean 104.7 RMS 69.44 (GeV) T Leading jet E 30 40 50 60 70 100 200 300 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 hratio0 Entries 8 Mean 104.7 RMS 69.44 Data-to-prediction ratio Syst. unc. (a) 30 40 50 60 100 200 300 Events per bin 0 10 20 30 40 50 60 70 3 10 × hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R hd En M R 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. hr En Me RM (GeV) T Leading jet E 30 40 50 60 70 100 200 300 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM hr En Me RM Data-to-prediction ratio Syst. unc. (b) (b) (a) FIG. 1. Leading jet ET distribution in the control region for (a) the Wð→eνÞþ ≥1jets sample and (b) the Wð→μνÞþ ≥1jets sample. D. Estimated background Systematic uncertainties on the predictions are indicated by shaded areas. The lower plots show the ratios of the data to the corresponding predictions. 30 40 50 60 100 200 300 Events per bin 0 100 200 300 400 500 600 700 800 900 3 10 × hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hstack_0 Entries 2021579 Mean 40.46 RMS 16.64 hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM hd En Me RM 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. V. UNFOLDING 1 jet ≥ ) + ν μ → W( (GeV) T Leading jet E 50 100 150 200 250 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (d) 2. Distributions of data for the W →eν channel overlaid with predicted background and signal (a) for each jet multiplicity a function of the leading-jet ET. The same distributions are reported in (b) and (d) for the W →μν channel. The predicted signa ed by using an ALPGEN+PYTHIA LO cross-section calculation multiplied by the K factor. The lower plots show the ratio betw nd prediction. The shaded regions represent the systematic uncertainties. 1 2 3 4 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν μ → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. N jets ≥ ) + ν μ → W( Inclusive jet multiplicity 1 2 3 4 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (b) 1 2 3 4 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν e → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. N jets ≥ ) + ν e → W( Inclusive jet multiplicity 1 2 3 4 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (a) Data-to-prediction ratio 0 50 100 150 200 250 300 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν e → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. 1 jet ≥ ) + ν e → W( (GeV) T Leading jet E 50 100 150 200 250 300 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. V. UNFOLDING simulation and are normalized to the LO calculations scaled by a K factor of 1.4 [3]. The observed and expected distributions of the inclusive jet multiplicity and the leading-jet ET for events passing the signal selection requirements are shown in Fig. 2. The expected signal yields are predicted with a MC calculation based on ALPGEN+PYTHIA propagated through the detector The signal distributions obtained by subtracting the estimated background are influenced by the acceptance, nonlinear response, and finite resolution of the detector. To correct for these effects, and to facilitate comparison with 1 2 3 4 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν e → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. N jets ≥ ) + ν e → W( Inclusive jet multiplicity 1 2 3 4 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (a) 1 2 3 4 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν μ → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. N jets ≥ ) + ν μ → W( Inclusive jet multiplicity 1 2 3 4 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (b) 50 100 150 200 250 300 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν e → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. 1 jet ≥ ) + ν e → W( (GeV) T Leading jet E 50 100 150 200 250 300 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (c) 50 100 150 200 250 300 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν μ → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. MEASUREMENT OF THE DIFFERENTIAL CROSS … PHYS. REV. D 98, 112005 (2018) D. Estimated background The data are represented with black points, while the signal and background predictions are represented with filled stacked histograms. Systematic uncertainties on the predictions are indicated by shaded areas. The lower plots show the ratios of the data to the corresponding predictions. TABLE I. Numbers of events in the data and total background for each inclusive jet multiplicity in the signal region of the electron and muon channels. The individual background estimates are expressed as percentages of the numbers of events in data and are evaluated as explained in Sec. IV. Sample Wð→eνÞþ ≥Njets Number of jets N ≥ 1 2 3 4 Events in data 477665 65029 9483 1642 Total background prediction 182000 24000 30800 2900 5700 440 1320 110 Multijet 30% 33% 32% 29% Z þ jets 5% 4% 4% 2% t¯t 1% 4% 19% 45% Wð→τνÞ þ jets 2% 1% 1% 1% Single top quark < 1% 2% 3% 2% WW, WZ, ZZ 1% 3% 2% 2% Sample Wð→μνÞþ ≥N jets Number of jets N ≥ 1 2 3 4 Events in data 229823 28038 3967 807 Total background prediction 39800 5600 7270 760 1860 150 550 40 Multijet 3% 3% 3% 2% Z þ jets 10% 9% 7% 4% t¯t 1% 6% 28% 57% Wð→τνÞ þ jets 2% 2% 1% 1% Single top quark 1% 3% 4% 3% WW, WZ, ZZ 1% 4% 4% 2% TABLE I. Numbers of events in the data and total background for each inclusive jet multiplicity in the signal region of the electron and muon channels. The individual background estimates are expressed as percentages of the numbers of events in data and are evaluated as l i d i S IV TABLE I. Numbers of events in the data and total background for each inclusive jet multiplicity in the signal region of the electron and muon channels. The individual background estimates are expressed as percentages of the numbers of events in data and are evaluated as explained in Sec. IV. V. UNFOLDING The response matrix for each distribution subjected to unfolding is built using the W þ jets sample generated with ALPGEN+ PYTHIA, which has approximately 10 times the size of the data sample. In the case of the jet-multiplicity distribution, unfolding is performed using bins that correspond to exclusive numbers of jets, except the last bin, i.e., the response matrix has bins corresponding to events with a W boson and exactly one, exactly two, exactly three, or at least four jets. The leading-jet ET distribution, for each of the two lepton channels, is unfolded considering bins designed to contain a sufficiently large number of events. The response matrices are determined by considering the jet with highest ET at the particle and detector levels inde- pendently, without any geometric matching between the two. The unfolded event phase space has been restricted to the fiducial region by applying an acceptance matrix after applying the inverse of the response matrix. The acceptance matrix is determined using the W þ jets sample generated with ALPGEN+PYTHIA for each unfolded distribution. The response and acceptance matrices are reported in Appendix B. V. UNFOLDING In the case of the jet-multiplicity distribution, unfolding is performed using bins that correspond to exclusive numbers of jets, except the last bin, i.e., the response matrix has bins corresponding to events with a W boson and exactly one, exactly two, exactly three, or at least four jets. The leading-jet ET distribution, for each of the two lepton channels, is unfolded considering bins designed to contain a sufficiently large number of events. The response matrices are determined by considering the jet with highest ET at the particle and detector levels inde- pendently, without any geometric matching between the two. The SVD-inversion results are rendered robust against fluctuations of the bin populations in data and MC by introducing a regularization condition, namely a “minimum curvature condition” [41] on the unfolded distribution, to avoid amplifying fluctuations coming from sparsely popu- lated MC and data bins. Regularization is characterized by a strength parameter. In this analysis, each distribution is unfolded by optimizing its strength parameter, as explained in Ref. [41]. In addition, it has been verified that the method employed in choosing each regularization parameter leads to a value that introduces the lowest systematic bias into the unfolded distribution. This is done using test distribu- tions similar to the observed distributions. The systematic uncertainty on the residual bias in the unfolding procedure is discussed in the next section. theory, the distributions are unfolded back to the particle- level separately for the two channels. The particle-level leptons and jets are reconstructed from all simulated particles with a lifetime of more than 10 ps, before the detector simulation, and by applying the same requirements as those used for the experimental data. The electron or muon from the W-boson decay is recombined with a radiated photon if the radial distance between the lepton and the photon [ΔRðl; γÞ] is less than 0.1. The neutrino momentum from the W-boson decay is used to calculate the missing transverse energy. Particle-level jets are con- structed by applying the JETCLU algorithm with cone radius 0.4 to the stable particles, from which the lepton (including the recombined photon) and neutrino from the W-boson decay are removed. The effects of the detector are described by a response matrix, determined from simulation, that maps all the generated events (at the particle level) to the reconstructed events (at the detector level). V. UNFOLDING (c) 50 100 150 200 250 300 Events per bin 1 10 2 10 3 10 4 10 5 10 6 10 7 10 -1 CDF Data, L=9.0 fb ) + jets (theo.) ν μ → W( ) + jets ν τ → Z+jets, W( WW, WZ, ZZ tt Single top Multijet Syst. unc. 1 jet ≥ ) + ν μ → W( (GeV) T Leading jet E 50 100 150 200 250 Data-to-prediction ratio 0 0.5 1 1.5 2 Data-to-prediction ratio Syst. unc. (d) (c) (d) FIG. 2. Distributions of data for the W →eν channel overlaid with predicted background and signal (a) for each jet multiplicity and (c) as a function of the leading-jet ET. The same distributions are reported in (b) and (d) for the W →μν channel. The predicted signal is obtained by using an ALPGEN+PYTHIA LO cross-section calculation multiplied by the K factor. The lower plots show the ratio between data and prediction. The shaded regions represent the systematic uncertainties. 112005-9 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. theory, the distributions are unfolded back to the particle- level separately for the two channels. The particle-level leptons and jets are reconstructed from all simulated particles with a lifetime of more than 10 ps, before the detector simulation, and by applying the same requirements as those used for the experimental data. The electron or muon from the W-boson decay is recombined with a radiated photon if the radial distance between the lepton and the photon [ΔRðl; γÞ] is less than 0.1. The neutrino momentum from the W-boson decay is used to calculate the missing transverse energy. Particle-level jets are con- structed by applying the JETCLU algorithm with cone radius 0.4 to the stable particles, from which the lepton (including the recombined photon) and neutrino from the W-boson decay are removed. The effects of the detector are described by a response matrix, determined from simulation, that maps all the generated events (at the particle level) to the reconstructed events (at the detector level). The response matrix for each distribution subjected to unfolding is built using the W þ jets sample generated with ALPGEN+ PYTHIA, which has approximately 10 times the size of the data sample. MEASUREMENT OF THE DIFFERENTIAL CROSS … MEASUREMENT OF THE DIFFERENTIAL CROSS … (GeV) T Leading jet E 50 100 150 200 250 300 Fractional uncertainty 0.15 − 0.1 − 0.05 − 0 0.05 0.1 0.15 0.2 0.25 ele_grallSYSup Entries 8 Mean 104.7 RMS 70.53 Total Acceptance MC background MJ background JES Unfolding 1 jet ≥ )+ ν e → W( (a) (GeV) T Leading jet E 50 100 150 200 250 300 Fractional uncertainty 0.15 − 0.1 − 0.05 − 0 0.05 0.1 0.15 0.2 0.25 muo_grallSYSup Entries 8 Mean 109.1 RMS 73.04 Total Acceptance MC background MJ background JES Unfolding 1 jet ≥ )+ ν μ → W( (b) FIG. 4. Fractional systematic uncertainties as functions of leading jet ET (a) in the W →eν channel and (b) in the W →μν channel. (GeV) T Leading jet E 50 100 150 200 250 300 Fractional uncertainty 0.15 − 0.1 − 0.05 − 0 0.05 0.1 0.15 0.2 0.25 ele_grallSYSup Entries 8 Mean 104.7 RMS 70.53 Total Acceptance MC background MJ background JES Unfolding 1 jet ≥ )+ ν e → W( (a) (GeV) T Leading jet E 50 100 150 200 250 300 Fractional uncertainty 0.15 − 0.1 − 0.05 − 0 0.05 0.1 0.15 0.2 0.25 muo_grallSYSup Entries 8 Mean 109.1 RMS 73.04 Total Acceptance MC background MJ background JES Unfolding 1 jet ≥ )+ ν μ → W( (b) (a) (b) FIG. 4. Fractional systematic uncertainties as functions of leading jet ET (a) in the W →eν channel and (b) in the W →μν channel. determined from data and validated using the CR. The uncertainty on this model includes contributions from the corrections applied to the data-driven sample as well as contributions from the multijet scale factor fe MJ. The corrections applied to the model are the nonelectron energy-scale correction and the trigger-bias correction. The uncertainties of the nonelectron energy-scale correc- tion are obtained by using the correction factors shifted by 1 standard deviation (10%–13%) and by smearing the missing transverse energy of the event (2%–13%), while those of the trigger-bias correction are derived by compar- ing data and predictions in two control regions restricted to mW T < 20 GeV=c2 and 20 < mW T < 40 GeV=c2 (1%–5%). The uncertainty on fe MJ is due to the contributions from CR background suppression (2%–5%). MEASUREMENT OF THE DIFFERENTIAL CROSS … The overall uncer- tainty on the inclusive cross sections ranges from 6% for Wð→eνÞþ ≥1 jet results to 21% for Wð→eνÞþ ≥4jets results. included [26,43]. The resulting uncertainties on the inclu- sive cross sections range from about 1% for Wþ ≥1 jet events to 11% for Wþ ≥4jets events in both the electron and the muon channels. The uncertainties in the inclusive cross sections from the unfolding procedure range from 0.1% to 7% for inclusive jet multiplicities between one and four, respectively. This uncertainty consists of a component due to approximations associated with the unfolding method and a component resulting from potential mismodelings in simulated W þ jets events used to determine the unfolding matrix. The residual bias in the unfolding procedure, described in Sec. V, is evaluated for each differential cross-section value using simulated experiments. For each observed distribu- tion, 1000 test distributions are generated and unfolded. The resulting distributions of the differences between generated and unfolded values are assumed to be a measure of the bias and are used as an estimate of the uncertainties of the unfolding procedure. The uncertainty in the simu- lated W þ jets sample comprises the uncertainties in the lepton acceptance (2.2%) and the uncertainties in the JES. In the muon channel, the major source of systematic uncertainty is the uncertainty on the MC prediction of the background [3%–14% of the Wð→μνÞþ ≥Njets cross sections for N ¼ 1–4]. This includes the uncertainty on the theoretical cross section used to normalize each back- ground process [6% for the WW, WZ, and ZZ processes, 3% for t¯t production, 11% for single-top-quark production, 20% and 40% for Z þ jets and Wð→τν þ jetsÞ processes, respectively, and 6% uncertainty on the integrated lumi- nosity of the sample]. In this channel, the uncertainty on the multijet background estimate gives the lowest contribution (1%–5%) to the overall uncertainty on the cross sections and is determined by varying the isolation requirement. The total uncertainty on the unfolded inclusive cross sections ranges from 7% for Wð→eνÞþ ≥1 jet events to 34% for Wð→eνÞþ ≥4jets events and from 5% for Wð→μνÞþ ≥1 jet results to 24% for Wð→μνÞþ ≥4jets results. Table II reports a summary of the systematic uncertainties. VI. SYSTEMATIC UNCERTAINTIES The systematic uncertainty on each of the unfolded measurements is assessed by repeating the unfolding procedure for variations of each parameter associated with systematic effects. Figures 3 and 4 show the systematic uncertainties obtained as differences between each set of unfolded data and the nominal result. These uncertainties include the contributions, discussed in Sec. IV, from the simulated background normalizations, the lepton acceptances, the JES, and the estimated multijet background yield. The first unfolding step consists of applying the inverted response matrix to the observed distribution. The matrix inversion is performed using the regularized singular-value decomposition (SVD) technique [41,42]. In the electron channel, the dominant source of system- atic uncertainty arises from the multijet background esti- mate. As discussed in Sec. IV, the multijet background is Inclusive jet multiplicity 1 2 3 4 Fractional uncertainty 0.4 − 0.3 − 0.2 − 0.1 − 0 0.1 0.2 0.3 0.4 Total Acceptance MC background MJ background JES Unfolding N jets ≥ )+ ν e → W( (a) Inclusive jet multiplicity 1 2 3 4 Fractional uncertainty 0.4 − 0.3 − 0.2 − 0.1 − 0 0.1 0.2 0.3 0.4 muo_grallSYSup_nj Entries 4 Mean 3.092 RMS 1.008 Total Acceptance MC background MJ background JES Unfolding N jets ≥ )+ ν μ → W( (b) FIG. 3. Fractional systematic uncertainties as functions of inclusive jet multiplicity (a) in the W →eν channel and (b) in the W →μν channel. Inclusive jet multiplicity 1 2 3 4 Fractional uncertainty 0.4 − 0.3 − 0.2 − 0.1 − 0 0.1 0.2 0.3 0.4 Total Acceptance MC background MJ background JES Unfolding N jets ≥ )+ ν e → W( (a) Inclusive jet multiplicity 1 2 3 4 Fractional uncertainty 0.4 − 0.3 − 0.2 − 0.1 − 0 0.1 0.2 0.3 0.4 muo_grallSYSup_nj Entries 4 Mean 3.092 RMS 1.008 Total Acceptance MC background MJ background JES Unfolding N jets ≥ )+ ν μ → W( (b) Inclusive jet multiplicity 1 2 3 4 Fractional uncertainty 0.4 − 0.3 − 0.2 − 0.1 − 0 0.1 0.2 0.3 0.4 Total Acceptance MC background MJ background JES Unfolding N jets ≥ )+ ν e → W( (a) Fractional uncertainty − (a) (b) FIG. 3. Fractional systematic uncertainties as functions of inclusive jet multiplicity (a) in the W →eν channel and (b) in the W →μν channel. 112005-10 PHYS. REV. D 98, 112005 (2018) VII. CHANNEL COMBINATION The cross sections are calculated by dividing the signal yields resulting from the unfolding by the integrated luminosity. The measurements from the muon and electron channels are combined. Assuming lepton universality, the combination is performed by using the asymmetric iterative best linear unbiased estimate method [44,45]. This method accounts for the correlations of asymmetric uncertainties. Additional sources of uncertainty are the contributions from electron and muon acceptances (2.2% in both chan- nels) and of the JES. The impact of the JES uncertainty is estimated by computing the cross section with the JES factors shifted by 1 standard deviation. The difference in the scale factors for simulated gluon and quark jets is also 112005-11 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. TABLE II. Summary of the systematic uncertainties. The uncertainties are listed as ranges where the impact of the uncertainty depends on the jet multiplicity. If the uncertainty has an impact on the shape of the leading jet ET distribution, a check mark symbol is placed in the column labeled “shape.” “EW and top-quark processes” refers to all the processes simulated with MC techniques, Z þ jets, Wð→τνÞ þ jets, WW, WZ, ZZ, t¯t, and single top quark. VII. CHANNEL COMBINATION Source Rate Shape Process affected Lepton acceptance 2.2% EW and top-quark processes MC background Z þ jets normalization 20% Z þ jets Wð→τνÞ þ jets normalization 40% Wð→τνÞ þ jets t¯t normalization 3% t¯t Single top-quark normalization 11% Single top quark WW, WZ, and ZZ normalization 6% WW, WZ, ZZ Luminosity 6% EW and top-quark processes MJ background Statistical uncertainty 0.1%–8% (1%–37%) Multijet electron (muon) sample Multijet Scale factor (fl MJ) 2%–5% (13%–68%) Multijet electron (muon) sample Nonelectron energy scale 10%–13% ✓ Multijet electron sample Nonelectron energy resolution 1%–13% ✓ Multijet electron sample Trigger bias correction 1%–5% ✓ Multijet electron sample Isolation requirement 1%–5% ✓ Multijet muon sample Jet-energy scale 1%–11% ✓ All backgrounds Quark/gluon JES 2.7%= ∓4.4% EW and top-quark processes Unfolding 0.1%–7% ✓ W þ jets Luminosity 6% W þ jets cross section 1 2 3 4 N jets) (pb) ≥ + W ( σ = N σ 2 − 10 1 − 10 1 10 2 10 -1 CDF Data, L=9.0 fb N jets ≥ )+ ν μ → W( N jets ≥ )+ ν e → W( Njets ≥ + ν l → W Inclusive jet multiplicity (N) 1 2 3 4 single channel / combination 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 combination unc. (a) 0 50 100 150 200 250 300 ) (pb/GeV) jet T /d(E 1 σ d 4 − 10 3 − 10 2 − 10 1 − 10 1 10 -1 CDF Data, L=9.0 fb 1 jet ≥ ) + ν μ → W( 1 jet ≥ ) + ν e → W( 1 jet ≥ + ν l → W (GeV) T Leading jet E 0 50 100 150 200 250 300 single channel / combination 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 combination unc. (b) FIG. 5. Measured cross sections in the Wð→eνÞ þ jets (dots) and Wð→μνÞ þ jets (squares) decay channels as functions of (a) the inclusive number of jets and (b) the inclusive leading jet ET in events with one or more jets. The hatched areas in the upper plots are the total uncertainties, reported separately for the electron and muon channels. The lower plots show the ratios between measured cross sections and the combined results. The bands correspond to 1σ systematic uncertainties of the combination. MEASUREMENT OF THE DIFFERENTIAL CROSS … MEASUREMENT OF THE DIFFERENTIAL CROSS … Systematic uncertainties related to JES, MC-based predic- tions, unfolding, and luminosity are considered to be 100% correlated between channels. Statistical, acceptance, and multijet background uncertainties are considered uncorre- lated between muons and electrons. The upper plots in Fig. 5 show the observed inclusive and differential cross sections multiplied by the branching fractions of W-boson decays into electrons or muons. The ratios and the combined results are shown in the lower panels. The uncertainties on the ratios are small because of correlations between the uncertainties on the individual and combined results. A detailed test of consistency with lepton univer- sality follows. TABLE III. Magnitude of the ratio of W →lν coupling constants, jgμj=jgej, measured from the ratio of the Wð→μνÞ þ jets and Wð→eνÞ þ jets cross sections for each inclusive jet multiplicity. Jet multiplicity jgμj=jgej ≥1jet 0.992 0.002 (stat) þ0.050 −0.053 (syst) ≥2jets 0.972 0.006 (stat) þ0.060 −0.064 (syst) ≥3jets 0.918 0.020 (stat) þ0.099 −0.116 (syst) ≥4jets 1.077 0.076 (stat) þ0.203 −0.243 (syst) and ΓðW →μνÞ and ΓðW →eνÞ are the decay widths of the W boson in the muon and the electron channels, respectively. Assuming that the couplings of the W bosons in the electroweak and top-quark background processes are those predicted by the standard model, the ratio jgμj=jgej is given by the ratio of the cross sections measured in the two channels, The resulting values of jgμj=jgej for each jet multiplicity are reported in Table III. The magnitudes of coupling ratios for various jet multiplicities are consistent with the previous CDF measurement, 0.991 0.012, obtained in the inclu- sive channel, i.e., Wþ ≥0jets [8]. Consistency between results of inclusive jet multiplicities measured here with those measured previously and with lepton universality promote confidence in the cross-section measurements and support channel combination. VII. CHANNEL COMBINATION 0 50 100 150 200 250 300 ) (pb/GeV) jet T /d(E 1 σ d 4 − 10 3 − 10 2 − 10 1 − 10 1 10 -1 CDF Data, L=9.0 fb 1 jet ≥ ) + ν μ → W( 1 jet ≥ ) + ν e → W( 1 jet ≥ + ν l → W (GeV) T Leading jet E 0 50 100 150 200 250 300 single channel / combination 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 combination unc. (b) 1 2 3 4 N jets) (pb) ≥ + W ( σ = N σ 2 − 10 1 − 10 1 10 2 10 -1 CDF Data, L=9.0 fb N jets ≥ )+ ν μ → W( N jets ≥ )+ ν e → W( Njets ≥ + ν l → W Inclusive jet multiplicity (N) 1 2 3 4 single channel / combination 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 combination unc. (a) (b) (a) FIG. 5. Measured cross sections in the Wð→eνÞ þ jets (dots) and Wð→μνÞ þ jets (squares) decay channels as functions of (a) the inclusive number of jets and (b) the inclusive leading jet ET in events with one or more jets. The hatched areas in the upper plots are the total uncertainties, reported separately for the electron and muon channels. The lower plots show the ratios between measured cross sections and the combined results. The bands correspond to 1σ systematic uncertainties of the combination. 112005-12 PHYS. REV. D 98, 112005 (2018) VIII. THEORETICAL PREDICTIONS predictions are generated using the CTEQ6.6 PDF set [47], with the same choice of renormalization and factorization scales as the ALPGEN+PYTHIA predictions, with the excep- tion of the LO prediction, for which μ0 equals HT=2, where HT is the scalar sum of the transverse momenta of all final-state particles. The differential cross-section measurements are com- pared with the predictions from various theoretical calculations. The ALPGEN+PYTHIA predictions use ALPGEN [28] to simulate the production of n partons in association with a W boson and PYTHIA [29] to perform the showering and hadronization. The CTEQ5L [33] parton distribution func- tions are used with the nominal choice of the renormaliza- tion and factorization scale (μ), i.e., μ0 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ m2 W þ P2 T p , where P2 T is the sum of the squared transverse momenta of all final-state partons from the same interaction point. The renormalization and factorization scale variations cover uncertainties connected with missing higher orders in the predictions. Jets are clustered with the JETCLU algorithm with a radius parameter of 0.4. For comparison of the MCFM predictions with measured cross sections at the particle level, hadronization is intro- duced using ALPGEN+PYTHIA and jets are clustered at both particle level and parton level using infrared- and collinear- safe algorithms [48]. Nonperturbative QCD (npQCD) corrections due to hadronization and the underlying event [30] are included in the MCFM predictions before com- parison with the measured cross sections. These npQCD corrections are shown in Fig. 6, where the contribution due to hadronization is opposite to that of the underlying event. The hadronization component is estimated by comparing the cross sections at the particle level with the ones at the parton level. The effects of the underlying event are evaluated by comparing cross sections at the particle level with and without the underlying event contribution. Jets are For comparison of the MCFM predictions with measured cross sections at the particle level, hadronization is intro- duced using ALPGEN+PYTHIA and jets are clustered at both particle level and parton level using infrared- and collinear- safe algorithms [48]. Nonperturbative QCD (npQCD) corrections due to hadronization and the underlying event [30] are included in the MCFM predictions before com- parison with the measured cross sections. These npQCD corrections are shown in Fig. 6, where the contribution due to hadronization is opposite to that of the underlying event. MEASUREMENT OF THE DIFFERENTIAL CROSS … D 98, 112005 (2018) T. AALTONEN et al. MEASUREMENT OF THE DIFFERENTIAL CROSS … σWþjetsBðW →μνÞ σWþjetsBðW →eνÞ ¼ ΓðW →μνÞ ΓðW →eνÞ ¼ g2μ g2e ; ð6Þ ð6Þ where σWþjets is the inclusive production cross section, BðW →μνÞ and BðW →eνÞ are the branching fractions Inclusive jet multiplicity 1 2 3 4 Corrections 0.7 0.8 0.9 1 1.1 1.2 1.3 jets) T npQCD Correction (anti-k Total Hadronization Underlying event (b) (GeV) T Leading jet E 50 100 150 200 250 300 Corrections 0.8 0.9 1 1.1 1.2 jets) T npQCD Correction (anti-k Total Hadronization Underlying event (d) Inclusive jet multiplicity 1 2 3 4 Corrections 0.7 0.8 0.9 1 1.1 1.2 1.3 npQCD Correction (SisCone jets) Total Hadronization Underlying event (a) Inclusive jet multiplicity 1 2 3 4 Corrections 0.7 0.8 0.9 1 1.1 1.2 1.3 jets) T npQCD Correction (anti-k Total Hadronization Underlying event (b) (GeV) T Leading jet E 50 100 150 200 250 300 Corrections 0.8 0.9 1 1.1 1.2 npQCD Correction (SisCone jets) Total Hadronization Underlying event (c) (GeV) T Leading jet E 50 100 150 200 250 300 Corrections 0.8 0.9 1 1.1 1.2 jets) T npQCD Correction (anti-k Total Hadronization Underlying event (d) FIG. 6. Nonperturbative QCD (npQCD) corrections applied to the MCFM theoretical predictions. The corrections as functions of the inclusive jet multiplicity [(a) and (b)] and the leading jet ET [(c) and (d)] are derived using SISCONE jets [(a) and (c)] and anti-kT jets [(b) and (d)]. The hadronization (dashed lines) and the underlying event (dot-dashed lines) contributions to the total corrections (solid lines) are shown. Inclusive jet multiplicity 1 2 3 4 Corrections 0.7 0.8 0.9 1 1.1 1.2 1.3 jets) T npQCD Correction (anti-k Total Hadronization Underlying event (b) Inclusive jet multiplicity 1 2 3 4 Corrections 0.7 0.8 0.9 1 1.1 1.2 1.3 npQCD Correction (SisCone jets) Total Hadronization Underlying event (a) (GeV) T Leading jet E 50 100 150 200 250 300 Corrections 0.8 0.9 1 1.1 1.2 npQCD Correction (SisCone jets) Total Hadronization Underlying event (c) Corrections Corrections FIG. 6. Nonperturbative QCD (npQCD) corrections applied to the MCFM theoretical predictions. The corrections as functions of the inclusive jet multiplicity [(a) and (b)] and the leading jet ET [(c) and (d)] are derived using SISCONE jets [(a) and (c)] and anti-kT jets [(b) and (d)]. The hadronization (dashed lines) and the underlying event (dot-dashed lines) contributions to the total corrections (solid lines) are shown. 112005-13 PHYS. REV. VIII. THEORETICAL PREDICTIONS Predictions computed with the MCFM 6.8 [46] generator are carried out at NLO for inclusive cross sections with number of jets N ¼ 1 and 2 but are limited to LO for N ¼ 3. No prediction is available for N ¼ 4. The MCFM FIG. 7. Measured inclusive jet cross sections (black dots) [σN ¼ σðWð→lνÞþ ≥NjetsÞ], as functions of the inclusive jet multiplicity for Wþ ≥N jet events compared to the theoretical predictions described in Sec. VIII. The panels show (a) the absolute comparisons, the ratios of the measured cross sections to (b) the ALPGEN+PYTHIA predictions and to (c) the MCFM theoretical predictions corrected for npQCD prediction using SISCONE and to (d) anti-kT jets. The shaded bands show the total systematic uncertainties, except for the 6% luminosity uncertainty. The dashed and solid lines indicate the PDF uncertainties and the uncertainties corresponding to the variation of the factorization and renormalization scale μ, respectively. FIG. 7. Measured inclusive jet cross sections (black dots) [σN ¼ σðWð→lνÞþ ≥NjetsÞ], as functions of the inclusive jet multiplicity for Wþ ≥N jet events compared to the theoretical predictions described in Sec. VIII. The panels show (a) the absolute comparisons, the ratios of the measured cross sections to (b) the ALPGEN+PYTHIA predictions and to (c) the MCFM theoretical predictions corrected for npQCD prediction using SISCONE and to (d) anti-kT jets. The shaded bands show the total systematic uncertainties, except for the 6% luminosity uncertainty. The dashed and solid lines indicate the PDF uncertainties and the uncertainties corresponding to the variation of the factorization and renormalization scale μ, respectively. 112005-14 112005-14 PHYS. REV. D 98, 112005 (2018) MEASUREMENT OF THE DIFFERENTIAL CROSS … and the predictions are particle-level cross sections restricted to the following requirements on the final-state particles: only one central (jηj < 1) lepton with ET > 25 GeV and at least one jet with ET > 25 GeV and pseudorapidity jηj < 2. The reconstructed transverse mass of the W boson is required to be greater than 40 GeV=c2. Jets are recon- structed using the JETCLU algorithm with a radius parameter of 0.4 in the measurement and in the ALPGEN+PYTHIA predictions, while for the MCFM predictions, the algorithms used are the anti-kT and cone algorithm. clustered at the parton level and at the particle level using both the anti-kT [49] and the SISCONE v.1.4.0-devel [50] infrared- and collinear-safe algorithms provided by the FASTJET v.2.4.1 package [51]. In both algorithms the jet radius is set to be 0.4 and for the SISCONE algorithm two jets are merged when they share 75% of the energy. The systematic uncertainties in the theoretical cross sections contain contributions from PDF and scale uncer- tainties. The PDF uncertainties are obtained by varying each of the eigenvalues in the CTEQ5L and CTEQ6.6 sets by 1 standard deviation [52]. The largest uncertainty in the theoretical predictions is due to the choice of the renorm- alization and factorization scales, which are kept equal to eachotherand arevariedbetweenthe extremes μ0=2 and2μ0. Figures 7(b)–(d) and 8(b)–(d) show that the ALPGEN+ PYTHIA predictions are affected by a large uncertainty due to variations of the renormalization and factorization scales. The data are consistent with the predictions within these uncertainties. The NLO MCFM predictions corrected for npQCD effects agree with the measurements despite the differences in the jet-reconstruction algorithms used in MCFM and this analysis. This observation would be more significant if similar infrared-safe reconstruction algo- rithms had been used for both the theory and the data analysis. However, as observed in Ref. [53], the differences between the SISCONE and anti-kT algorithms used for the IX. RESULTS AND COMPARISON WITH THEORETICAL PREDICTIONS The cross sections as functions of the jet multiplicity [σN ¼ σðWð→lνÞþ ≥NjetsÞ] and the leading-jet ET (dσ1=dEjet T ) are shown in Figs. 7 and 8. The ALPGEN+ PYTHIA and the MCFM predictions, corrected for npQCD effects, are included for comparison. Both the measurements FIG. 8. Measured differential cross sections (dσ1=dEjet T ) as functions of the leading jet ET for Wþ ≥1 jet events compared to the theoretical predictions described in Sec. VIII. The panels show (a) the absolute comparisons, the ratios of the measured cross sections to (b) the ALPGEN+PYTHIA predictions and to (c) the MCFM theoretical predictions corrected for npQCD prediction using SISCONE and to (d) anti-kT jets. The shaded bands show the total systematic uncertainties, except for the 6% luminosity uncertainty. The dashed and solid lines indicate the PDF uncertainties and the uncertainties corresponding to the variation of the factorization and renormalization scale μ, respectively. FIG. 8. Measured differential cross sections (dσ1=dEjet T ) as functions of the leading jet ET for Wþ ≥1 jet events compared to the theoretical predictions described in Sec. VIII. The panels show (a) the absolute comparisons, the ratios of the measured cross sections to (b) the ALPGEN+PYTHIA predictions and to (c) the MCFM theoretical predictions corrected for npQCD prediction using SISCONE and to (d) anti-kT jets. The shaded bands show the total systematic uncertainties, except for the 6% luminosity uncertainty. The dashed and solid lines indicate the PDF uncertainties and the uncertainties corresponding to the variation of the factorization and renormalization scale μ, respectively. 112005-15 X. SUMMARY AND CONCLUSIONS Measurements of differential inclusive cross sections for the production of jets in association with a W boson, using 9.0 fb−1 of p¯p collision data collected by the CDF experi- ment at the Tevatron, are reported. The differential cross sections as functions of jet multiplicity and leading-jet ET are measured independently for the Wð→eνÞ and Wð→μνÞ decay modes and are combined at the particle level after unfolding detector acceptance and resolution effects. Measurements are performed in the kinematic region El T > 25 GeV, jηlj < 1, Ejet T > 25 GeV, jηjetj < 2, and mW T > 40 GeV=c2 and jets are reconstructed with the JETCLU algorithm. Cross sections are compared with the theoretical predictions of the ALPGEN generator interfaced with PYTHIA (enhanced leading-order QCD predictions) and the MCFM generator (next-to-leading order QCD predictions) corrected for nonperturbative QCD effects. The theoretical predictions are mainly affected by the 112005-15 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. Inclusive jet multiplicity (N) 1 2 3 4 N-1 σ / N σ 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0.13 0.14 0.15 (JETCLU jets) -1 CDF Data, L = 9.0 fb ALPGEN+PYTHIA (JETCLU jets) MCFM (SisCone jets) jets) T MCFM (anti-k FIG. 9. Ratio between inclusive-jet differential cross sections (σN=σN−1 for N ¼ 2, 3, 4) in data (black dots), ALPGEN+PYTHIA predictions (triangles), and MCFM predictions corrected for npQCD effects using SISCONE cone (open squares) and anti- kT jets (solid squares). The σN=σN−1 MCFM prediction for N ¼ 3 is a LO ratio, while no MCFM is available for N > 3. Error bars show the total uncertainties. Inclusive jet multiplicity (N) 1 2 3 4 N-1 σ / N σ 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0.13 0.14 0.15 (JETCLU jets) -1 CDF Data, L = 9.0 fb ALPGEN+PYTHIA (JETCLU jets) MCFM (SisCone jets) jets) T MCFM (anti-k uncertainty on the factorization and renormalization scale. This uncertainty of the ALPGEN+PYTHIA predictions is significantly larger than the uncertainty on the measure- ments, whereas for the MCFM predictions it is comparable to the experimental uncertainty. The agreement with these predictions observed for the measurements reported here suggests that the NLO perturbative QCD calculations properly model the jet multiplicity and jet ET distributions of the W þ jets process. The ratio of the lepton coupling constants reported in Table III is consistent with lepton universality and validates the procedure used to evaluate the QCD background. The production of a W boson in association with jets is among the dominant backgrounds in current measurements and searches for nonstandard-model physics at the Large Hadron Collider. The proper modeling of this process, supported by our work, is therefore important to consolidate and enhance the physics reach of Large Hadron Collider studies. FIG. 9. Ratio between inclusive-jet differential cross sections (σN=σN−1 for N ¼ 2, 3, 4) in data (black dots), ALPGEN+PYTHIA predictions (triangles), and MCFM predictions corrected for npQCD effects using SISCONE cone (open squares) and anti- kT jets (solid squares). The σN=σN−1 MCFM prediction for N ¼ 3 is a LO ratio, while no MCFM is available for N > 3. Error bars show the total uncertainties. ACKNOWLEDGMENTS MCFM NLO predictions is indicative of the bias introduced by the use of JETCLU. These differences are observed to be smaller than the uncertainties in the measurements. Moreover, a similar agreement between data and theory has been observed previously in analogous comparisons [54]. MCFM NLO predictions is indicative of the bias introduced by the use of JETCLU. These differences are observed to be smaller than the uncertainties in the measurements. Moreover, a similar agreement between data and theory has been observed previously in analogous comparisons [54]. Figure 9 shows the ratios between inclusive jet multiplicity cross sections (σN=σN−1). The MCFM pre- diction for N ¼ 3 is calculated using a LO prediction for both the numerator (σ3) and the denominator (σ2), while NLO predictions are used for the numerator and the denominator for N ¼ 2. The theoretical predictions agree with the measurements. The cross-section ratios are sensitive to the strong-interaction coupling, and display no discernible dependence on the jet multiplicity, as expected. We thank the Fermilab staff and the technical staffs of the participating institutions for their vital contributions. This work was supported by the U.S. Department of Energy and National Science Foundation; the Italian Istituto Nazionale di Fisica Nucleare; the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Natural Sciences and Engineering Research Council of Canada; the National Science Council of the Republic of China; the Swiss National Science Foundation; the A. P. Sloan Foundation; the Bundesministerium für Bildung und Forschung, Germany; the Korean World Class University Program, the National Research Foundation of Korea; the Science and Technology Facilities Council and the Royal Society, UK; the Russian Foundation for Basic Research; the Ministerio de Ciencia e Innovación, and Programa Consolider-Ingenio 2010, Spain; the Slovak R&D Agency; the Academy of Finland; the Australian Research Council (ARC); and the EU community Marie Curie Fellowship Contract No. 302103. p y g p Figure 9 shows the ratios between inclusive jet multiplicity cross sections (σN=σN−1). The MCFM pre- diction for N ¼ 3 is calculated using a LO prediction for both the numerator (σ3) and the denominator (σ2), while NLO predictions are used for the numerator and the denominator for N ¼ 2. The theoretical predictions agree with the measurements. The cross-section ratios are sensitive to the strong-interaction coupling, and display no discernible dependence on the jet multiplicity, as expected. MEASUREMENT OF THE DIFFERENTIAL CROSS … PHYS. REV. D 98, 112005 (2018) (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 0 50 100 150 200 250 300 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (a) (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 200 250 300 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T W boson p 0 20 40 60 80 100 120 140 160 180 200 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (b) ) T E (electron, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Events per 0.1 radiants 0 20 40 60 80 100 120 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (electron, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (c) ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Events per 0.1 radiants 0 20 40 60 80 100 120 140 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (d) Events per 2 GeV Events per 5 GeV/c 10. Validation plots of the background model in the electron channel: comparison between the data and the prediction for th butions of (a) missing transverse energy =ET, (b) W-boson pT, (c) azimuthal distance between the electron and the ⃗=ET [Δϕðe; ⃗=ETÞ d) azimuthal distance between the leading jet and the ⃗=ET [Δϕðleading jet; ⃗=ETÞ] in the control region. MEASUREMENT OF THE DIFFERENTIAL CROSS … The data are represented by th points while the signal and the background predictions are represented by stacked histograms. Systematic uncertainties on th ctions are indicated by the shaded areas (see Sec. IV for discussion on systematic uncertainty). The lower plots show the ratio en the data and the corresponding predictions. , ( (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 0 50 100 150 200 250 300 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T W boson p 0 20 40 60 80 100 120 140 160 180 200 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (b) Events per 5 GeV/c (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 0 50 100 150 200 250 300 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (a) Events per 2 GeV (b) ) T E (electron, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Events per 0.1 radiants 0 20 40 60 80 100 120 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (electron, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (c) ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Events per 0.1 radiants 0 20 40 60 80 100 120 140 3 10 × 1 jet ≥ ) + ν e → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 Data-to-prediction ratio 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (d) (d) (c) FIG. 10. APPENDIX A: BACKGROUND VALIDATION PLOTS As a consistency check of the background models, data and predictions are compared in a control region where the background contributions are expected to be much larger than the signal, as explained in Sec. IV. In addition to those shown in Fig. 1 of Sec. IV C, other examples of back- ground-modeling validation plots for the electron and muon channels are shown in Figs. 10 and 11, respectively. The reasonably good agreement between the data and the predictions supports the assumption that the multijet model and the MC simulations adequately describe the contribu- tions from the background processes. 112005-16 MEASUREMENT OF THE DIFFERENTIAL CROSS … (b) (a) ( ) ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12 14 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (d) Events per 0.1 radiants Data-to-prediction ratio (muon,leading jet) φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35 40 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (muon,leading jet) φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (c) Events per 0.1 radiands Data-to-prediction ratio Events per 0.1 radiands Events per 0.1 radiants (c) (d) FIG. 11. Validation plots of the background model in the muon channel: comparison between the data and the prediction for the distributions of (a) missing transverse energy =ET, (b) W-boson pT, (c) azimuthal distance between the muon and the leading jet [Δϕðμ; leading jetÞ], and (d) azimuthal distance between the leading jet and the ⃗=ET [Δϕðleading jet; ⃗=ETÞ] in the control region. The data are represented by the black points while the signal and the background predictions are represented by stacked histograms. Systematic uncertainties on the predictions are indicated by the shaded areas (see Sec. IV for discussion on systematic uncertainty). The lower plots show the ratios between the data and the corresponding predictions. MEASUREMENT OF THE DIFFERENTIAL CROSS … (c) ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12 14 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. ) T E (leading jet, φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (d) Events per 0.1 radiands Events per 0.1 radiants Data-to-prediction ratio Data-to-prediction ratio Data-to-prediction ratio Data-to-prediction ratio 11. Validation plots of the background model in the muon channel: comparison between the data and the prediction for t butions of (a) missing transverse energy =ET, (b) W-boson pT, (c) azimuthal distance between the muon and the leading j μ; leading jetÞ], and (d) azimuthal distance between the leading jet and the ⃗=ET [Δϕðleading jet; ⃗=ETÞ] in the control region. The da epresented by the black points while the signal and the background predictions are represented by stacked histograms. Systemat rtainties on the predictions are indicated by the shaded areas (see Sec. IV for discussion on systematic uncertainty). The lower plo the ratios between the data and the corresponding predictions. , ( (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 Events per 4 GeV/c 0 2 4 6 8 10 12 14 16 18 20 22 24 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (b) Data-to-prediction ratio (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 Events per 2 GeV 0 2 4 6 8 10 12 14 16 18 20 22 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (a) Data-to-prediction ratio Data-to-prediction ratio Syst. unc. MEASUREMENT OF THE DIFFERENTIAL CROSS … Validation plots of the background model in the electron channel: comparison between the data and the prediction for the distributions of (a) missing transverse energy =ET, (b) W-boson pT, (c) azimuthal distance between the electron and the ⃗=ET [Δϕðe; ⃗=ETÞ], and (d) azimuthal distance between the leading jet and the ⃗=ET [Δϕðleading jet; ⃗=ETÞ] in the control region. The data are represented by the black points while the signal and the background predictions are represented by stacked histograms. Systematic uncertainties on the predictions are indicated by the shaded areas (see Sec. IV for discussion on systematic uncertainty). The lower plots show the ratios between the data and the corresponding predictions. 112005-17 PHYS. REV. D 98, 112005 (2018) T. AALTONEN et al. APPENDIX B: DETECTOR RESPONSE AND ACCEPTANCE MATRICES FOR THE They are nondiagonal matrices as shown in Figs. 12 and 1 where the fraction of detector-level events reconstructe (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 Events per 2 GeV 0 2 4 6 8 10 12 14 16 18 20 22 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV) T E 0 5 10 15 20 25 30 35 40 45 50 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (a) (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 Events per 4 GeV/c 0 2 4 6 8 10 12 14 16 18 20 22 24 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (GeV/c) T W boson p 0 20 40 60 80 100 120 140 160 180 200 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. (b) (muon,leading jet) φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35 40 3 10 × 1 jet ≥ ) + ν μ → W( -1 Data L=9.0 fb W+jets Z+jets, WW, WZ, ZZ , single top tt Multijet Syst. unc. (muon,leading jet) φ Δ 0 0.5 1 1.5 2 2.5 3 3.5 0.4 0.6 0.8 1 1.2 1.4 1.6 Data-to-prediction ratio Syst. unc. MEASUREMENT OF THE DIFFERENTIAL CROSS … Diagonal elements of the acceptance matrices for the particle-level measurement of σðWð→eνÞ þ NjetsÞ and σðWð→μνÞ þ NjetsÞ The uncertainties reported are only TABLE V. Diagonal elements of the acceptance matrices for the particle-level measurement of dσ1=dEjet T [where σ1 ¼ Wð→lνÞþ ≥1jet] for the electron (l ¼ e) and muon (l ¼ μ) channels. The uncertainties reported are only those from Monte Carlo statistics. Wð→eνÞ channel Wð→μνÞ channel 25 GeV ≤Ejet T < 35 GeV ð35.03 0.05Þ% ð35.09 0.05Þ% 35 G V Ejet 45 G V ð35 06 0 07Þ% ð35 12 0 07Þ% FIG. 13. Detector response matrices that describe the migra- tion between bins corrected by the unfolding procedure for the measurement of dσ1=dEjet T , where σ1 ¼ Wð→lνÞþ ≥1jet (a) for the electron (l ¼ e), and (b) for the muon (l ¼ μ) channels. FIG. 13. Detector response matrices that describe the migra- tion between bins corrected by the unfolding procedure for the measurement of dσ1=dEjet T , where σ1 ¼ Wð→lνÞþ ≥1jet (a) for the electron (l ¼ e), and (b) for the muon (l ¼ μ) channels. FIG. 12. Detector response matrices that describe the migration between bins corrected by the unfolding procedure for the measurement of σðWð→lνÞ þ NjetsÞ (a) in the electron channel (l ¼ e), and (b) in the muon channel (l ¼ μ). matrices are diagonal, and the entries are listed in Tables IV and V. About 35%–50% of the particle-level events are selected in each bin. The uncertainties reported include only the statistical contribution. TABLE V. Diagonal elements of the acceptance matrices for the particle-level measurement of dσ1=dEjet T [where σ1 ¼ Wð→lνÞþ ≥1jet] for the electron (l ¼ e) and muon (l ¼ μ) channels. The uncertainties reported are only those from Monte Carlo statistics. APPENDIX B: DETECTOR RESPONSE AND ACCEPTANCE MATRICES FOR THE PARTICLE-LEVEL RESULTS They are nondiagonal matrices as shown in Figs. 12 and 13, where the fraction of detector-level events reconstructed from each particle-level bin is presented. The acceptance matrices quantify the probabilities of an event to be detected and selected, as functions of the particle-level quantities N jets and Ejet T . The acceptance The detector response matrices account for migrations of events between the bins in which the cross sections are measured and the corresponding bins at particle level. 112005-18 MEASUREMENT OF THE DIFFERENTIAL CROSS … MEASUREMENT OF THE DIFFERENTIAL CROSS … PHYS. REV. D 98, 112005 (2018) matrices are diagonal, and the entries are listed in Tables IV and V. About 35%–50% of the particle-level events are selected in each bin. The uncertainties reported include only the statistical contribution. FIG. 12. Detector response matrices that describe the migration between bins corrected by the unfolding procedure for the measurement of σðWð→lνÞ þ NjetsÞ (a) in the electron channel (l ¼ e), and (b) in the muon channel (l ¼ μ). FIG. 13. Detector response matrices that describe the migra- tion between bins corrected by the unfolding procedure for the measurement of dσ1=dEjet T , where σ1 ¼ Wð→lνÞþ ≥1jet (a) for the electron (l ¼ e), and (b) for the muon (l ¼ μ) channels. TABLE IV. Diagonal elements of the acceptance matrices for the particle-level measurement of σðWð→eνÞ þ NjetsÞ and σðWð→μνÞ þ NjetsÞ. The uncertainties reported are only statistical. Wð→ Þ channel Wð→ Þ channel TABLE V. Diagonal elements of the acceptance matrices for the particle-level measurement of dσ1=dEjet T [where σ1 ¼ Wð→lνÞþ ≥1jet] for the electron (l ¼ e) and muon (l ¼ μ) channels. The uncertainties reported are only those from Monte Carlo statistics. Wð→eνÞ channel Wð→μνÞ channel 25 GeV ≤Ejet T < 35 GeV ð35.03 0.05Þ% ð35.09 0.05Þ% 35 GeV ≤Ejet T < 45 GeV ð35.06 0.07Þ% ð35.12 0.07Þ% 45 GeV ≤Ejet T < 60 GeV ð35.58 0.08Þ% ð35.5 0.08Þ% 60 GeV ≤Ejet < 80 GeV ð36 2 0 1Þ% ð36 4 0 1Þ% matrices are diagonal, and the entries are listed in Tables IV and V. About 35%–50% of the particle-level events are selected in each bin. The uncertainties reported include only the statistical contribution. FIG. 12. Detector response matrices that describe the migration between bins corrected by the unfolding procedure for the measurement of σðWð→lνÞ þ NjetsÞ (a) in the electron channel (l ¼ e), and (b) in the muon channel (l ¼ μ). FIG. 13. Detector response matrices that describe the migra- tion between bins corrected by the unfolding procedure for the measurement of dσ1=dEjet T , where σ1 ¼ Wð→lνÞþ ≥1jet (a) for the electron (l ¼ e), and (b) for the muon (l ¼ μ) channels. TABLE IV. APPENDIX C: CROSS-SECTION TABLES The measured inclusive cross sections for each jet multiplicity and the differential cross sections with respect to the transverse energy of the leading jet are reported in Tables VI and VII, respectively. TABLE VI. Measured inclusive Wð→lνÞþ ≥Njets cross sections for each jet multiplicity. Cross sections and uncertainties are expressed in pb. σN Statistical uncertainty Systematic uncertainty Luminosity uncertainty Wð→lνÞþ ≥1jet 60.2 0.1 þ2.6 −2.8 3.6 Wð→lνÞþ ≥2jets 7.31 0.04 þ0.44 −0.46 0.44 Wð→lνÞþ ≥3jets 0.84 0.02 þ0.10 −0.10 0.05 Wð→lνÞþ ≥4jets 0.10 0.01 þ0.02 −0.02 0.01 TABLE VI. Measured inclusive Wð→lνÞþ ≥Njets cross sections for each jet multiplicity. Cross sections and uncertainties are expressed in pb. σN Statistical uncertainty Systematic uncertainty Luminosity uncertainty TABLE VI. Measured inclusive Wð→lνÞþ ≥Njets cross sections for each jet multiplicity. Cross sections and uncertainties are expressed in pb. σN Statistical uncertainty Systematic uncertainty Luminosity uncertainty Wð→lνÞþ ≥1jet 60.2 0.1 þ2.6 −2.8 3.6 Wð→lνÞþ ≥2jets 7.31 0.04 þ0.44 −0.46 0.44 sive Wð→lνÞþ ≥Njets cross sections for each jet multiplicity. Cross sections and uncertainties are ð→lνÞþ ≥Njets cross sections for each jet multiplicity. Cross sections and uncertainties are TABLE VII. Measured differential cross section dσ1=dEjet T for each Ejet bin. Cross sections and uncerta TABLE VII. Measured differential cross section dσ1=dEjet T for each Ejet bin. Cross sections and uncertainties are expressed in pb/GeV. dσ1=dEjet T Statistical uncertainty Systematic uncertainty Luminosity uncertainty 25 GeV ≤Ejet T < 35 GeV 2.763 0.007 þ0.120 −0.121 0.166 35 GeV ≤Ejet T < 45 GeV 1.331 0.004 þ0.055 −0.055 0.080 45 GeV ≤Ejet T < 60 GeV 0.628 0.003 þ0.026 −0.026 0.038 60 GeV ≤Ejet T < 80 GeV 0.259 0.002 þ0.011 −0.011 0.016 80 GeV ≤Ejet T < 110 GeV 0.095 0.001 þ0.0038 −0.0039 0.005 110 GeV ≤Ejet T < 150 GeV 0.0243 0.0004 þ0.0011 −0.0011 0.0015 150 GeV ≤Ejet T < 200 GeV 0.0055 0.0001 þ0.0003 −0.0003 0.0004 200 GeV ≤Ejet T < 300 GeV 0.00075 0.00002 þ0.00004 −0.00004 0.00005 [1] Transverse energy (momentum) is the energy (momentum) projected into the plane transverse to the beam direction; a detailed explanation is given in Sec. II. [7] R. Aaij et al. (LHCb Collaboration), Measurement of forward W and Z boson production in association with jets in proton-proton collisions at ﬃﬃﬃs p ¼ 8 TeV, J. High Energy Phys. 05 (2016) 131. [2] T. Aaltonen et al. MEASUREMENT OF THE DIFFERENTIAL CROSS … Wð→eνÞ channel Wð→μνÞ channel 25 GeV ≤Ejet T < 35 GeV ð35.03 0.05Þ% ð35.09 0.05Þ% 35 GeV ≤Ejet T < 45 GeV ð35.06 0.07Þ% ð35.12 0.07Þ% 45 GeV ≤Ejet T < 60 GeV ð35.58 0.08Þ% ð35.5 0.08Þ% 60 GeV ≤Ejet T < 80 GeV ð36.2 0.1Þ% ð36.4 0.1Þ% 80 GeV ≤Ejet T < 110 GeV ð38.5 0.2Þ% ð38.4 0.2Þ% 110 GeV ≤Ejet T < 150 GeV ð41.7 0.3Þ% ð41.7 0.3Þ% 150 GeV ≤Ejet T < 200 GeV ð45.4 0.6Þ% ð45.5 0.6Þ% 200 GeV ≤Ejet T < 300 GeV ð49.5 1.3Þ% ð51.4 1.3Þ% TABLE IV. Diagonal elements of the acceptance matrices for the particle-level measurement of σðWð→eνÞ þ NjetsÞ and σðWð→μνÞ þ NjetsÞ. The uncertainties reported are only statistical. Wð→eνÞ channel Wð→μνÞ channel N ¼ 1jet ð35.33 0.04Þ% ð35.38 0.04Þ% N ¼ 2jets ð36.9 0.1Þ% ð37.0 0.1Þ% N ¼ 3jets ð37.4 0.2Þ% ð37.6 0.2Þ% N ≥4jets ð38.3 0.3Þ% ð38.2 0.2Þ% 112005-19 T. AALTONEN et al. PHYS. REV. 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https://openalex.org/W4392763102	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0299204&type=printable	English	null	SAQ training on sprint, change-of-direction speed, and agility in U-20 female football players	PloS one	2,024	cc-by	12,420	SAQ training on sprint, change-of-direction speed, and agility in U-20 female football players Young-Soo Lee1, Dayoung LeeID2, Na Young Ahn3 1 Department of Physical Education, Sejong University, Seoul, South Korea, 2 Department of Sport Science, Kangwon National University, Chuncheon, South Korea, 3 Department of Sport and Event Management, Bournemouth University, Poole, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ldy227@kangwon.ac.kr * ldy227@kangwon.ac.kr Editor: Luca Paolo Ardigò, Universita degli Studi di Verona, ITALY Editor: Luca Paolo Ardigò, Universita degli Studi di Verona, ITALY Editor: Luca Paolo Ardigò, Universita degli Studi di Verona, ITALY Received: April 7, 2022 Accepted: February 6, 2024 Published: March 13, 2024 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0299204 Abstract The purpose of the study was to investigate the effects of an 8-week speed, agility, and quickness (SAQ) training on performance changes in linear sprint speed, change-of-direc- tion (COD) speed, and reactive agility of U-20 female football players. Nineteen female foot- ball players randomly served as either experimental (n = 9) or control groups (n = 10). The players were tested for physical fitness tests: linear sprint speed including both short and long distances (5- and 10-m sprints without a ball and 20- and 30-m sprints with and without dribbling), COD speed (arrowhead agility test with and without dribbling a ball, Southeast Missouri [SEMO] agility test, and 22-m repeated slalom dribbling test), and reactive agility. Significant group × time interactions were observed for sprint over long distances and COD speed but not for short sprint and reactive agility performances. Paired t-tests revealed con- siderable improvements in all performances from the pre-test to post-test for the SAQ group, except for the arrowhead agility (left; p = .07). The control group only exhibited signifi- cant improvements in 10-m sprint performance after general football training. Eight weeks of SAQ training were effective at enhancing acceleration, maximum sprint speed, and agility performances amongst highly trained U-20 female football players. OPEN ACCESS Citation: Lee Y-S, Lee D, Ahn NY (2024) SAQ training on sprint, change-of-direction speed, and agility in U-20 female football players. PLoS ONE 19(3): e0299204. https://doi.org/10.1371/journal. pone.0299204 PLOS ONE RESEARCH ARTICLE PLOS ONE PLOS ONE Introduction Copyright: © 2024 Lee 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. Football is a physically intense sport with intermittent and rapid actions. For example, pro- fessional players engage in 5–6 training sessions and play 1–2 games a week during a season [1]. During a game, players run approximately 10 km involving varying levels of intensity and sprint every 70 seconds at a high-intensity [2]. They also constantly carry out dynamic and brief actions (e.g., passing, kicking, trapping, dribbling, tackling) and without a ball (e.g., high-speed running, accelerations, decelerations, and change-of-direction [COD]) [3–5]. Given the demands, players are required to possess multifaceted physical features. These include muscular strength and endurance, repeated sprint ability, sprinting speed, agility, to name a few, to perform high intensity actions with and without possession of a ball through- out a game [6–8]. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: The authors received no specific funding for this work. 1 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Researchers have emphasised the significance of players’ high-speed movements, charac- terised by constant changes in velocity and direction, which account for less than 12% of the overall football performance; these movements in turn greatly influence the outcome of a game [2,9]. Acceleration and deceleration speed, maximum sprint speed, COD speed, and reactive agility are the contributing factors to high-intensity performances during a game. Researchers have emphasised the significance of players’ high-speed movements, charac- terised by constant changes in velocity and direction, which account for less than 12% of the overall football performance; these movements in turn greatly influence the outcome of a game [2,9]. Acceleration and deceleration speed, maximum sprint speed, COD speed, and reactive agility are the contributing factors to high-intensity performances during a game. Acceleration and deceleration speed involves sprinting momentum over linear course (10- to 30-m), where the maximum acceleration speed can occur during the early stages of sprint start (0- to 5-m). Introduction COD speed is another aspect of agility that requires players to coordinate pre-planned and sports-specific movements maintaining body control [10]. While COD speed relies on players’ physical capacity to change directions, agility requires players to rap- idly process and react to diverse stimuli, performing perceptual and cognitive processing [11]. Hence, these high-speed actions are independent qualities and unrelated to each other [10,12–13]. For instance, some players excel in sprinting may have weaknesses in decision- making or precise reaction to stimuli, whereas others may maintain maximum sprint speed and control during sudden changes in direction, accelerations, and decelerations [11]. As a result, coaches and researchers strive to develop appropriate training regimes that strengthen the incorporated effects of acceleration and deceleration speed, COD speed, and agility. This approach aims to achieve high levels of physical performance in complex and dynamic movements [14]. SAQ training was specifically designed to enhance players’ COD speed and agility perfor- mances and improve high-speed running and sprint capabilities during a game [15]. Combin- ing a range of movement tasks, including lateral shuffles, forward and backward running, and ladder drills, SAQ training enables players to enhance their rapid transitions, motor coordina- tion, and reaction time. In other words, SAQ training focuses on performing movement tasks at a high rate in short-time (quickness) incorporated both straight-line speed and multidirec- tional sprints (COD speed) across various distances to enhance maneuverability [16]. Researchers have thus integrated SAQ training into football-specific exercises and condition- ing programmes and examined its effects on physical performance of football players [7,8,12,17–20]. In their study of young football players, Jovanovic and associates [12] found that an 8-week SAQ training programmes implemented during the season resulted in improved acceleration and short sprint performance compared to the group without the inter- vention. Milanović and associates [7] also found that a 12-week combined SAQ and flexibility training programms led to significant enhancements in speed, especially at short distances of 5 and 10 m, compared to a group that underwent regular training only. Furthermore, Formenti and colleagues stressed that SAQ training with a combination of closed (sprinting and COD) and open (balance and reactive movements) drills is effective when both football-specific and non-sport specific stimuli are incorporated, considering youth players’ perceptual response [21]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Introduction Finally, Azmi and Kusnani [22] trained football players for 8 weeks and concluded that the SAQ training method effectively improved sprint, agility, reaction time, and power in foot- ball players. SAQ training was specifically designed to enhance players’ COD speed and agility perfor- mances and improve high-speed running and sprint capabilities during a game [15]. Combin- ing a range of movement tasks, including lateral shuffles, forward and backward running, and ladder drills, SAQ training enables players to enhance their rapid transitions, motor coordina- tion, and reaction time. In other words, SAQ training focuses on performing movement tasks at a high rate in short-time (quickness) incorporated both straight-line speed and multidirec- tional sprints (COD speed) across various distances to enhance maneuverability [16]. y Although Yap and colleagues [23] have highlighted the importance of developing physical fitness, such as sprint speed and agility, for both female and male players to excel in high- intensity actions during a game, much of the research in this area has focused on male football players. To our knowledge, only a few researchers have empirically tested the propositions of Yap and Colleagues [23] showing positive outcomes of the SAQ exercises among female foot- ball players, both in youth [24] and adult [25] categories. Polman and associates [25] highlighted as much in their study of female football players, arguing that SAQ training using speed-ladder, hurdles, and reaction balls improved the physical and biomechanical character- istics of female football players. Considering physical qualities are cultivated from a young age, Although Yap and colleagues [23] have highlighted the importance of developing physical fitness, such as sprint speed and agility, for both female and male players to excel in high- intensity actions during a game, much of the research in this area has focused on male football players. To our knowledge, only a few researchers have empirically tested the propositions of Yap and Colleagues [23] showing positive outcomes of the SAQ exercises among female foot- ball players, both in youth [24] and adult [25] categories. Polman and associates [25] highlighted as much in their study of female football players, arguing that SAQ training using speed-ladder, hurdles, and reaction balls improved the physical and biomechanical character- istics of female football players. Participants Nineteen female football players (age = 18.89 ± 0.80 yr; height = 164.54 ± 4.11 cm; body mass = 58.28 ± 6.57 kg; and body mass index or BMI = 21.52 ± 3.46 kg/m2) competing at the national level volunteered to participate in the study. Prior to conducting the study, a power analysis was performed using GPower software [28]. The analysis indicated that for a two- tailed t-test with moderate effect size of .3, power of 80%., and alpha level at .05, a minimum sample size of 176 per group was recommended. However, due to the scarcity of highly trained players, our participant pool for the study was smaller. Further, while an experimental design with equivalent group is ideal to compare pre-test and post-test effects, practical difficulties led us utilise a nonequivalent control group, with the exception of one player in the study. All play- ers were members of a single regional intercollegiate football team registered at a local football association and had at least 5 years of football experience. None of the players had had any seri- ous injuries 6 months prior to the initial testing, and none had ever participated in SAQ train- ing. Based on the uneven and even numbers of player jerseys, each player was randomly assigned to either an SAQ training group (SAQ; n = 9; age = 19 ± 0.86 yr; height = 165.62 ± 4.10 cm; body mass = 59.91 ± 7.78kg; and BMI = 21.84 ± 4.62 kg/m2) or general training group (GTG; n = 10; age = 18.80 ± 0.78 yr; height = 163.58 ± 4.09 cm; body mass = 56.83 ± 5.26kg; and BMI = 21.23 ± 2.19 kg/m2). No large changes in body mass, height, or injuries were observed over the duration of the study. Table 2 presents the descriptive data of the SAQ and GTG groups. The study was approved by the ethics committee of Sejong University to ensure the safety and privacy of human subjects. Before providing written consent, all players were informed of the potential risks and benefits of participation and that they could withdraw from the study at any time. Introduction Considering physical qualities are cultivated from a young age, PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 2 / 25 PLOS ONE SAQ such information holds particular significance for young female players aiming to improve sustainable high-intensity running over long distances and COD speed. Further, despite the importance of and benefits from SAQ training, little is known about its effects in highly trained female youth football players, with an 8-week intervention [see 26 for classification of athletes]. Previous studies on male football players have reported notable improvements in short sprint performance and COD ability throughout the 8 weeks of SAQ training [12,22]. Similarly, Mathisen and Svein [24] found that SAQ training over the course of 8 weeks improved linear sprint speed and agility performance in female youth football players. When implementing a training method, 5–6 weeks are enough to induce significant physiological and neuromuscular adaptations in highly trained football players during pre-season and in-season [19,27]. Given the limited empirical evidence, the aim of the study was to investigate the effects of an 8-week SAQ training in the selected physical capabilities of female football players. We hypothesised that implementing the SAQ training over 8 weeks will improve linear sprint speed, COD speed, and reactive agility. Training Over the 8 weeks, all players performed either the SAQ training protocol or substitute drills involving ball skills, such as dribbling, passing, and shooting. These trainings occurred 3 times a week for 40 minutes per session, with at least 24 hours of recovery between each conditioning session, and the sessions were scheduled 2–3 days apart. Both groups of players underwent the same volume of a specific physical conditioning (16 hours) but at a different level of intensity. The experimental group performed the SAQ training with a progressive intensity from 80% to 100%, whereas the control group exercised normal football drills at various intensities within the range of 80% to 100%. The researchers ensured that all players reached at least 80% of intensity during conditioning sessions. Following this, both groups maintained the same vol- ume and intensity for the remaining practice. Each training session lasted up to 120 minutes and consisted of the programmed conditioning training, technical and tactical drills, and small-sided games or match-play. Regarding the intensity of the training sessions, we consis- tently monitored the participants’ heart rate (HR) and rating of perceived exertion (RPE). However, we did not document these records as they were not within the scope of the study. The overview of the 8-week SAQ training, along with the programmes for the control group, is presented in Table 1. The overview of the 8-week SAQ training, along with the programmes for the control group, is presented in Table 1. Design and procedures In a two-group, randomised controlled design, the effects of an 8-week SAQ training on accel- eration, linear sprint speed, COD speed, and reactive agility of female football players were assessed by comparing two experimental conditions: an intervention and control group. Per- formance was evaluated using the following tests: 5- and 10-m sprints without a ball and 20- and 30-m sprints with and without dribbling a ball for speed parameters; arrowhead agility tests with and without dribbling a ball, the Southeast Missouri (SEMO) agility test, and 22-m PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 3 / 25 PLOS ONE SAQ repeated slalom dribble test for COD speed parameters; and reaction agility test for agility assessment. repeated slalom dribble test for COD speed parameters; and reaction agility test for agility assessment. All experimental procedures took place for 8 consecutive weeks, from February (the pre- season) to April (the beginning of the in-season) in 2018. All physical conditioning and tests took place on a full-size (90 x 120 m) outdoor artificial turf pitch between 3:30 PM to 5:30 PM and were guided by experienced football coaches certified by a continental football asso- ciation. All players were familiarised with the different training protocols and testing proce- dures prior to the initiation of the study and wore football boots throughout the training and testing sessions. To ensure consistency, all players were instructed to maintain normal die- tary intakes consisting of a standardised breakfast, lunch, and dinner, as well as their regular lifestyles during the study. This approach aimed to minimise any uncontrolled impacts on the results. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Testing protocol Performance testing was conducted twice, before and after the 8-week SAQ training during regular training hours, with a 24-hour recovery between the training and testing. Prior to test- ing, all players performed a standard warm-up, consisting of 10–15 minutes of jogging, dynamic stretching with progressive speed runs, and ball contact exercises. The measurements included acceleration and maximum sprint speed, COD speed, and reactive agility. Each player was instructed to give their maximal effort during performance testing. All players com- pleted a maximum of 2 trials for every measure to become familiar themselves with a battery of physical fitness tests, with a minimum of 5 minutes of recovery time between each test. At the end of testing, all players performed the same cool-down for 10–15 minutes involving low- intensity running and static stretching. On the first day of testing, the body mass and height of each player was measured using a stadiometer and electronic scale (FA-94H, Fanics and DB- 150A, CAS, South Korea, respectively). The BMI was also calculated as weight/height squared (kgm–2), and there were no significant differences between the groups on anthropometric data. Speed testing. Speed refers to one’s ability to move a body as fast as possible, which is often measured by linear sprinting speed [29]. Whilst 5- to 10-m sprints are considered as PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 4 / 25 PLOS ONE SAQ Table 1. An overview of the 8-week SAQ and general training programmes. Weeks SAQ Training General Training 1–2 Intensity 80% Speed Wall Drill 3 Count–A Skip, A Run, and B Run COD Speed Hop Scotch Agility Ladder Drill Reactive Agility Reaction Ball Drop Passing With inside and laces of a foot or head On and partly above the ground Driven and lofted passes (with a spin) In pairs or groups of 3–6 1–3 touches Moving to receive the second ball Ball Controlling With inside, outside, and sole of a foot for receiving ground balls With feet, thigh, chest, and head With a 90˚ or 180˚ turn In pairs or groups of 3–4 In a fluid motion Dribbling With inside and outside of a foot Fake moves–Step-Over, Scissors, etc. In a different pace In a different direction–forward, etc. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Testing protocol Turning and cutting At speed Kicking With inside, outside, laces of a foot Chipping and crossing Short and long distances In pairs or groups of 3–4 Shooting With inside and outside of a foot or head With instep drive on a half or side volley In the near, middle, and far post From distance and difficult positions Quick turn towards a goal Solo and combination play to finish 1–2 touches 3–4 Intensity 90% Speed Speed Ladder Drill Speed Training Band COD speed Agility Ladder Drill Reactive Agility Mirror Game 5–6 Intensity 90% Speed Speed Hurdle Drill COD speed Slalom Sprint Agility Hurdle Drill Reactive Agility Reaction Training 7–8 Intensity 100% Speed Sprint Shuttle Run COD Speed Illinois Agility Run T-Drill Reactive Agility Reaction Sprint Note: SAQ = speed, agility, and quickness. COD = change-of-direction. https://doi.org/10.1371/journal.pone.0299204.t001 Table 1. An overview of the 8-week SAQ and general training programmes. indicators of acceleration speed [8,19], sprints over 10 m are used to assess high-running veloc- ity. In football, where the average sprint distance is around 20 m and rarely exceeds 30 m [30], maximum sprint speed was measured by 20- and 30-m sprints [31]. To evaluate players’ sprinting capabilities, all players performed 12 straight-line sprints: two trials over 5-, 10-, 20-, and 30-m without dribbling a ball, and another 2 trials while dribbling a ball over each distance of 20 and 30 m. 5-m and 10-m Sprint Tests. All players began by standing upright, with the toe of the front foot positioned right behind the start line. The players sprinted as quickly as possible through the end line and rested 2 minutes in between. The same starting and recovery strat- egies were used for subsequent analysis. Sprint times were recorded by the first author using timing gates placed on the start and finish lines in units of 0.01 seconds (Weltek, TK- 9920, Star Sports, South Korea), and the best record for each test was attained for data analysis. 20-m and 30-m Sprint Tests. All players completed two sets of maximal straight sprints without a ball over 20- and 30-m, with the same starting and recovery strategies as employed in the short sprint tests. The same photoelectric cells were used to measure maximum sprint speed, and the best result for each test was retained for further analysis. Testing protocol 5 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ 20-m and 30-m Sprint Tests with a Ball. The dribbling speed test reflects the entities of maxi- mum sprint speed and dribbling skill under time pressure. All players were instructed to drib- ble a standard ball to the finish point, while maintaining maximum velocity throughout. The same starting, recovery, and recording strategies were adopted from the previous sprint tests. To ensure validity, a trial was considered valid when the players completed at least 4 touches for 20-m and 6 touches for 30-m sprint [31]. Otherwise, the trial was discarded and reat- tempted, and the fastest time was taken for further analysis. COD speed and agility testing. Agility is defined as one’s ability to quickly change direc- tion in response to a stimulus (reactive agility), while COD speed coordinates pre-planned, sports-specific movements, maintaining body control [10]. Following the speed testing, all players underwent the arrowhead agility test, both with and without dribbling, as well as the SEMO agility test and the 22-m repeated slalom dribbling test, which are highly reliable and widely used to measure COD speed [32,33]. When testing, the same procedure was used for the starting position and recovery phase as in the speed testing (i.e., a 2-min rest interval between 2 trials and a 5-min between each test), with the exception of the reactive agility test. COD Speed Tests. To measure COD speed without a ball, all players were instructed to sprint and perform COD movements maximally through a certain sequence of cones as depicted in Figs 1 and 2. Fig 1. Arrowhead agility test. https://doi.org/10.1371/journal.pone.0299204.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 6 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ Fig 2. SEMO agility test. https://doi org/10 1371/journal pone 0299204 g002 https://doi.org/10.1371/journal.pone.0299204.g002 The players completed the arrowhead agility test twice, both in the left and right directions [see 33]. For the SEMO agility test, the players were required to perform forward sprinting, lat- eral shuffling, and diagonal backpedaling while turning around the cones [see 34]. Six trials without dribble were completed, and the attempt was considered invalid if the players stepped on, moved over, or knocked down a cone. Each trial was timed by the first author using a handheld stopwatch with a precision of 0.01 seconds [35], and the fastest times were recorded for data analysis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Testing protocol COD Speed Tests with a Ball. The dribbling agility test evaluates both COD speed and drib- bling performance under control. Each player performed 4 trials of the arrowhead agility test, dribbling a ball through each direction (Fig 1). For the 22-m repeated slalom dribble test [see 32 for details], the players were instructed to dribble slalom as quickly as possible through a series of 14 cones placed 2 m apart, as shown in Fig 3. 7 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ Fig 3. 22-m slalom repeated dribble test. https://doi.org/10.1371/journal.pone.0299204.g003 Fig 3. 22-m slalom repeated dribble test. https://doi.org/10.1371/journal.pone.0299204.g003 Upon reaching the last cone, the players made a 180º turn and dribbled back through the cones to the starting line. The trial was disregarded and reattempted if the players disrupted a cone. Each player completed 6 trials, and all tests were conducted by the same rater using a handheld stopwatch, and the best time was attained for further analysis. Reactive Agility Testing. Reactive agility refers to unplanned, dynamic movement patterns in response to sport-specific cues [20]. These tasks involve open skill agility, requiring players to quickly and efficiently read and react in a game situation, to take an appropriate action based on stimuli [11]. Following the completion of COD speed testing, all players were asked to sprint as fast as possible to assess how promptly they produce unplanned directional changes while processing sport-specific cues. The testing protocol was adopted from previous research, as depicted in Fig 4 [see 20]. On command, the players moved 5 m forward, passing through a trigger gate at a maximal speed. Upon passing through the trigger gate, they changed direction either to the left or right side based on cues provided by the researcher. The players then performed a 45º cut and sprinted an additional 5 m through a target gate while still maintaining their full speed. Each player completed 2 trials, with a 3-minute rest between each attempt. The same method was used for the starting position as used in the speed testing. Times from the start to finish lines were measured by the same researcher using a handheld stopwatch, and the best result was used for data analysis. Statistical analyses Data are presented as mean ± standard deviation. The data were stored in Microsoft Excel and analysed using the R language (version 4.2.3 by R Core Team, Vienna, Austria) with R Studio (version 2023.09.0+463; R Studio PBC, Boston, USA). Our final dataset is presented in S1 File. The assumptions of normality and homogeneity of variances were checked by Shapiro-Wilk and Levene’s test on the residual terms of the change scores, while the sphericity assumption was not applicable because there were only two levels of the within-subjects factor (i.e., time). For the inferential analysis, two-way (group × time) mixed factorial analyses of variance PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 8 / 25 PLOS ONE SAQ Fig 4. Reaction agility test. https://doi.org/10.1371/journal.pone.0299204.g004 https://doi.org/10.1371/journal.pone.0299204.g004 (ANOVA) were employed to investigate both the interactions and main effects of training and time. In case of a significant interaction, pairwise comparisons were not applied because the factors entailed only two levels. Instead, independent or paired t-tests were conducted to deter- mine any significant differences between the two groups or test occasions within each group. For specific conditions that did not satisfy the homogeneity of variance, the Mann-Whitney test was used to assess significant differences between the groups during the post-training assessment. Cohen’s d and partial eta squared (η2) were used to determine the magnitude of mean differences in t-tests and ANOVAs, respectively. Effect sizes were evaluated based on the following criteria [36]: values of .2, .5, and .8 for small, medium, and large effects of d, and .01, .06, and .14 considered as the cut-off for small, medium, and large values of η2, respectively. Statistical significance was set at p < .05. Table 2 presents the summary statistics for all vari- ables used in the study. (ANOVA) were employed to investigate both the interactions and main effects of training and time. In case of a significant interaction, pairwise comparisons were not applied because the factors entailed only two levels. Instead, independent or paired t-tests were conducted to deter- mine any significant differences between the two groups or test occasions within each group. For specific conditions that did not satisfy the homogeneity of variance, the Mann-Whitney test was used to assess significant differences between the groups during the post-training assessment. Cohen’s d and partial eta squared (η2) were used to determine the magnitude of mean differences in t-tests and ANOVAs, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE PLOS ONE SAQ Table 2. Changes in measure with confidence intervals and correlation matrix. PLOS ONE Changes in measure with confidence intervals and correlation matrix. i bl Ch i Note: SAQ = speed, agility, and quickness. GTG = general training group. n = sample size. LL = lower limit. UL = upper limit. BMI = body mass index. COD = change- of-direction. SEMO = Southeast Missouri. L = left. R = right. The values in the brackets represent the 95% confidence interval (CI) for each change in measure for both groups and mean difference between the groups. p < .05. https://doi.org/10.1371/journal.pone.0299204.t002 Note: SAQ = speed, agility, and quickness. GTG = general training group. n = sample size. LL = lower limit. UL = upper limit. BMI = body mass index. COD = change- of-direction. SEMO = Southeast Missouri. L = left. R = right. The values in the brackets represent the 95% confidence interval (CI) for each change in measure for both groups and mean difference between the groups. p < 05 PLOS ONE variables Change in measure SAQ (n = 9) [LL, UL] GTG (n = 10) [LL, UL] Mean Difference [LL, UL] 1 2 3 4 5 6 7 8 9 10 11 12 13 Age (y) 19.00 ± .86 18.80 ± .78 Height (cm) 165.62 ± 4.10 163.58 ± 4.09 Body mass (kg) 59.91 ± 7.78 56.83 ± 5.26 BMI (kg/m–2 ) 21.8 ± 4.62 21.2 ± 2.19 1 5-m sprint -.10 ± .07 [-.16, -.05] -.03 ± .10 [-.11, .04] -.06 ± .09 [-11, -.02] — 2 10-m sprint -.16 ± .09 [-.23, -.08] -.09 ± .10 [-.16, -.01] -.12 ± .10 [-.17, -.07] -.09 — 3 20 m sprint -.16 ± .15 [-.28, -.04] .06 ± .12 [-.02, .16] -.04 ± .18 [-.12, .04] .23 .15 — 4 30-m sprint -.25 ± .15 [-.37, -.14] .02 ± .21 [-.12, .17] -.10 ± .23 [-.21, .01] .68 * .39 .41 — 5 20-m dribble -.21 ± .14 [-.32, -.10] .01 ± .21 [-.14, .15] -.09 ± .20 [-.19, .01] .37 -.02 .43 .40 — 6 30-m dribble -.24 ± .24 [-.43, -.05] .08 ± .24 [-.09, .25] -.07 ± .29 [-.21, .07] -.15 .57 * .24 .16 .17 — 7 Arrowhead agility (L) -.11 ± .16 [-.24, .01] .26 ± .27 [.06, .46] .08 ± .29 [-.05, .22] .37 -.10 .72 * .42 .67 .02 — 8 Arrowhead agility (R) -.21 ± .13 [-.31, -.11] .15 ± .27 [-.03, .35] -.02 ± .28 [-.15, .11] .57 * -.07 .47 * .45 .56 * .13 .80 *** — 9 SEMO agility -.64 ± .37 [-.93, -.35] .56 ± .47 [.22, .90] -.01 ± .74 [-.36, .35] .49 * .02 .64 .61 .48 * .27 .74 * .77 * — 10 Arrowhead dribble (L) -.25 ± .24 [-.44, -.06] .49 ± .46 [.15, .82] .13 ± .53 [-.11, .39] .17 .36 .42 .29 .54 * .55 * .34 .45 * .54 * — 11 Arrowhead dribble (R) -.25 ± .17 [-.38, -.11] .53 ± .46 [.20, .86] .16 ± .53 [-.09, .42] .36 .20 .43 .54 * .37 .22 .65 .66 .55 * .35 — 12 22-m slalom dribble -1.82 ± .80 [-2.44, -1.20] -.01 ± .71 [-.51, .50] -.86 ± 1.19 [-1.44, -.29] .44 .20 .39 .55 * .48 * .47 * .43 .57 * .58 ** .54 * .68 — 13 Reactive agility -.13 ± .09 [-.20, -.06] -.05 ± .22 [-.22, .10] -.09 ± .17 [-.18, -.01] -.06 -.04 .36 -.12 .11 .12 .34 .34 .18 -.01 .16 .22 — Table 2. Statistical analyses Effect sizes were evaluated based on the following criteria [36]: values of .2, .5, and .8 for small, medium, and large effects of d, and .01, .06, and .14 considered as the cut-off for small, medium, and large values of η2, respectively. Statistical significance was set at p < .05. Table 2 presents the summary statistics for all vari- ables used in the study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 9 / 25 PLOS ONE SAQ Table 3. Results of two-way mixed analyses of variances. DVs 2 × 2 mixed ANOVA (df1 = 1; df2 = 17) Main Effects Interaction Group Time Group × Time F p(partial η2) F p(partial η2) F p(partialη2) Speed 5-m sprint .04 .83(.01) 11.06 .01(.39) 2.93 .10(.14) 10-m sprint 1.60 .22(.08) 30.88 .01(.64)* 2.15 .16(.11) 20 m sprint 0.47 .50(.02) 2.23 .15(.11) 13.45 .01(.44) 30-m sprint 1.82 .19(.09) 7.30 .01(.30)* 11.25 .01(.39)** 20-m dribble 3.06 .09(.15) 6.00 .02(.26)* 6.82 .01(.28)* 30-m dribble .54 .47(.03) 1.95 .18(.10) 8.33 .01(.32)* COD Speed Arrowhead agility (L) 1.00 .33(.05) 1.95 .18(.10) 12.56 .01(.42) Arrowhead agility (R) .01 .91(.01) .37 .54(.02) 14.27 .01(.45) SEMO agility .03 .85(.01) .16 .69(.01) 37.78 .01(.69)* Arrowhead dribble (L) 8.85 .01(.34) 1.84 .19(.09) 18.47 .01(.52)* Arrowhead dribble (R) .15 .69(.01) 3.00 .10(.15) 23.12 .01(.57)* 22-m slalom dribble .06 .80(.01) 27.64 .01(.61)* 27.56 .01(.61)* Reactive Agility Reactive agility 1.98 .17(.10) 5.69 .02(.25)* .89 .35(.05) Note: ANOVA = analysis of variance df = degrees of freedom DV = dependent variable COD = change-of-direction SEMO = Southeast Missouri L = left R = right Table 3. Results of two-way mixed analyses of variances. Times are expresses in second. https://doi.org/10.1371/journal.pone.0299204.t003 Speed All group × time interactions were statistically significant for longer linear sprints, with large effects on speed (Table 3 and Figs 5–8). All group × time interactions were statistically significant for longer linear sprints, with large effects on speed (Table 3 and Figs 5–8). The main effect of time was also statistically significant for 5-, 10-, and 30-m sprints without a ball, as well as 20-m sprints with dribbling, with large effects. However, there was no signifi- cant effect on performance based on the group. Paired t-tests demonstrated significant improvements in all speed measures in the SAQ group after 8 weeks of training. On the other hand, the control group only showed greater reductions in 10-m sprint time after regular foot- ball training [see Table 4]. Further analysis showed that the SAQ group had significantly better performance in all speed measures at the post-testing, except for 5- and 10-m sprints, with large effects ranging from 1.05 to 1.42, compared with the control group. grees of freedom. DV = dependent variable. COD = change-of-direction. SEMO = Southeast Missouri. L = left. R = right. Results With the normality assumption confirmed, the independent t-tests were conducted to com- pare the means of the pre-test between the two groups. The results showed that the groups were equal (all p > .05), except for the 22-m repeated dribble test. To assess the training effect of this variable, the Mann-Whitney test was used to evaluate the differences between the groups at the post-test. 10 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 The results of the mixed-model ANOVA revealed that the group × time interactions were sig- nificant for all other measures, with large effects (Table 3 and Figs 9–13). Subsequent analysis using paired t-tests revealed significant improvements in all COD speed measures, except for the arrowhead agility (left) in the SAQ group. In contrast, the control group showed significant increases in testing time in the arrowhead agility (left), the Note: ANOVA = analysis of variance. df = degrees of freedom. DV = dependent variable. COD = change-of-direction. SEMO = Southeast Missouri. L = left. R = right. Fig 5. Interaction plot for 20-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m sprint (p < .01) during the 8-week training period, while no main effects were found. Post hot analysis showed a greater improvement in the SAQ group compared to the GTG group (p < .01). https://doi.org/10.1371/journal.pone.0299204.g005 Fig 5. Interaction plot for 20-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m sprint (p < .01) during the 8-week training period, while no main effects were found. Post hot analysis showed a greater improvement in the SAQ group compared to the GTG group (p < .01). https://doi.org/10.1371/journal.pone.0299204.g005 Fig 5. Interaction plot for 20-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m sprint (p < .01) during the 8-week training period, while no main effects were found. Post hot analysis showed a greater improvement in the SAQ group compared to the GTG group (p < .01). https://doi.org/10.1371/journal.pone.0299204.g005 SEMO agility, and arrowhead dribbling tests for both sides, with large effects (Table 4). Finally, the independent t-tests indicated that performance improved in the SEMO agility and the arrowhead agility with dribbling (left) in the SAQ group at the post-test. The Mann- Whitney test also revealed that the post-test scores of the 22-m slalom dribbling were signifi- cantly lower in the SAQ group compared to the control group (U = 16, z = -2.36, p = 0.01; Table 4). SEMO agility, and arrowhead dribbling tests for both sides, with large effects (Table 4). Finally, the independent t-tests indicated that performance improved in the SEMO agility and the arrowhead agility with dribbling (left) in the SAQ group at the post-test. The Mann- Whitney test also revealed that the post-test scores of the 22-m slalom dribbling were signifi- cantly lower in the SAQ group compared to the control group (U = 16, z = -2.36, p = 0.01; Table 4). COD speed The results of the mixed-model ANOVA revealed that the group × time interactions were sig- nificant for all other measures, with large effects (Table 3 and Figs 9–13). Subsequent analysis using paired t-tests revealed significant improvements in all COD speed measures, except for the arrowhead agility (left) in the SAQ group. In contrast, the control group showed significant increases in testing time in the arrowhead agility (left), the PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 11 / 25 PLOS ONE SAQ Fig 5. Interaction plot for 20-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m sprint (p < .01) during the 8-week training period, while no main effects were found. Post hot analysis showed a greater improvement in the SAQ group compared to the GTG group (p < .01). https://doi.org/10.1371/journal.pone.0299204.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Reactive agility A 2 × 2 mixed ANOVA was conducted to analyse the reactive agility test results. The analysis revealed that the group × time interaction was not significant (Table 3), while there was a sig- nificant main effect of time, with large effects, but not for the effect for group. A paired t-test was run to further explore the differences between the two testing sessions for each group. Sig- nificant enhancements were apparent in the reactive agility test for the SAQ group after train- ing, while no significant differences were found for the control group. Lastly, an independent t-test indicated no significant differences between the groups (Table 4). PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 12 / 25 PLOS ONE SAQ Fig 6. Interaction plot for 30-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m sprint (p < .01) during the 8-week training period, while the main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g006 Fig 6. Interaction plot for 30-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m sprint (p < .01) during the 8-week training period, while the main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g006 Fig 6. Interaction plot for 30-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m sprint (p < .01) during the 8-week training period, while the main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g006 Fig 6. Interaction plot for 30-m sprint. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m sprint (p < .01) during the 8-week training period, while the main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g006 https://doi.org/10.1371/journal.pone.0299204.g006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Discussion The purpose of the study was to evaluate the effects of implementing an 8-week traditional and ball-oriented training combined with SAQ training on high-intensity performance in U- 20 female football players competing at the national level. The main findings were as that: (a) the players in the SAQ group significantly enhanced sprint speed over long distances and COD ability, compared to those in the control group, and (b) the SAQ training programme resulted in greater improvements in all tested variables from the pre-test to the post-test, except for the arrowhead agility test (left). Although some significant changes occurred between the pre-test and the post-test for the control group, the players in this group actually increased testing time in the arrowhead agility (left), arrowhead agility with dribble for both sides, and the SEMO agility, with the only exception of a decrease in 10-m sprint time. Therefore, the results of this study demonstrate the benefits of combining SAQ training with regular football practices for improving sprinting speed, COD speed, and reactive agility per- formance of players. Therefore, the results of this study demonstrate the benefits of combining SAQ training with regular football practices for improving sprinting speed, COD speed, and reactive agility per- formance of players. 13 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ Fig 7. Interaction plot for 20-m dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m dribble (p < .01) after the 8-week training. The main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g007 Fig 7. Interaction plot for 20-m dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m dribble (p < .01) after the 8-week training. The main effects for time were significant for the GTG group only. Fig 7. Interaction plot for 20-m dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m dribble (p < .01) after the 8-week training. The main effects for time were significant for the GTG group only. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Discussion https://doi.org/10.1371/journal.pone.0299204.g007 https://doi.org/10.1371/journal.pone.0299204.g007 Maximum sprint speed is an essential physical element for football players [15]. The results showed that only the experimental group, who underwent 8 weeks of SAQ training, signifi- cantly reduced sprint times across all speed tests. Although there were significant differences between the two groups in all speed tests at post-testing, except for the short sprints, and between the pre-post assessments in 5-, 10-, 30-m sprints without a ball and 20-m sprint with dribbling (all p < .05), such improvements were only observed in the SAQ group, not in the control group (Tables 3 and 4). These findings are consistent with previous research [7,12,17], which demonstrated significant enhancements in acceleration and maximum sprint speed over short distances (5- and 10-m). Similarly, the results obtained in this study provide support for our experimental hypothesis that an 8-week SAQ training method improves sprint perfor- mances over 20- and 30-m with and without dribbling. Although there is some evidence sug- gesting that a 12-week SAQ training significantly reduces sprint times over short [19] and long distances [20], it was unclear whether 8 weeks of an SAQ training programme would yield similar results. The results of the current study show that 8 weeks of SAQ protocol can lead to meaningful changes in acceleration speed and high-running velocity. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 14 / 25 PLOS ONE SAQ Fig 8. Interaction plot for 30-m dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m dribble (p < .01) after the 8-week training period, while no main effects were found. https://doi.org/10.1371/journal.pone.0299204.g008 Fig 8. Interaction plot for 30-m dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for 30-m dribble (p < .01) after the 8-week training period, while no main effects were found. https://doi.org/10.1371/journal.pone.0299204.g008 https://doi.org/10.1371/journal.pone.0299204.g008 However, it should be noted that the control group also showed significant improvements in 10-m sprint performance after engaging in general football training. One possible explana- tion for the enhanced values may be due to different mechanisms at play during the 5 and 10 m sprints [17]. As suggested by Milanović et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE PLOS ONE SAQ Table 4. Means, standard deviations, and results of independent and paired t-tests for the SAQ and GTG group. Variables SAQ (n = 9) GTG (n = 10) Posttest scores Means ± SD Paired t-test df = 8 Means ± SD Paired t-test df = 9 Independent t-test df = 17 Pre Post t p(d) Pre Post t p(d) t p(d) Speed 5-m sprint 1.45 ± .08 1.35 ± .05 4.55 .01(1.51) 1.41 ± .08 1.38 ± .07 1.00 .34(.31) -1.00 .33(.45) 10-m sprint 2.30 ± .06 2.14 ± .09 5.12 .01(1.70)* 2.30 ± .09 2.21 ± .04 2.87 .02(.89)* -2.08 .06(.92) 20 m sprint 3.74 ± .14 3.57 ± .12 3.29 .01(1.09)** 3.67 ± .20 3.74 ± .17 -1.71 .12(.54) -2.32 .03(1.06)* 30-m sprint 5.25 ± .17 4.99 ± .15 5.09 .01(1.69)*** 5.22 ± .13 5.24 ± .29 -.41 .68(.13) -2.29 .03(1.05)* 20-m dribble 4.19 ± .12 3.97 ± .13 4.52 .01(1.50)** 4.20 ± .16 4.21 ± .26 -.10 .92(.03) -1.11 .02(1.11)* 30-m dribble 5.73 ± .23 5.49 ± .10 2.92 .01(.97) 5.64 ± .39 5.73 ± .20 -1.09 .30(.34) -1.42 .01(1.42) COD Speed Arrowhead agility (L) 9.41 ± .24 9.29 ± .28 2.04 .07(.68) 9.09 ± .29 9.35 ± .38 -3.01 .01(.95)* -0.36 .72(.16) Arrowhead agility (R) 9.32 ± .27 9.10 ± .27 4.98 .01(1.66)* 9.12 ± .21 9.27 ± .32 -1.83 .09(.58) -1.45 .16(.66) SEMO agility 12.16 ± .24 11.52 ± .36 5.15 .01(1.71)* 11.53 ± .48 12.09 ± .56 -3.79 .01(1.20)** -1.27 .01(1.21)* Arrowhead dribble (L) 10.96 ± .28 10.70 ± .34 3.10 .01(1.03)* 11.14 ± .58 11.64 ± .50 -3.34 .01(1.05) -4.64 .01(2.13)* Arrowhead dribble (R) 10.99 ± .42 10.74 ± .49 4.31 .01(1.43) 10.68 ± .44 11.22 ± .55 -3.67 .01(1.16) -1.96 .06(.90) 22-m slalom dribble† 14.93 ± .89 13.10 ± .72 6.80 .01(2.26)* 13.95 ± .44 13.95 ± .59 .01 .99(.01) — — Reactive Agility Reactive agility 2.28 ± .86 2.21 ± .06 4.28 .01(1.42) 2.28 ± .09 2.22 ± .16 .81 .43(.25) -1.37 .18(.63) Note: SAQ = speed, agility, and quickness. GTG = general training group. n = sample size. SD = standard deviation. df = degrees of freedom. COD = change-of- direction. SEMO = Southeast Missouri. L = left. R = right. Mdn = median. Q1 = Quartile 1. Q3 = Quartile 3. ndard deviations, and results of independent and paired t-tests for the SAQ and GTG group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Discussion [7], football players are involved in high-inten- sity activities and directional changes that last 2–4 seconds and require short sprints (e.g., no more than 15 m). Thus, while an SAQ training protocol appeared to be effective in enhancing all speed performance in this study, further investigation is still necessary to fully understand the differences in sprint times across various distances. COD speed is the ability of players to accelerate, decelerate, cut, and turn rapidly when changing direction. This plays an important role in optimising the high-intensity perfor- mance and reducing the risks of injuries [12]. In the present study, there were significant group × time interactions across the tests. Supporting our hypothesis, the results demon- strated greater improvements in the arrowhead agility for both sides (with and without PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 15 / 25 PLOS ONE Amongst others (e.g., the Illinois agility test, T-test, 505 test, and PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 16 / 25 PLOS ONE SAQ Fig 9. Interaction plot for arrowhead agility (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead agility test (left) after 8 weeks training (p < .01). No significant main effects were observed. https://doi.org/10.1371/journal.pone.0299204.g009 Fig 9. Interaction plot for arrowhead agility (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead agility test (left) after 8 weeks training (p < .01). No significant main effects were observed. Fig 9. Interaction plot for arrowhead agility (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead agility test (left) after 8 weeks training (p < .01). No significant main effects were observed. https://doi.org/10.1371/journal.pone.0299204.g009 https://doi.org/10.1371/journal.pone.0299204.g009 Zigzag test), COD performance with and without a ball in this study was evaluated by the arrowhead agility, the SEMO, and 22-m slalom dribble tests, which are considered as reliable and valid methods for assessing pre-planned, multidirectional movements in football [32,33]. As revealed in the paired t-tests, it can be concluded that SAQ training was beneficial for improving COD speed of football players to perform faster and more agile, pre-planned, and multidirectional movements during a game. Reactive agility represents open skills that require players to read accurately and respond quickly to situational cues to perform actions efficiently [10]. As such, visual scanning, deci- sion-making, and reaction time are the major aspects of reactive agility, all of which are critical for information processing during a football match [20]. In this study, no significant interac- tions or differences between the groups were found; yet, the results were similar to previous research indicating greater improvements in reactive agility performance in the SAQ group [17]. The findings are particularly in line with previous research, which reduced time in the reactive agility test through SAQ training [20]. PLOS ONE Note: SAQ = speed, agility, and quickness. GTG = general training group. n = sample size. SD = standard deviation. df = degrees of freedom. COD = change-of- direction. SEMO = Southeast Missouri. L = left. R = right. Mdn = median. Q1 = Quartile 1. Q3 = Quartile 3. * Times are expresses in second. p †Nonparametric test was performed using the Mann-Whitney test indicating that the posttest scores of the 22-m slalom dribble test were significantly lower for the SAQ group (Mdn = 12.88; Q1 = 12.67, Q3 = 13.88) than for the GTG group (Mdn = 13.92; Q1 = 13.43, Q3 = 14.53), U = 16, z = -2.36, p = 0.01. †Nonparametric test was performed using the Mann-Whitney test indicating that the posttest scores of the 22-m slalom dribble test were significantly lower for the SAQ group (Mdn = 12.88; Q1 = 12.67, Q3 = 13.88) than for the GTG group (Mdn = 13.92; Q1 = 13.43, Q3 = 14.53), U = 16, z = -2.36, p = 0.01. dribbling), SEMO agility, and 22-m slalom dribble performances for the SAQ group after 8 weeks of training, compared to the control group, who underwent general football training. Moreover, there were significant differences between the two groups in the arrowhead agility test (left) after each programmed training, while significant decreases occurred between the pre-post assessments in 22-m slalom dribble test for the SAQ group (Table 4). Follow-up analyses, however, showed significant differences between the two groups in the SEMO, arrowhead dribble test (left), and 22-m slalom dribble tests at post-testing (Table 4). Interest- ingly, the control group demonstrated significant increases in the arrowhead agility (left), the SEMO, and arrowhead dribble tests. On the contrary, the SAQ training programme induced significant changes in all COD abilities for the experimental group, except for the arrowhead agility (left). These findings support results of previous investigations [17,22] that found significant improvements in a T-test after an SAQ intervention. In a study of female basketball players, Moselhy [37] assessed COD speed using other parameters, such as the Illinois agility test while Jovanovic et al. [12] used vertical, lateral, and/or horizontal jumps to measure agility of football players, all of which showed significant enhancements in COD abilities through implementing an SAQ training programme. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE The pre-post differences in the reactive agility test support our experimental hypothesis that an 8-week SAQ training significantly leads to PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 17 / 25 PLOS ONE SAQ Fig 10. Interaction plot for arrowhead agility (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead agility test (right) during the 8-week training period (p < .01), while no main effects were found. Pre to post changes were found in the SAQ group only (p < .01). https://doi.org/10.1371/journal.pone.0299204.g010 Fig 10. Interaction plot for arrowhead agility (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead agility test (right) during the 8-week training period (p < .01), while no main effects were found. Pre to post changes were found in the SAQ group only (p < .01). https://doi.org/10.1371/journal.pone.0299204.g010 Fig 10. Interaction plot for arrowhead agility (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead agility test (right) during the 8-week training period (p < .01), while no main effects were found. Pre to post changes were found in the SAQ group only (p < .01). faster performance in the reactive agility test. Consequently, it can be suggested that SAQ training is an effective method for improving the perceptual and decision-making components of reactive agility in female football players. faster performance in the reactive agility test. Consequently, it can be suggested that SAQ training is an effective method for improving the perceptual and decision-making components of reactive agility in female football players. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Implications, limitations, and conclusion We conclude with a discussion of the implications and limitations of our research. The results from the present study highlight the importance and effectiveness of incorporating SAQ train- ing into football-specific practices, particularly enhancing the high-intensity performance in U-20 female football players during the pre- and in-season. From a practical standpoint, foot- ball coaches and conditioning practitioners can implement a periodised SAQ conditioning over 8 weeks, which can enhance players’ ability to sprint and perform multidirectional move- ments (linear, lateral, and diagonal) without losing maximal running speed and body control. By combining SAQ exercises with regular football training, significant improvements in sprint performance over short distances and COD speed can be achieved. It is worth noting that PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 18 / 25 PLOS ONE SAQ Fig 11. Interaction plot for SEMO agility. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the SEMO agility test after the 8-week training (p < .01), while no main effects were observed. https://doi org/10 1371/journal pone 0299204 g011 Fig 11. Interaction plot for SEMO agility. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the SEMO agility test after the 8-week training (p < .01), while no main effects were observed. Fig 11. Interaction plot for SEMO agility. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the SEMO agility test after the 8-week training (p < .01), while no main effects were observed Fig 11. Interaction plot for SEMO agility. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the SEMO agility test after the 8-week training (p < .01), while no main effects were observed. https://doi.org/10.1371/journal.pone.0299204.g011 although spring speed and COD speed are separate and independent qualities [38,39], a strong association between higher levels of sprint speed over short distances, including acceleration speed enhanced COD speed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 https://doi.org/10.1371/journal.pone.0299204.g012 Implications, limitations, and conclusion Additionally, it is also crucial to note that acceleration, decelera- tion, and COD speed involve distinct techniques [40,41], where separate training and evalua- tion sessions should be taken to assess players’ physical abilities in sprinting over short and long distances, and their agility in changing direction. As with all research, our study is subject to a number of limitations that can represent ave- nues for future research. First, we focused on female youth football players who compete at the national level. Thus, generalisation of the results to other populations in different team sports (rugby or hockey), performance levels (elite or amateur) or age groups (U-12 or U-15) should be done with caution due to the different nature of various sports and variations in physical demands. Considering individual variations and specific player characteristics, future research can explore the applicability of SAQ training to different age groups and sports and investigate its effects on sprint and agility performance. Further, our sample 19 / 25 PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 PLOS ONE SAQ Fig 12. Interaction plot for arrowhead dribble (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead dribble test (left; p < .01), while the main effects for group were found, indicating a different mean change between the SAQ and GTG groups. https://doi.org/10.1371/journal.pone.0299204.g012 Fi 12 I t ti l t f h d d ibbl (L) N t SAQ d ilit d i k t i i GTG l t i i Th lt f Fig 12. Interaction plot for arrowhead dribble (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead dribble test (left; p < .01), while the main effects for group were found, indicating a different mean change between the SAQ and GTG groups. https://doi.org/10.1371/journal.pone.0299204.g012 Fig 12. Interaction plot for arrowhead dribble (L). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Implications, limitations, and conclusion The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead dribble test (right) after 8 weeks training (p < .01), while no main effects were found. The significant main effects of time and group were also found. Specifically, there was a significant improvement in COD speed in the 22-m slalom dribbling test (Fig 14), and another significant difference between groups in the arrowhead dribble (left). Fig 13. Interaction plot for arrowhead dribble (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead dribble test (right) after 8 weeks training (p < .01), while no main effects were found. The significant main effects of time and group were also found. Specifically, there was a significant improvement in COD speed in the 22-m slalom dribbling test (Fig 14), and another significant difference between groups in the arrowhead dribble (left). https://doi.org/10.1371/journal.pone.0299204.g013 the study. These measures are commonly used to assess exercise intensity, and their absence may limit the comprehensive understanding of the participants’ physiological responses dur- ing training. Additionally, we note that the validity of the arrowhead test has not been specifi- cally tested in previous studies [32,33], although its reliability has been reported. Similarly, the SEMO agility test has not been assessed for validity and reliability, but it is widely used in foot- ball, while dribbling and slalom tests have been found to be both valid and reliable [42]. In light of the current findings, researchers could focus on evaluating the validity and reliability of both the arrowhead and SEMO tests. Finally, as the present study indicates, coaches and conditioning practitioners can implement SAQ exercises 2–3 times a week to transfer training effects to sprint performance over short and long distances, as well as COD abilities of players. Enhanced acceleration, sprint speed, COD ability, and reactive agility can contribute to better athletic performance in high-intensity activities and ballistic and dynamic movements during a football game [35]. Together, as shown in this study, an SAQ training programme with a gradual increase in intensity is more effective for improving physical capacities committed to high-intensity performance than regular football training alone. Implications, limitations, and conclusion The results of a two-way mixed analysis of variance (ANOVA) showed a significant group × time interaction for the arrowhead dribble test (left; p < .01), while the main effects for group were found, indicating a different mean change between the SAQ and GTG groups. https://doi.org/10.1371/journal.pone.0299204.g012 represents a specific group of female footballers in East Asia, and the study was cross-sec- tional, with the small sample size. Indeed, the scarcity of highly trained female football play- ers presents challenges in achieving a large sample size of participants with the desired level of training. Thus, the results may not be generalisable to other geographic settings and time periods. To remedy this shortcoming and increase the potential for generalisability, we encourage future researchers to conduct additional trials in diverse geographical contexts and sporting settings. We would also like to acknowledge the inherent limitations of using a stopwatch for timing. Despite our effort to minimise potential impacts on the reliability of results, the use of a stop- watch can introduce some variability, even with the same rater. Hence, future researchers could explore alternative methods for timing, such as electronic timing systems or motion cap- ture technology (e.g., speed gate). Doing so will provide more precise measurements. Further- more, HR and RPE were consistently monitored as indicators of training intensity. However, the lack of documentation and recording of these indicators can be considered a limitation of PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 20 / 25 PLOS ONE SAQ Fig 13. Interaction plot for arrowhead dribble (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for the arrowhead dribble test (right) after 8 weeks training (p < .01), while no main effects were found. The significant main effects of time and group were also found. Specifically, there was a significant improvement in COD speed in the 22-m slalom dribbling test (Fig 14), and another significant difference between groups in the arrowhead dribble (left). htt //d i /10 1371/j l 0299204 013 Fig 13. Interaction plot for arrowhead dribble (R). Note: SAQ = speed, agility, and quickness training group. GTG = general training group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 Acknowledgments The author(s) would like to acknowledge all the participants for their contributions to the study. The author(s) declare no conflicts of interest associated with this publication, and there is no specific funding for this work. Implications, limitations, and conclusion In summary, 8 weeks of SAQ training appear to induce substantial changes in sprint and COD speed in highly trained female youth football players. PLOS ONE \| https://doi.org/10.1371/journal.pone.0299204 March 13, 2024 21 / 25 PLOS ONE SAQ Fig 14. Interaction plot for 22-m slalom dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m dribble (p < .01) after the 8-week training. The main effects for time were significant for the GTG group only. Fig 14. Interaction plot for 22-m slalom dribble. Note: SAQ = speed, agility, and quickness training group. GTG = general training group. The results of a two-way mixed analysis of variance (ANOVA) indicated a significant group × time interaction for 20-m dribble (p < .01) after the 8-week training. The main effects for time were significant for the GTG group only. https://doi.org/10.1371/journal.pone.0299204.g014 References 1. Bangsbo J, Mohr M, Krustrup P. Physical and metabolic demands of training and match-play in the elite football player. Journal of Sports Sciences. 2006; 24(7):665–74. https://doi.org/10.1080/ 02640410500482529 PMID: 16766496 2. Stølen T, Chamari K, Castagna C, Wisløff U. Physiology of soccer. Sports Medicine. 2005; 35(6):501– 36. 3. Bangsbo J, Nørregaard L, Thorsoe F. Activity profile of competition soccer. Canadian Journal of Sport Sciences. 1991; 16(2):110–6. PMID: 1647856 4. 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https://openalex.org/W2071646533	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0020538&type=printable	English	null	Caterpillars and Fungal Pathogens: Two Co-Occurring Parasites of an Ant-Plant Mutualism	PloS one	2,011	cc-by	7,931	Caterpillars and Fungal Pathogens: Two Co-Occurring Parasites of an Ant-Plant Mutualism Olivier Roux1, Re´gis Ce´re´ghino2,3, Pascal J. Solano4, Alain Dejean1,3 ane (UMR-CNRS 8172), Campus Agronomique, Kourou, France, 2 CNRS, UMR 5245, EcoLab (Laboratoire d’Ecologie Fonctionnelle), ulouse, UPS, Toulouse, France, 4 Aniane, France 1 CNRS, E´cologie des Foreˆts de Guyane (UMR-CNRS 8172), Campus Agronomique, Kourou, France, 2 CNRS, UMR 5245, EcoLab (Laboratoire d’Ecologie Fonctionnelle), Toulouse, France, 3 Universite´ de Toulouse, UPS, Toulouse, France, 4 Aniane, France Introduction driven by chemical compounds [13–16]; however to enter into the domatia of certain myrmecophyte species, founding ant queens must be the right size or be able to recognize and to gnaw an entrance hole into the prostomata or thinner area, generally devoid of vessels [10],[17–19]. Mutualisms are interspecific interactions involving two or more species where each partner obtains resources that it would obtain less efficiently if alone, and so derives a net fitness benefit [1–3]. These mutualistic partnerships are transmitted from one genera- tion to the next in one of two ways. In vertical transmission, hosts transmit symbiont offspring directly to their own offspring [4],[5], while in horizontal transmission the partners need to renew their association after each reproductive event [3],[6]. Biotic pollina- tion, seed dispersal by animals, ant-plant associations and interactions between rhizobia or mycorrhiza and plant roots are transmitted horizontally and can be mutualistic [4],[6–9]. Nevertheless, these mechanisms do not keep the mutualism between myrmecophytes and plant-ants free from conflict, competition and/or exploitation by other ants or by non-ant species. In this context of competition for resources, the abilities of species are generally unequal, leading to hierarchically-organized systems with dominant and subordinate species. To survive, weaker competitors must develop colonization strategies, be resistant to perturbations, manage with fewer resources or have good longevity with the aim of conserving their access to the ‘‘niche’’ [20–22]. One alternative way to obtain an advantage over a better competitor is to be the first to obtain access to resources and to monopolize them. Often this advantage allows poor competitors to persist longer in habitats than they would otherwise [23],[24]. This phenomenon is known as ‘‘temporal priority’’ and has been documented in many taxa such as mycorrhizal fungi [25–27], plants [28],[29], amphib- ians [24],[30] and insects [23],[31]. Myrmecophytes (or ant-plants) are involved in mutualisms with a limited number of so-called plant-ants that they shelter in domatia (i.e. hollow branches or thorns and leaf pouches) and usually provide with food through extra-floral nectaries (EFNs) and/or food bodies (FBs). In return, plant-ants protect their host plant from herbivores, competitors, encroaching vines and fungal pathogens [10]. Because the transmission of ant-plant mutualisms is horizontal, myrmeco- phytes have evolved several types of selective filters enabling them to host those ant species most favorable to their fitness [11],[12]. Abstract Copyright: 2011 Roux et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support for this study was provided by the Programme Amazonie II of the French Centre National de la Recherche Scientifique (project 2ID) and the Programme Convergence 2007–2013, Re´gion Guyane, from the European Community (project DEGA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: olivier.roux@ird.fr PLoS ONE \| www.plosone.org Abstract In mutualisms, each interacting species obtains resources from its partner that it would obtain less efficiently if alone, and so derives a net fitness benefit. In exchange for shelter (domatia) and food, mutualistic plant-ants protect their host myrmecophytes from herbivores, encroaching vines and fungal pathogens. Although selective filters enable myrmecophytes to host those ant species most favorable to their fitness, some insects can by-pass these filters, exploiting the rewards supplied whilst providing nothing in return. This is the case in French Guiana for Cecropia obtusa (Cecropiaceae) as Pseudocabima guianalis caterpillars (Lepidoptera, Pyralidae) can colonize saplings before the installation of their mutualistic Azteca ants. The caterpillars shelter in the domatia and feed on food bodies (FBs) whose production increases as a result. They delay colonization by ants by weaving a silk shield above the youngest trichilium, where the FBs are produced, blocking access to them. This probable temporal priority effect also allows female moths to lay new eggs on trees that already shelter caterpillars, and so to occupy the niche longer and exploit Cecropia resources before colonization by ants. However, once incipient ant colonies are able to develop, they prevent further colonization by the caterpillars. Although no higher herbivory rates were noted, these caterpillars are ineffective in protecting their host trees from a pathogenic fungus, Fusarium moniliforme (Deuteromycetes), that develops on the trichilium in the absence of mutualistic ants. Therefore, the Cecropia treelets can be parasitized by two often overlooked species: the caterpillars that shelter in the domatia and feed on FBs, delaying colonization by mutualistic ants, and the fungal pathogen that develops on old trichilia. The cost of greater FB production plus the presence of the pathogenic fungus likely affect tree growth. Citation: Roux O, Ce´re´ghino R, Solano PJ, Dejean A (2011) Caterpillars and Fungal Pathogens: Two Co-Occurring Parasites of an Ant-Plant Mutualism. PLoS ONE 6(5): e20538. doi:10.1371/journal.pone.0020538 Editor: Corrie S. Moreau, Field Museum of Natural History, United States of America Editor: Corrie S. Moreau, Field Museum of Natural History, United States of America Received February 8, 2011; Accepted May 3, 2011; Published May 31, 2011 Received February 8, 2011; Accepted May 3, 2011; Published May 31, 2011 Copyright: 2011 Roux 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. The caterpillars’ diet To assess if the caterpillars feed exclusively on the FBs, in 2001 and 2002, we selected 83 saplings on which we had found at least Figure 1. Trichilium of Cecropia. A- Azteca alfari workers removing food bodies (arrow) from a Cecropia obtusa trichilium. B- Pseudocabima guianalis caterpillars occupying the upper part of a C. obtusa. They gnawed an entrance hole into the prostomata (p) in order to enter into the hollow internodes. Secreted strands of silk cover the trichilium and keep the withered stipules (DS) from falling. C- Trichilium of C. obtusa infested by Fusarium moniliforme. D- Penetration by a filament of F. moniliforme (arrows) inside a food body (FB). E–F. moniliforme spreading throughout a food body (FB and black arrows). The cells of the trichilium at the base of the FB seem to react to the presence of fungus (white arrow). doi:10.1371/journal.pone.0020538.g001 The myrmecophyte Cecropia obtusa (Cecropiacae), the focal species of this study, is mutualistically associated with several Azteca species (Dolichoderinae) whose founding queens and workers recognize the zone where the prostomata is situated and so establish colonies in the internodal domatia [13],[40]. In addition to shelter, the plant provides the Azteca colonies with food in the form of glycogen-rich Mu¨llerian bodies produced by the trichilia, pads of dense trichomes situated at the base of the leaf petiole, and lipid-rich pearl bodies produced beneath young leaves. Mutualistic Azteca workers generally protect their host trees from defoliating insects, encroaching vines and fungal pathogens [17],[41–43], but this is not the case for saplings [44],[45]. Because the transmission of the Azteca–Cecropia mutualism is horizontal, the size of the internodes plus the production rate of the food bodies do not permit Azteca colonies to develop before the saplings reach ca. 1 m in height [44],[46]. Before Cecropia saplings reach this minimum size, herbivores and parasites may use this absence of mutualistic plant-ants to opportunistically take over the Cecropia’s resources; whereas, after this period of time, potential invaders must overcome the biotic defenses conferred by plant- ants [44]. We noted that recently-perturbed areas are rapidly occupied by thousands of C. obtusa saplings, permitting some caterpillars to live in the domatia and feed on the FBs, and that caterpillar presence was associated with the development of a fungus on the old trichilia. Ethics Statement This study was conducted according to relevant national and international guidelines. Study sites We conducted this study between 2000 and 2009 in French Guiana near the Petit Saut dam (5u039390 N-53u029360W) and near the Montagne des singes (5u04919.60N-52u41942.50W). We selected and tagged C. obtusa that were ca. 1.15 m to 1.30 m tall (N = 610) between 2000 and 2006 in a cleared 1.5 ha zone situated near the dam, and on plants growing along a straight, recently-opened dirt road near the Montagne des singes (N = 64) between 2006 and 2009. In these areas, C. obtusa is mostly associated with Azteca alfari and A. ovaticeps whose colonies exploit Mu¨llerian bodies (Fig. 1A), tend hemipterans in the host tree domatia and prey on insects landing on the leaves [42]. These insects, often less numerous and less aggressive than ants, must find weaknesses in the mutualism to be able to colonize their host tree and exploit its resources. Using chemical mimicry or camouflage to counter ant aggressiveness is one solution for getting past ant defenses [38],[39]; however, being the first to arrive to colonize new treelets might also represent a serious advantage because the biotic defense provided by mutualistic plant-ants is not yet in place and nearly leaves the plant without indirect defenses. Introduction Host- plant selection by founding ant queens, for example, seems to be It is well known that mutualistic plant-ant species compete for their host-plant [14],[32–34]. Moreover, mutualistic ants are not PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e20538 1 Two Parasites of an Ant-Plant Mutualism the only ones competing for this resource. Indeed, parasites of ant-myrmecophyte mutualisms-mostly ant species-are able to colonize the myrmecophytes, but do not provide them with protection [8],[9]. This was first shown for Pseudomyrmex nigropilosus that colonizes myrmecophytic Acacia and consumes their EFNs and FBs, but exhibits no defensive behavior [35]. Some non-ant insects are also able to colonize and parasitize myrmecophytes, benefiting from the shelter and food provided by the plant in different ways; for example, the larvae of the clerid beetle Phyllobaenus sp. parasitize myrmecophytic Piper trees, feeding on both the FBs and on mutualistic ants. The fitness of the host trees is reduced due to a greater investment in FB production and a decrease in biotic protection by the guest ants, with a subsequent increase in herbivory [36]. Also, females of the chrysomelid beetle Coelomera sp. open an entrance hole in the prostomata of Cecropia trees in the same manner as mutualistic Azteca ants. They then lay eggs in the domatia and the larvae feed on young leaves [37]. PLoS ONE \| www.plosone.org The caterpillars Observations made on the 83 saplings sheltering caterpillars permitted us to note that the first instar caterpillars lived under a silk shelter that they built between the stipules developing around the terminal bud, the trunk and the youngest leaf. They only left this shelter between 15:00 and 18:00 to feed on the FBs produced daily during that time period by the youngest trichilia, or sometimes by the other trichilia. As the shoot grows, the stipules, which normally drop off, are trapped by the silk (Fig. 1B; Fig. S1a). From their third instar, ca. 1.5-cm-long caterpillars, like ants, gnawed the prostomata in order to shelter in the last internode. They wove a silk shield above the upper part of the tree trunk and the youngest trichilium (Fig. 1B, Fig. S1b-d), and left the domatia only to feed on the FBs under the shelter of strands of silk. Pupation occurred inside the domatia. Larval and pupal development took about 30 days. Just-emerging moths leave the trunk by flying out through the stomata that are widened by the caterpillars when they are in their last larval stage. We also tested, in 2003–2004, if the presence of ants and caterpillars affected the presence of defoliating insects by scoring the amount of herbivory on the oldest leaf on 90 saplings (30 sheltered A. alfari, 30 sheltered caterpillars, and the 30 others were unoccupied), each bearing at least five leaves. We chose the oldest leaves because they provide an idea of the history of the defoliation over the preceding ca. 18 months which corresponds to the lifespan of C. obtusa leaves [42]. We evaluated the percentage of foliar surface eaten by insects (FSE) using the following scale: (1) leaf intact; (2) slightly attacked: 0%,FSE#25%; (3) somewhat attacked: 25%,FSE#50%; (4) very attacked: 50%,FSE#75%; and (5) extremely attacked: FSE.75%. We compared the results using the Kruskal-Wallis test. To verify how caterpillars can delay or even prevent ant colonization and vice versa, every 8 months between June 2006 and June 2008, we noted which ant species or if caterpillars sheltered in the domatia and fed on the FBs on the 64 C. obtusa situated near the Montagne des singes. An additional survey was conducted in July 2009. In July 2008, we measured the height of the trees that had sheltered (1) Azteca colonies during the experimental period (A. ovaticeps: N = 10; A. Fungal infestation of the trichilia Between 2003 and 2005, we recorded the number of trees whose trichilia had a fungal infestation out of 610 C. obtusa saplings sheltering an Azteca colony (N = 349), caterpillars (N = 83), or not occupied (N = 178). We scored the presence versus absence of fungal infestation on the trees as ‘‘1’’ and ‘‘0’’, respectively, and compared the results using the Kruskal-Wallis test and Dunn’s post-hoc test. To analyze how ant or caterpillar presence affected fungal development, we took samples of 20 trichilia with and 20 without developed mycelium from C. obtusa in all cases (i.e. trichilia taken from trees sheltering ants, caterpillars or unoccupied) and cultivated the mycelium in aseptic conditions in Sabouraud’s nutritive substrate (N = 120 trichilia). We analyzed these samples under a microscope to verify how the mycelium develops on the trichilia. We first fixed the sampled trichilia with FAA (formalin, acetic acid, alcohol), and then embedded them in paraffin. We stained tissue sections with basic fuchsin light-green or toluidine blue contrasted with sodium molybdate. Food body production, herbivory, ant and caterpillar presence and tree growth To quantify FB production, in 2003 we selected 30 saplings, each bearing at least five leaves: 10 sheltered A. alfari, 10 sheltered caterpillars and the remaining 10 were unoccupied. Because FB production increases when they are removed by ants or experimentally [17], we conducted experiments where both ants and caterpillars were prevented access to the upper zone of the trees where most of the FB production occurs. We did this by placing a ring of TanglefootH around the trunk under the lowermost leaf to isolate the top of each sapling, and thus prevent the ants and caterpillars from having access to the leaves and to the FBs. We then plugged the entrance holes situated in this upper, isolated section with a spot of TanglefootH. The ants and caterpillars could, nevertheless, move freely in and out of the domatia using the lower entrance holes giving them access to the lower part of the trunk and to older leaves with inactive trichilia. We placed aluminum foil shelters around the isolated trichilia to protect them from flying insects and to gather the FBs that dropped off [48],[51]. For each sapling and during 20 days, at ca. 19:00, we removed the FBs produced that day and that had fallen from the least mature trichilia onto the aluminum foil, and counted them. We compared the results using a repeated measures ANOVA followed by a Newman-Keuls’ post-hoc test for multiple comparisons (GraphPad Prism 4.03 software). Voucher specimens of the adult moths obtained after the metamorphosis of the caterpillars were identified as Pseudocabima guianalis (Lepidoptera, Pyralidae, Phyticinae) and were deposited at the Systematic Entomology Laboratory of the United States Department of Agriculture (USDA), Beltsville, Maryland. Fungal samples were identified as Fusarium moniliforme (Deuteromycetes) and were deposited at the Laboratoire de biologie et taxonomie des microchampignons, Muse´um National d’Histoire Naturelle, Paris, France. The caterpillars alfari: N = 22), (2) neither ants nor caterpillars (N = 10), or (3) caterpillars during the entire experi- mental period or that had been replaced by an Azteca colony only during the last part of the experimental period (N = 14). All of these trees are the same age as they developed just after the dirt road was built near the Montagne des singes and have a similar exposure to the sun and to rain. We compared the results using an ANOVA and Newman-Keuls’ post-hoc test. Two Parasites of an Ant-Plant Mutualism Two Parasites of an Ant-Plant Mutualism one caterpillar. Preliminary studies conducted during a 24-hour period permitted us to learn that FBs are produced during the afternoon, between 15:00 and dusk (see also [47–50]), and that caterpillars are active during FB production. During a more comprehensive survey, we observed their behavior for 5 minutes per sapling in the morning between 8:00 and 11:00 as well as during FB production in the afternoon between 15:00 and 18:00. Observations were also made at night between 21:00 and 5:00 to ensure that the caterpillars were not active nocturnally. The caterpillars’ diet To broaden our understanding of the biological interactions and coexistence of these caterpillars within the Azteca-Cecropia mutualism, we conducted a correlation study where we posed the following questions. (1) Do caterpillars mainly or even exclusively feed on the FBs and, if so, does this activity increase FB production as is known for mutualistic plant-ants [36]? (2) Is caterpillar presence associated with the greater herbivory of Cecropia saplings? (3) Does this presence favor fungal development on the trichilia with deleterious consequences for the plant? (4) Can Azteca ants prevent colonization by caterpillars, or, inversely can caterpillars delay or even prevent colonization by ants thus allowing them to exploit Cecropia saplings longer? Figure 1. Trichilium of Cecropia. A- Azteca alfari workers removing food bodies (arrow) from a Cecropia obtusa trichilium. B- Pseudocabima guianalis caterpillars occupying the upper part of a C. obtusa. They gnawed an entrance hole into the prostomata (p) in order to enter into the hollow internodes. Secreted strands of silk cover the trichilium and keep the withered stipules (DS) from falling. C- Trichilium of C. obtusa infested by Fusarium moniliforme. D- Penetration by a filament of F. moniliforme (arrows) inside a food body (FB). E–F. moniliforme spreading throughout a food body (FB and black arrows). The cells of the trichilium at the base of the FB seem to react to the presence of fungus (white arrow). doi:10.1371/journal.pone.0020538.g001 PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e20538 PLoS ONE \| www.plosone.org 2 Fungal presence Fusarium moniliforme was present on 323 of the 610 C. obtusa saplings (53.0%), sometimes completely covering the trichilia (Fig. 1C). The percentage of infested individuals was significantly lower among saplings sheltering an Azteca colony than those sheltering P. guianalis caterpillars or not occupied, while the difference between the latter two cases was not significant (Fig. 2). Normally, FBs are sub-spherical and homogeneous with reserve cells bordered by a cell wall and a thin cuticle. The penetration of the F. moniliforme mycelium into an FB occurs once it is already formed, but not necessarily completely developed. In Figure 1D, a filament of F. moniliforme can be seen entering into an FB. The bordering cells are in necrosis, as are the first reserve cells. The trichomes around the FB react strongly to the presence of the fungus, saturating their cell walls with lignin (Fig. 1E); whereas the Fusarium moniliforme was present on 323 of the 610 C. obtusa saplings (53.0%), sometimes completely covering the trichilia (Fig. 1C). The percentage of infested individuals was significantly lower among saplings sheltering an Azteca colony than those sheltering P. guianalis caterpillars or not occupied, while the difference between the latter two cases was not significant (Fig. 2). May 2011 \| Volume 6 \| Issue 5 \| e20538 PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e20538 3 Two Parasites of an Ant-Plant Mutualism Figure 2. Trichilium infested by Fusarium moniliforme. Percentages of Cecropia obtusa saplings whose trichilia were attacked by Fusarium moniliforme in three situations: saplings sheltering an Azteca colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings (N = number of saplings in each case). Statistical comparisons; Kruskal-Wallis test: H3 610 = 338.1; P,0.001; Dunn’s multiple comparison test, different letters indicate significant differences at P,0.001. doi:10.1371/journal.pone.0020538.g002 Two Parasites of an Ant-Plant Mutualism Figure 2. Trichilium infested by Fusarium moniliforme. Percentages of Cecropia obtusa saplings whose trichilia were attacked by Fusarium moniliforme in three situations: saplings sheltering an Azteca colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings (N = number of saplings in each case). Statistical comparisons; Kruskal-Wallis test: H3 610 = 338.1; P,0.001; Dunn’s multiple comparison test, different letters indicate significant differences at P,0.001. doi:10.1371/journal.pone.0020538.g002 FB cells show no reaction either in the cell wall or in the cytoplasm. Once inside the FB, the mycelium progressively invades all of the cells, down to the base of the FB (Fig. Ant and caterpillar occupancy of Cecropia obtusa trees Ant and caterpillar occupancy of Cecropia obtusa trees Out of the 610 C. obtusa saplings studied near the Petit Saut dam, only 349 (57.2%) sheltered A. alfari or A. ovaticeps colonies. Among the others, 178 (29.2%) were totally unoccupied, while the remaining 83 (13.6%) sheltered three to six P. guianalis caterpillars at different larval stages (Fig. S1c). For the 64 C. obtusa surveyed during 3 years near the Montagne des singes, at the start of the survey the percentage of C. obtusa sheltering P. guianalis caterpillars was by far superior (39.1% or 25 trees out of 64; Fig. 3), illustrating that there are variations between areas. When present, caterpillars were also more numerous with some trees sheltering up to 12 caterpillars. Saplings were also associated with the two Cecropia-ant species typical of the area, A. alfari and A. ovaticeps, as well as, unexpectedly, the fire ant Solenopsis saevissima (tree Nu7). Also, six trees were unoccupied at the start of the survey, and three of the 25 saplings bearing caterpillars were also occupied by A. alfari (trees Nu 24, 43 and 52). This dual hosting was also observed later in the survey for two additional trees (trees Nu 14 and 41), but after a few months, all five trees were occupied only by Azteca colonies. Note that, in the end, tree Nu 52 was colonized by A. ovaticeps. Over the course of the different surveys, unoccupied trees were colonized by caterpillars (four cases) or directly by Azteca ants (trees Nu 19 and 25). Although S. saevissima workers exploited the FBs and were aggressive towards flying insects landing on their host tree foliage, tree Nu7 was colonized in the end by caterpillars (Fig. 3). While the two Azteca species occupied more and more trees over time, the number of trees sheltering P. guianalis caterpillars first increased and then decreased. They were replaced by A. alfari or A. ovaticeps colonies on 15 and 10 trees, respectively. At the end Fungal presence 1E). The first cells of the inner trichilium react to the presence of the mycelium when the FB is highly invaded. They seem to contain more tannin and are more elongated. of the experiment-so 3 years after the beginning of the survey-six trees still sheltered caterpillars. During this entire lapse of time, the trees occupied by A. alfari or A. ovaticeps were never colonized by caterpillars. In July 2009, four Cecropia trees still sheltered caterpillars. Food body production, herbivory and tree growth We noted a proliferation of the mycelium in in vitro cultures in Petri dishes with both healthy and infected trichilia, betraying the presence of spores in all cases (i.e. trichilia taken from trees sheltering ants, caterpillars or unoccupied). FB production was significantly higher for saplings sheltering Azteca ants than for those sheltering caterpillars and for the latter compared to unoccupied saplings (Fig. 4). We did not note significant differences in the percentage of foliar surface eaten by defoliating insects between the C. obtusa sheltering an Azteca colony, caterpillars, or not occupied by either ants or caterpillars (Kruskal-Wallis test, H2 90 = 1.813; P.0.05). Nevertheless, caterpillar presence affected tree growth as those sheltering Azteca colonies during this experimental period were significantly taller at the end of the survey than those sheltering caterpillars or those that were unoccupied (Fig. 5). The differences were not significant between trees sheltering colonies of the two Azteca species, or between trees sheltering caterpillars or that were unoccupied. grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 Figure 3. Host successional patterns for Cecropia treelets. successional patterns for each Cecropia sapling monitored during year survey on the dirt road near the Montagne des singes. D g g doi:10.1371/journal.pone.0020538.g003 This type of increase in FB production is usually stimulated when mutualistic ants remove the FBs because the space thus made available reduces the pressure on the trichilia and favors the production of the next group of FBs [17],[54]. In the absence of mutualistic ants, FB production remains low, but is high enough to be attractive to founding ant queens [17],[54]. Because our experimental design did not allow Azteca ants, caterpillars or other insects access to the trichilia, FB production should have been reduced to the same level as on unoccupied trees. Yet, this was not the case, suggesting that a factor other than FB removal plays a role, such as the plant obtaining nutrients from its ant or caterpillar inhabitants [55]. When parasitic ants are present, the establishment of mutualistic species is durably prevented, and plant fitness is lessened due to increased herbivory [8]. The presence of non-ant parasites does not imply the exclusion of mutualistic ants, but host tree leaves can suffer herbivory if these insects feed on the plant [37] or on plant- ants that are therefore unable to protect their host trees [36]. However, here, the presence of P. guianalis caterpillars was not associated with greater herbivory compared to trees sheltering Azteca colonies or unoccupied trees because saplings rely on secondary anti-defoliator compounds and structures for their protection [37],[44],[45]. Even though the saplings did not suffer greater herbivory, the protective mutualism is very disrupted as, when present, Azteca colonies significantly limit the development of the Fusarium mycelium. Indeed, we show that in the absence of mutualistic ants, Fusarium developed on the trichilia of both unoccupied trees and trees sheltering caterpillars. Moreover, this fungus is known to produce growth-inhibiting mycotoxines that are also responsible for necrosis in plants [56],[57] and insects [58],[59]. Consequent- ly, likely due to the presence of this pathogen plus the cost of producing FBs, the growth rate of the trees that sheltered caterpillars during the survey conducted at the Montagne des singes was affected if compared to those that sheltered Azteca colonies during the same period (Fig. 5). Discussion All in all, these results constitute a new step in understanding the nature of the parasitism of the Azteca-Cecropia mutualism by non- ant insects. Like Coelomera chrysomelid beetles [37] and mutualistic ants, P. guianalis caterpillars at their third larval stage recognize the prostomata and gnaw an entrance hole to shelter in the host tree domatia. The difference with the damage caused by Coelomera is that it is direct as they feed on young leaves [37], whereas P. guianalis caterpillars, like mutualistic ants, feed on the FBs produced by the plant. They are indirectly detrimental to their host tree because they allow Fusarium to develop on the trichilium. Moreover, although it is possible that female moths select the most productive trees for their offspring [52], the caterpillars seems to induce an increase in FB production, as do Azteca workers and clerid beetle (Phyllobaenus sp.) larvae on Piper [36]. However, unlike some mutualistic ants [53], P. guianalis caterpillars do not provide any services in return for this increase in FB production. PLoS May 2011 \| Volume 6 \| Issue 5 \| e20538 May 2011 \| Volume 6 \| Issue 5 \| e20538 PLoS ONE \| www.plosone.org 4 Two Parasites of an Ant-Plant Mutualism grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 Therefore, mutualistic Azteca likely control the extent of the fungal infection in the same way that, by defending myrmeco- phytic Piper from stem-boring insects, Pheidole ants reduce fungal infections [60]. On the other hand, when deprived of their mutualistic Crematogaster ants, myrmecophytic Macaranga suffer from both shoot borers and pathogenic fungi [61]. Indeed, ants’ antifungal activity is well known [37],[60],[62] and can be due to chemicals produced by the venom, the metapleural or the mandibular glands [63–66] or results from the activity of symbiotic bacteria [67]. On the plant side, it has been noted that some myrmecophyte species have lost their intrinsic physiological defenses against fungal infection [60],[68]. The spores of Fusarium can be disseminated by both wind and insects, particularly Lepidoptera larvae that are resistant [58], explaining why the P. guianalis caterpillars were not infected by Fusarium, while the host plant trichilia were. Because we did not note a difference in the amount of herbivory between ant-inhabited and ant-free Cecropia, one can hypothesize that Fusarium might be the main selective driving force in the present situation. In that case, the earlier the C. obtusa treelets shelter mutualistic Azteca colonies, the more they will grow due to the antifugal activity of the ants (particularly by suppressing spore germination [69]). Later, as the trees grow and their ability to synthesize secondary antiherbivore compounds lessens, Azteca workers, that belong to larger and larger colonies, Figure 3. Host successional patterns for Cecropia treelets. Host successional patterns for each Cecropia sapling monitored during the 3- year survey on the dirt road near the Montagne des singes. Dashes correspond to trees sheltering both caterpillars and A. alfari. Trees were May 2011 \| Volume 6 \| Issue 5 \| e20538 May 2011 \| Volume 6 \| Issue 5 \| e20538 PLoS ONE \| www.plosone.org 5 Two Parasites of an Ant-Plant Mutualism Figure 4. Food body production. Comparison of the mean food body production per leaf and per day (6SE) by the youngest trichilia on Cecropia obtusa saplings during 20 successive days in three situations: saplings sheltering an Azteca alfari colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings (10 individuals in each case). Statistical comparisons; repeated measures ANOVA: F2 30 = 64.81; P,0.001; Newman-Keuls’ post-hoc test: different letters indicate significant differences at P,0.001. doi:10.1371/journal.pone.0020538.g004 Figure 4. Food body production. grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 Comparison of the mean food body production per leaf and per day (6SE) by the youngest trichilia on Cecropia obtusa saplings during 20 successive days in three situations: saplings sheltering an Azteca alfari colony, saplings sheltering Pseudocabima guianalis caterpillars, and unoccupied saplings (10 individuals in each case). Statistical comparisons; repeated measures ANOVA: F2 30 = 64.81; P,0.001; Newman-Keuls’ post-hoc test: different letters indicate significant differences at P,0.001. doi:10.1371/journal.pone.0020538.g004 When caterpillars do successfully colonize a tree, several overlapping generations can be observed-the youngest sheltering under the stipules developing around the terminal bud, the trunk and the youngest leaf, and the oldest in the host tree domatia. During the hours of FB production, both young and old caterpillars share the FBs on the trichilia. Smaller caterpillars likely benefit from the silk woven above the trichilia by larger mates as protection from competing ants, predators and/or parasitoids which seem repelled, and so do not walk on it (pers. obs.). The overlap between different generations of caterpillars plus the fact that certain trees can be occupied during several years imply that female P. guianalis moths lay eggs on trees already sheltering caterpillars. will take over and provide their host trees with biotic protection from herbivorous insects [11],[17],[42]. We cannot exclude that the occupancy by caterpillars could be favored by the plant’s characteristics or micro-environmental conditions rather than by the competitive and colonizing abilities of the insects. Nevertheless, because C. obtusa is a pioneer species that develops in large numbers in recently cleared areas, some insect species with a high rate of dispersal can be the first to reach the resources provided by the trees (see [51] for Reduvidae feeding on C. obtusa FBs before the installation of Azteca colonies). It is thus likely that a temporal priority enabled P. guianalis caterpillars to install themselves on certain trees prior to the arrival of the plant’s mutualistic Azteca ants with which they are involved in competitive exclusion (see also [23],[24] for a temporal priority concerning ant-myrmecophyte mutualisms). When an incipient Azteca colony successfully colonizes their host tree, large caterpillars seem to deny the first workers access to the Figure 5. Height of Cecropia treelets. Mean height of trees (6SE) that sheltered colonies of one of the two Azteca species, Pseudocabima guianalis caterpillars, or that sheltered neither Azteca nor caterpillars during the experimental period. References 19. Brouat C, Garcia N, Andary C, McKey D (2001) Plant lock and ant key: pairwise coevolution of an exclusion filter in an ant-plant mutualism. Proc R Soc London, B 268: 2131–2141. 1. Bronstein JL (1994) Our current understanding of mutualism. Quart Rev Biol 69: 31–51. 2. Bronstein JL, Dieckmann U, Ferrie`re R (2004) Coevolutionary dynamics and the conservation of mutualisms. In: Ferrie`re R, Dieckmann U, Couvet D, eds. Evolutionary Conservation Biology. Cambridge: Cambridge University Press. pp 305–326. 20. MacArthur RH, Wilson EO (1967) The theory of island biogeography: Princeton University Press. 224 p. 21. Grubb PJ (1977) The maintenance of species richness in plant communities: the importance of the regeneration niche. Biol Rev 52: 107–145. pp 3. Herre EA, Knowlton N, Mueller UG, Rehner SA (1999) The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends Ecol Evol 14: 49–53. 22. Tilman D (1994) Competition and biodiversity in spatially structured habitats. Ecology 75: 2–16. 4. Zientz E, Feldhaar H, Stoll S, Gross R (2005) Insights into the microbial world associated with ants. Arch Microbiol 184: 199–206. 23. Palmer TM, Young TP, Stanton ML (2002) Burning bridges: priority effects and the persistence of a competitively subordinate acacia-ant in Laikipia, Kenya. Oecologia 133: 372–379. 5. Klein RW, Kovac D, Schellerich A, Maschwitz U (1992) Mealybug-carrying by swarming queens of a southeast asian bamboo-inhabiting ant. Naturwissenschaf- ten 79: 422–423. 24. Wilbur HW, Alford RA (1985) Priority effects in experimental pond communities: responses of Hyla to Bufo and Rana. Ecology 66: 1106–1114. 6. Wilkinson DM, Sherratt TN (2001) Horizontally acquired mutualisms, an unsolved problem in ecology. 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Acknowledgments We are grateful to the Laboratoire Environnement de Petit Saut for its logistical help, A. Yockey-Dejean for proof-reading the manuscript, M. A. Solis (USDA, Beltsville, Maryland, USA) and E. Bury (Muse´um National d’Histoire Naturelle, Paris, France) for identifying the P. guianalis and F. moniliforme, respectively. We would also like to thank anonymous referees for their helpful comments. References Oikos 117: 310–320. 10. Rico-Gray V, Oliveira P, eds (2007) The ecology and evolution of ant-plant interactions. Chicago: The University of Chicago press. 331 p. 10. Rico-Gray V, Oliveira P, eds (2007) The ecology and evolutio i i Chi Th U i i f Chi 331 interactions. Chicago: The University of Chicago press. 331 p. 28. Bertness MD, Shumway SW (1993) Competition and facilitation in marsh plants. Amer Nat 142: 718–724. 11. Davidson DW, McKey D (1993) The evolutionary ecology of symbiotic ant- plant relationships. J Hymenopt Res 2: 13–83. 29. Ladd B, Facelli JM (2008) Priority effects produced by plant litter result in non- additive competitive effects. Oecologia 157: 687–696. p p J y p 12. Ward PS (1993) Systematic studies on Pseudomyrmex acacia-ants ( p p y p 12. Ward PS (1993) Systematic studies on Pseudomyrmex acacia-ants (Hymenoptera: Formicidae: Pseudomyrmecinae). J Hymenopt Res 2: 117–168. 30. Lawler SP, Morin PJ (1993) Temporal overlap, competition, and priority effects in larval anurans. Ecology 74: 174–182. 13. Longino JT (1989) Geographic variation and community structure in an ant- plant mutualism: Azteca and Cecropia in Costa Rica. Biotropica 21: 126–132. . Longino JT (1989) Geographic variation and community structur gy 31. Hodge S, Arthur W, Mitchell P (1996) Effects of temporal priority on interspecific interactions and community development. Oikos 76: 350–358. 14. Vasconcelos HL (1993) Ant colonization of Maieta guianensis seedlings, an Amazon ant-plant. Oecologia 95: 439–443. 32. Davidson DW, Snelling RR, Longino JT (1989) Competition among ants for myrmecophytes and the significance of plant trichomes. Biotropica 21: 64–73. 15. Edwards DP, Hassall M, Sutherland WJ, Yu DW (2006) Assembling a mutualism: ant symbionts locate their host plants by detecting volatile chemicals. Insect Soc 53: 172–176. 33. Yu DW, Davidson DW (1997) Experimental studies of species-specificity in Cecropia-ant relationships. Ecol Mon 67: 273–294. 16. Jurgens A, Feldhaar H, Feldmeyer B, Fiala B (2006) Chemical composition of leaf volatiles in Macaranga species (Euphorbiaceae) and their potential role as olfactory cues in host-localization of foundress queens of specific ant partners. Biochem Syst Ecol 34: 97–113. 34. Fonseca CR, Benson WW (2003) Ontogenetic succession in Amazonian ant trees. Oikos 102: 407–412. 35. Janzen DH (1975) Pseudomyrmex nigropilosa: a parasite of a mutualism. Science 188: 936–937. 17. Davidson DW (2005) Cecropia and its biotic defenses. In: Berg CC, Rosselli PF, eds. Flora Neotropica Monograph. BronxNew York: The New York Botanical Garden. pp 214–226. 36. grouped to ensure the legibility of the figure, and so do not correspond to their geographic distribution. doi:10.1371/journal.pone.0020538.g003 Statistical comparisons (normality and equal variance tests passed); ANOVA: F2 56 = 8.56; P,0.0001. Newman-Keuls’ post-hoc test: different letters indicate significant differences at P,0.05. doi:10.1371/journal.pone.0020538.g005 Figure 5. Height of Cecropia treelets. Mean height of trees (6SE) that sheltered colonies of one of the two Azteca species, Pseudocabima guianalis caterpillars, or that sheltered neither Azteca nor caterpillars during the experimental period. Statistical comparisons (normality and equal variance tests passed); ANOVA: F2 56 = 8.56; P,0.0001. Newman-Keuls’ post-hoc test: different letters indicate significant differences at P,0.05. doi:10.1371/journal.pone.0020538.g005 PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e20538 6 Two Parasites of an Ant-Plant Mutualism most productive trichilia by weaving silk above them. They, thus, indirectly slow down colony growth as these foraging workers only have access to the lower, less-productive trichilia often already covered by F. moniliforme. Consequently, the development of the colonies depends mostly on the ants attending hemipterans in the internodes of the host trees (if any). So, although caterpillars can delay Azteca colonization, the Cecropia trees are finally exclusively occupied by Azteca ants. Furthermore, once Azteca colonizes a Cecropia tree, the workers exploit the FBs on the upper, most- productive trichilia and patrol the foliage, rendering the situation irreversible by preventing colonization by caterpillars. Indeed, the Azteca workers, that are able to capture insects the size of a female P. guianalis moth [42], probably destroy any insect eggs that have been successfully laid on their host plant’s foliage (see [70] and references therein). Supporting Information Figure S1 Caterpillars on Cecropia treelets. a Upper part of a young Cecropia obtusa sheltering Pseudocabina guianalis caterpil- lars. Strands of silk produced by the caterpillars keep the stipules of two leaves against the trunk (yellow arrow). An entrance hole gnawed by a caterpillar is visible (white arrow). Note that the leaves were not attacked by defoliating insects. b A forth instar caterpillar eating food bodies on the youngest trichilia on a tree, some strands of silk are visible. c Three larval stages eating food bodies on the same trichilia. d A forth instar caterpillar eating food bodies on a trichilia that began to be infected by Fusarium moniliforme. e Several caterpillars at different stages on a trichilia, some strands of silk are visible. In conclusion, P. guianalis caterpillars are able to ‘‘break the code’’ [36] of the Azteca-Cecropia mutualism by recognizing the prostomata and exploiting the resources Cecropia normally supplies to mutualistic Azteca; they even induce greater FB production. Although no higher herbivory rates were noted, these caterpillars are ineffective in keeping a fungus from developing on the trichilia of their host trees, something that mutualistic Azteca ants can do. By denying mutualistic ants access to FBs and young leaves, P. guianalis caterpillars become a more formidable competitor of mutualistic ants and so are parasites of both Cecropia saplings and the Azteca-Cecropia mutualism. Author Contributions Conceived and designed the experiments: AD PJS. Performed the experiments: AD PJS. Analyzed the data: OR RC AD. Contributed reagents/materials/analysis tools: AD. Wrote the paper: OR AD. Conceived and designed the experiments: AD PJS. Performed the experiments: AD PJS. Analyzed the data: OR RC AD. Contributed reagents/materials/analysis tools: AD. Wrote the paper: OR AD. Two Parasites of an Ant-Plant Mutualism Two Parasites of an Ant-Plant Mutualism 54. Folgarait PJ, Johnson HL, Davidson DW (1994) Responses of Cecropia to experimental removal of Mullerian bodies. Funct Ecol 8: 22–28. 38. Maschwitz U, Schroth M, Ha¨nel H, Pong TY (1984) Lycaenids parasitizing symbiotic plant-ant partnerships. Oecologia 64: 78–80. y p p p g 39. 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https://openalex.org/W2890445289	https://europepmc.org/articles/pmc6129583?pdf=render	English	null	Factors Explaining Language Performance After Training in Elders With and Without Subjective Cognitive Decline	Frontiers in aging neuroscience	2,018	cc-by	10,724	Factors Explaining Language Performance After Training in Elders With and Without Subjective Cognitive Decline Ramón López-Higes1, Jose M. Prados1, Susana Rubio-Valdehita1, Inmaculada Rodríguez-Rojo1,2, Jaisalmer de Frutos-Lucas2,3, Mercedes Montenegro4, Pedro Montejo4, David Prada4 and María L. D. Losada1 1 Department of Experimental Psychology, Complutense University of Madrid, Madrid, Spain, 2 Laboratory of Cognitive and Computational Neuroscience, Center of Biomedical Technology, Polytechnical University – Complutense University, Madrid, Spain, 3 Centre for the Prevention of Cognitive Impairment, Madrid, Spain, 4 Biological and Health Psychology Department, Universidad Autónoma de Madrid, Madrid, Spain The present study explores if cognitive reserve, executive functions, and working memory capacity are predictive of performance in the language domain (speciﬁcally in sentence comprehension and naming) after a cognitive training intervention. Sixty- six Spanish older adults voluntarily participated in the study, classiﬁed either as older adults with subjective cognitive decline according to Jessen et al.’s (2014) criteria (n = 35; 70.94 ± 4.16 years old) or cognitively intact (n = 31; 71.34 ± 4.96 years old). Written sentence comprehension and visual confrontation naming were assessed both immediately after recruitment (at the baseline), and then 6 months later, once each participant had completed his/her cognitive training (a well-known program in Spain, called UMAM; English translation: Madrid City Council Memory Unit Program). Cognitive reserve, executive functions (cognitive ﬂexibility and controlled interference efﬁciency), and working memory capacity were measured for all participants at the baseline. Results pointed out that the subjective cognitive decline group presented greater beneﬁts in the language domain than cognitively intact participants. We also observed that lower executive functioning and working memory capacity at the baseline predicted larger beneﬁts in language performance after training, but only in the group of cognitively intact older adults. However, selected predictors hardly explained subjective cognitive decline participants’ results in language performance after training. Citation: López-Higes R, Prados JM, Rubio-Valdehita S, Rodríguez-Rojo I, de Frutos-Lucas J, Montenegro M, Montejo P, Prada D and Losada MLD (2018) Factors Explaining Language Performance After Training in Elders With and Without Subjective Cognitive Decline. Front. Aging Neurosci. 10:264. doi: 10.3389/fnagi.2018.00264 ORIGINAL RESEARCH published: 03 September 2018 doi: 10.3389/fnagi.2018.00264 Keywords: subjective cognitive decline, older adults, cognitive training, sentence comprehension, naming, executive functions, working memory, cognitive reserve Edited by: Edited by: P. Hemachandra Reddy, Texas Tech University Health Sciences Center, United States Reviewed by: Patrizia Giannoni, University of Nîmes, France Bhoomika Kar, Allahabad University, India Correspondence: Ramón López-Higes rlopezsa@psi.ucm.es Edited by: P. Hemachandra Reddy, Texas Tech University Health Sciences Center, United States Reviewed by: Patrizia Giannoni, University of Nîmes, France Bhoomika Kar, Allahabad University, India *Correspondence: Ramón López-Higes rlopezsa@psi.ucm.es Received: 12 April 2018 Accepted: 14 August 2018 Published: 03 September 2018 Citation: López-Higes R, Prados JM, Rubio-Valdehita S, Rodríguez-Rojo I, de Frutos-Lucas J, Montenegro M, Montejo P, Prada D and Losada MLD (2018) Factors Explaining Language Performance After Training in Elders With and Without Subjective Cognitive Decline. Front. Aging Neurosci. 10:264. doi: 10.3389/fnagi.2018.00264 Received: 12 April 2018 Accepted: 14 August 2018 Published: 03 September 2018 INTRODUCTION Nowadays there is an increasing interest in non-pharmacological intervention eﬀects on older adult cognition. Some studies have shown that cognitive training (CT) is beneﬁcial for older adults’ memory (Gross et al., 2012; Rosi et al., 2017) but also for other domains in this population, as for attention (Rahe et al., 2015), working memory, reasoning and language comprehension (Carretti et al., 2013; Karbach and Verhaeghen, 2014), processing speed and September 2018 \| Volume 10 \| Article 264 1 Frontiers in Aging Neuroscience \| www.frontiersin.org Language After Training in Elders López-Higes et al. for results against the existence of such eﬀects). Carretti et al. (2013) have investigated the eﬃcacy of a verbal WM training program in CI older adults, considering the speciﬁc training-related beneﬁts in a verbal WM task (criterion) and the transfer eﬀects on measures of WM updating, reasoning, and on language comprehension. They found that the trained older adults performed better than controls in the criterion task and retained this beneﬁt 6 months later. Transfer eﬀects were seen in reasoning and language comprehension performance and were substantially maintained at the 6-month follow-up. In the same line, a study conducted by Payne and Stine- Morrow (2017) examined the eﬀects of a novel home-based computerized CT program focused in verbal WM in healthy older adults in comparison with an active component-control group. Participants in the WM training group showed non-linear improvements in performance on trained verbal WM tasks. In contrast with the control group, WM training participants also showed improvements on untrained verbal WM tasks and across untrained dimensions of language, including sentence memory, verbal ﬂuency, and comprehension of syntactically ambiguous sentences. reasoning (Willis and Caskie, 2013), or executive functions (Jackson et al., 2012; Mowszowski et al., 2016); CT is also beneﬁcial for older adults with subjective cognitive decline (SCD) or people with mild cognitive impairment (Martin et al., 2011; Rebok et al., 2014; Smart et al., 2017). Another issue that requires attention is the one concerning the factors that are strongly associated with greater beneﬁts after CT application. For example, lifestyle factors, occupation attainment or education (cognitive reserve proxies) inﬂuence individuals’ cognition along life (Stern, 2012). In this vein, Mondini et al. INTRODUCTION (2016) have shown that cognitive reserve (CR) can modulate the general cognitive status (measured by Mini-Mental State Examination; MMSE onward) after a CT program and should be considered as a predictor of CT eﬃcacy in older adults with mild to moderate dementia. In Spain a recently published study (Lojo- Seoane et al., 2018) have shown the inﬂuence of CR on episodic memory, WM, and general cognitive performance in adults with subjective cognitive complaints evaluated at baseline and at a follow-up after an interval of about 18 months. The results exhibited the positive eﬀect of CR on cognitive performance at baseline and at follow-up, conﬁrming the mediating role of WM on episodic memory and general cognitive performance. Another study (Clark et al., 2016) has indicated that educational attainment modulates the eﬀectivity of training in cognitively intact (CI) older adults, being participants with low educational level the ones with the best performance after training. These and other examples reveal that there are important factors at the baseline which might modulate CT gains, both general or/and in speciﬁc cognitive domains (Kraemer et al., 2002; Ranganath et al., 2011). In a study conducted by Bamidis et al. (2015) with a community-dwelling sample of CI and cognitively impaired older adults, the authors reported a robust modulation (inverse) eﬀect of executive functions’ baseline performance on training beneﬁts in global cognition. Additionally, López-Higes et al. (2018) have shown a compensation eﬀect regarding general cognitive status in SCD participants with lower interference eﬃciency at the baseline. Following the administration of a task- switching intervention in three diﬀerent populations (children, young, and older adults), Karbach et al. (2017) demonstrated that this training program led to a reduction of age diﬀerences. They also observed that baseline abilities predicted both training and transfer gains. Speciﬁcally their results showed a compensation eﬀect, since those participants with lower abilities were the ones who improved the most. However, there are other studies that have found opposite results. For example, Guye et al. (2017) have demonstrated very limited evidence for individual diﬀerences as predictors of training outcomes, although they found that cognitive performance at baseline was related to training improvement. In particular, those with higher abilities seemed to beneﬁt more, indicating a magniﬁcation eﬀect, which was especially apparent in their young adult population. INTRODUCTION An additional related issue that has generated a considerable debate is the question of whether and how CT may produce improvements in untrained tasks (see for example Karbach In Spain, a well-known multifactorial CT program, called UMAM by its name in Spanish [(programa de la Unidad de Memoria del Ayuntamiento de Madrid); English translation: Madrid City Council Memory Unit Program; Montejo et al., 2013] has proven its eﬃcacy on subjective and objective memory measures (and also in mood) in older adults without cognitive impairment, both in the post-training assessment and at 6- month follow-up (Montejo, 2015). The beneﬁts of the UMAM CT program on other cognitive domains, such as language, have been explored in a previous study comparing APOE ε4 carriers and non-carriers (López-Higes et al., 2017). That study revealed that there were consistent treatment beneﬁts in complex sentence comprehension, but only in the APOE ε4 non-carrier group. p y g p The present study explores the role of CR and executive functions at the baseline as factors which might modulate the eﬃcacy of the UMAM program on complex sentence comprehension and naming in CI and in SCD older adults. The three main executive functions that have been used as predictors in this study: (a) alternation (cognitive ﬂexibility), (b) updating and monitoring of WM representations, and (c) inhibition (as they appear in Miyake et al., 2000 model). In the Miyake and Friedman (2012) model, the part equivalent to the inhibition of the previous model would be a common part of all the executive functions, while the other two, updating and monitoring as well as alternation, would maintain a corresponding speciﬁc part within the model. Comprehension and naming are abilities related to successful daily living, since they are needed in various everyday activities and are relevant to older adults’ health and well-being. If older adults with SCD have higher risk of developing dementia (Jessen et al., 2014), it is of great interest to investigate beneﬁts of CT in this population in comparison with CI participants, as well as to ﬁnd which factors might be associated with greater gains subsequently. Frontiers in Aging Neuroscience \| www.frontiersin.org INTRODUCTION Considering a recent work where results evidenced that the UMAM CT program was most beneﬁcial for SCD participants in explaining that their eﬃciency in inhibition support their An additional related issue that has generated a considerable debate is the question of whether and how CT may produce improvements in untrained tasks (see for example, Karbach and Verhaeghen, 2014; Au et al., 2015, 2016; Greenwood and Parasuraman, 2016, for evidence in favor to transfer eﬀects; see also, Melby-Lervåg and Hulme, 2013 or Simons et al., 2016, September 2018 \| Volume 10 \| Article 264 2 Language After Training in Elders López-Higes et al. institute in Madrid) and, were also enrolled in the UMAM program. general cognitive performance, while the Cis’ WM capacity was the most important performance predictor (López-Higes et al., 2018), our hypothesis are as follows: (1) We expected that CR will not have a signiﬁcant role as predictor of CT outcomes in language in any group; (2) We hypothesized that sentence comprehension and naming will be most improved in the SCD’s participants after the CT intervention than in those pertaining to the CI’s older group; (3) Provided that the UMAM program is multifactorial, we anticipate a positive eﬀect on participants’ executive functioning which, in turn, will explain linguistic performance; Finally, we assume that program eﬀects will be diﬀerent depending on the group considered. In short, within the group of older adults with SCD a greater weight of inhibition is expected as a predictor/mediator of linguistic performance, while in the group of CI participants the variable with the greater weight will be a measure related to updating and monitoring representations in WM. General inclusion criteria to participate in the study were: (1) Normal performance in the Logical Memory delayed recall subtest of the Wechsler Memory Scale – Third Edition (Wechsler, 1997; more than 10 units for people with 16 years of formal education or more; and more than six units for people with 8–15 years of formal education); (2) Yesavage Geriatric Depression Scale (GDS-15; Sheikh and Yesavage, 1986) lower or equal to 9; (3) MMSE (Folstein et al., 1975; Spanish adaptation by Lobo et al., 1999) higher than 24 points at the baseline. All participants had normal or corrected hearing and vision. INTRODUCTION p p g Thirty-ﬁve participants were identiﬁed as older adults with SCD (10 males and 25 females) according to Jessen et al.’s (2014) criteria, that is: (a) they had requested medical advice services about their memory complaints; (b) they presented self- perception of cognitive decline, mainly associated with memory loss; (c) they felt that their subjective decline aﬀected daily life activities; (d) they set the onset of their subjective decline within the last 5 years, and (e) concerns associated with their subjective decline were conﬁrmed by a reliable informant. The remaining 31 participants (12 males and 19 females) did not meet criteria for SCD, and they formed a group of CI older adults. The selection process described along these last lines is summarized in Figure 1. Participants Sixty-six older Spanish-speaking adults voluntarily participated in the study. They were recruited from the Center for the Prevention of Cognitive Impairment (a public health FIGURE 1 \| Flowchart showing the inclusion/selection criteria of the 66 participants. All of them were given two neuropsychological assessments: at baseline (0 months) and at the endpoint (6 months). The UMAM cognitive training program was applied between the baseline and the endpoint period. (1) Wechsler Memory Scale III. (2) Mini-Mental State Examination. (3) Geriatric Depression Scale-Short Version. (4) Subjective Cognitive Decline. (5) Cognitively Intact. FIGURE 1 \| Flowchart showing the inclusion/selection criteria of the 66 participants. All of them were given two neuropsychological assessments: at baseline (0 months) and at the endpoint (6 months). The UMAM cognitive training program was applied between the baseline and the endpoint period. (1) Wechsler Memory Scale III. (2) Mini-Mental State Examination. (3) Geriatric Depression Scale-Short Version. (4) Subjective Cognitive Decline. (5) Cognitively Intact. Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org September 2018 \| Volume 10 \| Article 264 3 Language After Training in Elders López-Higes et al. TABLE 1 \| Descriptive statistics for socio-demographic variables (age, years of formal education) as well as scores of, global cognitive status (MMSE), depressive symptomatology (GDS-15), cognitive reserve, executive functions and working memory capacity in both groups at baseline. objectives and working on with speciﬁc exercises involving attention (focused attention, attentive listening) or perception (a basic process in the registration phase of memorization). Exercises include visual and auditory material. Language acts as mediator in the processes related to codiﬁcation and retrieving information from memory. Through language exercises the program aims to increase verbal ﬂuency as well as encourage evocation. g p ( g , y l cognitive status (MMSE), depressive e, executive functions and working I SCD SD Mean SD 4.16 71.39 4.96 5.88 13.02 6.05 1.19 28.45 1.50 2.20 2.55 2.39 3.85 13.59 4.37 1.08 2.39 0.93 7.17 4.71 6.66 2.07 11.95 2.25 atistics for socio-demographic education) as well as scores , depressive symptomatology ns and WM capacity in both r adults had signiﬁcant lower a group than CI participants 016; observed power = 0.669]. The UMAM Cognitive Training Program The UMAM Cognitive Training Program The UMAM program was initially designed and implemented in 1995 by the Memory Training Unit of the City Council of Madrid (Unidad de Memoria del Ayuntamiento de Madrid: UMAM). More than 20,000 older adults have beneﬁted from this CT program. This multifactorial intervention consists of 30 sessions that has a duration of 90 min each. Twenty- eight sessions are carried out along 3 months followed by two additional maintaining-booster sessions. Training is organized in groups of 12–18 participants that come to the center in three mornings per week. A typical session begins with 10 min of relaxation, 15 min for homework and other activation activities, followed by 60 min of CT of speciﬁc activities, and concluded with suggested homework for the next day (5 min). Frontiers in Aging Neuroscience \| www.frontiersin.org Participants The present study complied with the ethical standards of the Declaration of Helsinki and was approved by the Ethical Committee of the San Carlos Clinical Hospital in Madrid, which is one of the main medical institutions that participates in the current research project. From the ﬁrst session, it is highlighted the importance of reﬂecting on one’s own daily memory failures (causes and related variables; metamemory) and on the methods used to avoid or overcome them. The UMAM program has been analyzed during its years of implementation, always obtaining good results. The most recent evaluations of this program, such as that observed by of Montejo (2003) where 1,083 users over 65 years of age show that the UMAM program produced a 40% improvement in objective memory in 77% of the cases, and on a subjective level they improved until the 75%, a percentage that was even maintained along the 6 months follow up. In another sample of 2,553 users (Montejo et al., 2013) the normal pre-treatment memory level was 24.8%, reaching a post level of 56%. As mentioned before, the program has proven its eﬃcacy in older adults without cognitive impairment, both in the post-training assessment and at 6-month follow-up (Montejo, 2015). Participants n groups arose for any other ed about the objectives of participate after signing a The present study complied he Declaration of Helsinki cal Committee of the San d, which is one of the main ates in the current research v v CI SCD Mean SD Mean SD Age 70.94 4.16 71.39 4.96 Years of formal education 14.38 5.88 13.02 6.05 MMSE 28.94 1.19 28.45 1.50 GDS-15 1.53 2.20 2.55 2.39 Cognitive reserve 15.40 3.85 13.59 4.37 Ratio TMT 2.29 1.08 2.39 0.93 Interference 9.07 7.17 4.71 6.66 Digit reordering 12.68 2.07 11.95 2.25 The main strategies that are worked on in the program are visualization, association, categorization, and repetition. 2. Module of memory concepts. In this module professionals present the most important aspects of memory functioning, highlighting those whose knowledge may have an impact on daily life: types and phases of memory, memory in the elderly and factors that aﬀect memory performance. 3. Application module to daily life. It is about practicing and adapting memory strategies to the most frequent daily forgetfulness. In addition, speciﬁc techniques are taught and practiced for some memory problems: forgetting where something has been put, forgetting names, understanding and remembering texts, etc. The UMAM program includes techniques that are of interest for the daily forgetfulness in this population. The method of the 3 “R” is emphasized, since it is a procedure to improve the comprehension and memory of texts, as well as the use of an speciﬁc technique to recall names. Another aspect that we also deal with is the use of external aids, resources such as diaries, alarms or warnings, telephone directory, putting notes, lists, labels, etc. Table 1 shows the descriptive statistics for socio-demographic variables (age, years of formal education) as well as scores of global cognitive status (MMSE), depressive symptomatology (GDS-15), CR, executive functions and WM capacity in both groups at the baseline. SCD older adults had signiﬁcant lower scores in interference eﬃcacy as a group than CI participants [F(1, 64) = 5.948; p = 0.018; η2 p = 0.016; observed power = 0.669]. No signiﬁcant diﬀerences between groups arose for any other variable. All participants were informed about the objectives of the study and were invited to participate after signing a written informed consent form. Dependent Variables (Naming and Sentence Comprehension) CR, etc.), as well as those taking into account a measure of training eﬀects (diﬀerences between post- and pre-measure in each dependent variable), were computed by means of General Linear Model ANOVAs. Eﬀect sizes were estimated by means of partial eta-square (η2 p); observed power was also given when is needed. In all the analyses we used IBM SPSS statistical program v. 22.0. Regarding predictor variables, we obtained a total score for each participant reﬂecting an estimation of her/his CR. With respect to digit reordering, we considered the number of series correctly ordered by each participant as a measure of his/her WM capacity (reﬂecting updating and monitoring of WM representations). With respect to TMT, we used the B/A ratio score (proposed by Lamberty et al., 1994) since it tries to eliminate the eﬀect of the visual search and the perceptomotor speed also present in part B, to measure only the eﬀect of the executive function, ﬁnding signiﬁcant correlations with other tasks that measure alternation or task- switching ability (Arbuthnott and Frank, 2000). Other authors (Maruta et al., 2011) have considered that part B is a common and sensitive measurement of cognitive ﬂexibility (see also Lezak, 1995). That is why we have assumed that the ratio B/A is a more ﬁne-grained score of that function. Finally, with regards to the Stroop test, we used the Interference index proposed by Chafetz and Matthews (2004). In order to test the role of diﬀerent predictors regarding CT outcomes in the language domain we have followed a procedure described in Mondini et al. (2016). The measures employed in the analyses resulted from subtracting the baseline scores to the endpoint scores in each of the selected dependent variables. All resulting models were compared using four indexes providing goodness- of-ﬁt measures: Bayesian Information Criterion (BIC), Akaike Information Criterion (AIC), Bayes Factor (BF), and R2. Higher BF and R2 values and lower BIC and AIC indexes, denote better model ﬁt to data. BIC, AIC and R2 were computed using IBM SPSS statistical software v. 20.0, but the Bayes Factor index was computed with version 0.9.8 of the BayesFactor package developed by Perception and Cognition Lab, Department of Psychological Sciences at the University of Missouri1. The Boston Naming Test (Kaplan et al., 1983) was selected to explore word retrieval by visual confrontation naming. Materials P di V Predictor Variables (CR and Executive Functions) Cognitive reserve was estimated using the CR Questionnaire (CRQ; Cuestionario de Reserva Cognitiva; Rami et al., 2011), a brief questionnaire well suited for clinical context. Inhibition eﬃciency and alternation (cognitive ﬂexibility) were assessed by the Stroop test (Golden, 1978) and the Trail Making Test parts A and B (TMT-A and TMT-B; Reitan, 1994), respectively. The assessment protocol also included a digit reordering task (MacDonald et al., 2001), which involves maintaining and manipulating information in WM (Hill et al., 2010). The UMAM program is organized in modules: 1. Module of cognitive stimulation and learning of speciﬁc strategies. This module stimulates basic cognitive processes such as attention, concentration, or perception. The most important memory strategies are taught and practiced: visualization, association, categorization, elaboration, etc. Basic cognitive processes are stimulated by pursuing speciﬁc September 2018 \| Volume 10 \| Article 264 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 Language After Training in Elders López-Higes et al. Dependent Variables (Naming and Sentence Comprehension) Sentence comprehension was assessed by means of a sentence–picture simple veriﬁcation task included in the ECCO_Senior test (Exploración Cognitiva de la Comprensión de Oraciones para mayores; English translation: Cognitive Assessment of Sentence Comprehension for seniors; López-Higes et al., 2012). This test includes 36 semantically reversible sentences that are either congruent or incongruent with the picture that accompanies them. Spanish is a Subject–Verb–Object (SVO) language (e.g., El hombre persigue al perro; English translation: The man chases the dog). Non-canonical word order (as for example in passive sentences: The man is kicked by the woman) or number of propositions in a sentence (one in The girl kissed her boyfriend vs. two in The woman kissed by her grandmother was sitting in a chair) are factors that makes sentences’ processing and interpretation more diﬃcult (Thompson and Shapiro, 2007). Procedure An extensive neuropsychological assessment of each participant, including diﬀerent cognitive domains (memory, executive functions, language), was conducted at two diﬀerent times, one immediately after recruitment (baseline) and then 6 months later (post-training measure or endpoint). Neuropsychological assessments were conducted by an experienced psychologist or psychiatrist at the Center for the Cognitive Impairment Prevention. In the ﬁrst session, the participants completed the screening tests (MMSE, GDS-15) and CRQ. In addition they were informed about the main goals of the study and signed an informed consent document. All the remaining neuropsychological and cognitive tests were applied in two additional sessions of 50 min each (approximately). Although there was a ﬁxed block of tests arranged for each session, the order in which the tests were presented was randomized in each session for each participant. Tests were applied and scored following the instructions provided in the users’ manuals. 1http://pcl.missouri.edu/bf-reg September 2018 \| Volume 10 \| Article 264 Training Outcomes Categorization Once diﬀerences in measures between post and pre-training were computed for all participants, it was possible to recode values in ﬁve categories of outcomes: (a) a positive value greater or equal to one SD was considered as moderate improvement, (b) positive values between 0 and 1 SD were mild improvement, (c) a value equal to zero meant null improvement, (d) negative values between 0 and 1 SD formed a category of mild worsening, and (e) negative values greater or equal to 1 SD were considered moderate worsening. Table 3 summarizes percentages in these ﬁve categories by group. Pearson Chi-square did not reach statistical signiﬁcance in any case [χ2(4) = 0.664, p = 0.324; χ2(4) = 1.500, p = 0.827; χ2(4) = 4.857, p = 0.302]. Finally, when visual confrontation naming was considered (Table 6), the best model for the CI group was the 3rd (BIC = 143.11; AIC = 138.90; BF = 2.75; R2 = 0.17; p < 0.033), with interference as the only signiﬁcant predictor (t = −2.40, p = 0.023). None of the regression models computed for this dependent measure in the SCD group reached statistical signiﬁcance. TABLE 3 \| Percentages of cases across outcome categories in CI and in SCD groups. CI SCD Non-canonical sentences Null improvement 9.7% 20.0% Mild worsening 16.1% 14.3% Moderate worsening 19.4% 11.4% Mild improvement 35.5% 20.0% Moderate Improvement 19.4% 34.3% Sentences with two propositions Null improvement 25.8% 31.4% Mild worsening 22.6% 28.6% Moderate worsening 3.2% 5.7% Mild improvement 25.8% 20.0% Moderate improvement 22.6% 14.3% BNT Null improvement 29.0% 13.2% Mild worsening 25.8% 18.4% Moderate worsening 0.0% 2.6% Mild improvement 32.3% 42.1% Moderate improvement 12.9% 23.7% TABLE 3 \| Percentages of cases across outcome categories in CI and in SCD groups Training Outcomes Differences Intragroup analysis in the CI group showed that the diﬀerence between pre- and post-training measures in sentences including two propositions reached statistical signiﬁcance [F(1, 30) = 5.025; p = 0.033; η2 p = 0.145; observed power = 0.583]. However, in the SCD group the analysis revealed that performance in naming improved after cognitive training [F(1, 34) = 12.612; p = 0.001; η2 p = 0.240; observed power = 0.934]. Eﬀect sizes in both cases were large, but especially in SCD older adults. g Considering sentences with two propositions, the model with the best goodness-of-ﬁt for the CI group was the 9th (Table 5) with TMT ratio and digit reordering as predictors (BIC = 130.15; AIC = 124.55; BF = 12.51; R2 = 0.34; p < 0.003). Both variables made a signiﬁcant contribution to the model, but with diﬀerent signs, thus in diﬀerent directions (TMT ratio: t = 2.16, p = 0.039; digit reordering: t = −2.58, p = 0.016). Model 12th also had a good ﬁt, and included as predictors CR, TMT ratio and digit reordering (BIC = 131.29; AIC = 124.28; BF = 10.62; R2 = 0.39; p < 0.003). CR did not contribute in a signiﬁcant manner to the model (t = −1.43, p = 0.165), but TMT ratio and digit reordering did it in the same direction than in the previous model (TMT ratio: t = 2.28, p = 0.031; digit reordering: t = −2.32, p = 0.028; see Figure 2). In the SCD group the 1st model, with CR as the only predictor (see again Table 6), only approached signiﬁcance (BIC = 126.31; AIC = 122.42; BF = 1.13; R2 = 0.08; p < 0.052). CR did not have a signiﬁcant role (t = −1.58, p = 0.126). No signiﬁcant diﬀerences between groups were observed in any of the scores representing the eﬀect of training (post – pre) in the three dependent measures. Prediction Models and Beneﬁts in Linguistic Performance After CT At the baseline the CI group presented a signiﬁcant better performance in naming [F(1, 64) = 8.174; p = 0.006; η2 p = 0.106; observed power = 0.805] than the SCD group. Diﬀerences in sentences not ﬁtted to canonical word order in Spanish almost reached statistical signiﬁcance [F(1, 64) = 3.724; p = 0.058; η2 p = 0.051; observed power = 0.477]. Regarding the main objective of the study, Table 4 shows that for CI participant’s performance on sentences not ﬁtted to the canonical word order in Spanish the model with best goodness of ﬁt was the 2nd (BIC = 124.01; AIC = 119.81; BF = 2.20; R2 = 0.15; p < 0.026), with TMT ratio as the only signiﬁcant predictor (t = 2.26, p = 0.032). In SCD older adults the best model was the 7th, which includes CR and digit reordering (BIC = 127.87; AIC = 122.68; BF = 1.92; R2 = 0.11; p < 0.030). However, nor CR (t = −1.79, p = 0.086) neither digit reordering (t = 0.55, p = 0.583) reached statistical signiﬁcance. RESULTS First, we computed descriptive statistics by group across dependent variables in the baseline and in post-training phases and across ﬁve categories of outcomes (percentages). We conducted a repeated measures ANOVA to explore intragroup diﬀerences between the baseline and the endpoint across dependent variables. Diﬀerences between groups at the baseline across dependent measures or across other relevant factors (age, Baseline Comparisons Between Groups Table 2 shows means and standard deviations (SD) by group across language performance measures (pre and post). TABLE 2 \| Mean and standard deviation (in brackets) in dependent measures at the baseline (pre-) and at the endpoint (post-training) for both groups. Baseline (pre) Endpoint (post) Non-canonical sentences Sentences with two propositions BNT Non-canonical sentences Sentences with two propositions BNT CI 14.91 (1.91) 15.41 (2.30) 54.26 (3.79) 15.26 (1.93) 16.32 (1.77) 55.06 (3.65) SCD 13.79 (2.29) 14.72 (2.63) 50.39 (6.31) 14.31 (1.86) 14.74 (2.21) 52.15 (4.86) Frontiers in Aging Neuroscience \| www.frontiersin.org 5 September 2018 \| Volume 10 \| Article 264 Language After Training in Elders López-Higes et al. Frontiers in Aging Neuroscience \| www.frontiersin.org How Single Predictors Explain Language performance After CT CI group SCD group Model BIC AIC BF R2 p BIC AIC BF R2 p 0 Intercept 125.65 122.85 128.34 125.75 1 CR 129.00 124.79 2.84 0.00 0.813 127.29 123.40 1.06 0.10 0.037 2 TMT 124.01 119.81 2.20 0.15 0.025 131.42 127.53 3.04 0.00 0.642 3 Inter 128.89 124.69 2.68 0.01 0.692 129.14 125.26 1.22 0.06 0.115 4 DR 127.80 123.59 2.06 0.03 0.262 130.59 126.70 3.02 0.00 0.307 5 CR + TMT 127.32 121.71 1.10 0.16 0.077 130.37 125.19 2.17 0.10 0.102 6 CR + Inter 132.15 126.55 5.52 0.01 0.861 129.80 124.62 1.07 0.17 0.077 7 CR + DR 131.19 125.59 4.04 0.04 0.533 127.87 122.68 1.92 0.11 0.029 8 TMT + Inter 126.55 120.94 1.19 0.18 0.052 132.27 127.09 1.82 0.10 0.265 9 TMT + DR 126.79 121.18 1.09 0.17 0.059 133.60 128.42 6.19 0.01 0.516 10 Inter + DR 130.84 125.24 1.20 0.05 0.447 130.72 125.54 2.84 0.07 0.122 11 CR + TMT + Inter 129.53 122.52 1.56 0.19 0.097 132.91 126.43 2.05 0.18 0.150 12 CR + TMT + DR 130.16 123.15 1.90 0.17 0.128 130.83 124.35 3.79 0.11 0.060 13 TMT + Inter + DR 129.08 122.07 1.35 0.20 0.079 133.78 127.30 3.77 0.10 0.218 14 CR + TMT + Inter + DR 132.21 123.80 2.41 0.21 0.134 133.10 125.33 1.53 0.27 0.077 CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor. Bolded values correspond to models whit better ﬁt to data. TABLE 4 \| Linear regression models for sentences not ﬁtted to canonical word order in Spanish included in ECCO_Senior. CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor. Bolded values correspond to models whit better ﬁt to data. TABLE 5 \| Linear regression models for sentences with two propositions included in ECCO_Senior. How Single Predictors Explain Language performance After CT CI group SCD group Model BIC AIC BF R2 p BIC AIC BF R2 p 0 Intercept 135.80 133.00 126.83 124.24 1 CR 136.73 132.53 1.18 0.08 0.116 126.31 122.42 1.13 0.08 0.051 2 TMT 133.37 129.17 2.98 0.18 0.016 130.09 126.20 2.79 0.01 0.833 3 Inter 139.14 134.94 2.90 0.00 0.804 127.71 123.82 2.40 0.02 0.120 4 DR 131.55 127.35 5.02 0.21 0.006 130.11 126.22 2.60 0.01 0.876 5 CR + TMT 133.53 127.92 3.63 0.26 0.011 129.57 124.39 2.54 0.09 0.145 6 CR + Inter 140.07 134.47 2.64 0.08 0.282 128.78 123.59 1.26 0.16 0.098 7 CR + DR 133.37 127.77 3.85 0.26 0.010 129.21 124.03 2.46 0.09 0.122 8 TMT + Inter 136.71 131.11 1.17 0.18 0.053 130.98 125.80 5.11 0.02 0.295 9 TMT + DR 130.15 124.55 12.51 0.34 0.002 133.35 128.17 5.27 0.02 0.964 10 Inter + DR 134.92 129.31 1.91 0.21 0.021 130.86 125.67 5.43 0.02 0.277 11 CR + TMT + Inter 136.12 129.11 2.05 0.28 0.020 132.05 125.57 2.50 0.16 0.198 12 CR + TMT + DR 131.29 124.28 10.62 0.39 0.002 132.46 125.98 4.71 0.09 0.234 13 TMT + Inter + DR 133.16 126.15 5.51 0.35 0.005 134.13 127.65 9.83 0.02 0.459 14 CR + TMT + Inter + DR 133.29 124.88 7.10 0.42 0.003 134.86 127.09 3.92 0.17 0.272 CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor Bolded values correspond to models whit better ﬁt to data TABLE 4 \| Linear regression models for sentences not ﬁtted to canonical word order in Spanish included in ECCO_Senior. How Single Predictors Explain Language performance After CT CI group SCD group Model BIC AIC BF R2 p BIC AIC BF R2 p 0 Intercept 135.80 133.00 126.83 124.24 1 CR 136.73 132.53 1.18 0.08 0.116 126.31 122.42 1.13 0.08 0.051 2 TMT 133.37 129.17 2.98 0.18 0.016 130.09 126.20 2.79 0.01 0.833 3 Inter 139.14 134.94 2.90 0.00 0.804 127.71 123.82 2.40 0.02 0.120 4 DR 131.55 127.35 5.02 0.21 0.006 130.11 126.22 2.60 0.01 0.876 5 CR + TMT 133.53 127.92 3.63 0.26 0.011 129.57 124.39 2.54 0.09 0.145 6 CR + Inter 140.07 134.47 2.64 0.08 0.282 128.78 123.59 1.26 0.16 0.098 7 CR + DR 133.37 127.77 3.85 0.26 0.010 129.21 124.03 2.46 0.09 0.122 8 TMT + Inter 136.71 131.11 1.17 0.18 0.053 130.98 125.80 5.11 0.02 0.295 9 TMT + DR 130.15 124.55 12.51 0.34 0.002 133.35 128.17 5.27 0.02 0.964 10 Inter + DR 134.92 129.31 1.91 0.21 0.021 130.86 125.67 5.43 0.02 0.277 11 CR + TMT + Inter 136.12 129.11 2.05 0.28 0.020 132.05 125.57 2.50 0.16 0.198 12 CR + TMT + DR 131.29 124.28 10.62 0.39 0.002 132.46 125.98 4.71 0.09 0.234 13 TMT + Inter + DR 133.16 126.15 5.51 0.35 0.005 134.13 127.65 9.83 0.02 0.459 14 CR + TMT + Inter + DR 133.29 124.88 7.10 0.42 0.003 134.86 127.09 3.92 0.17 0.272 CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor. Bolded values correspond to models whit better ﬁt to data. TABLE 5 \| Linear regression models for sentences with two propositions included in ECCO_Senior. TABLE 5 \| Linear regression models for sentences with two propositions included in ECCO_Senior. How Single Predictors Explain Language performance After CT When sentences not ﬁtted to canonical word order in Spanish are taken into account (see Table 4 again) there was a clear diﬀerence between groups, since in CI participants TMT ratio explains a 15% of criterion’s total variance, whereas CR was the variable which explains more than the other predictors (10%) in the SCD group. Regarding sentences with two propositions, digit reordering was the predictor with the highest explanation power in the CI group (it explains by itself 21% of the total variance), whereas CR (once again) explains only 8% (Table 5) in SCD participants. As Table 6 shows, Stroop interference was the predictor that best explained naming performance after CT in CI participants (17%), and consistently with previous results, CR explained 7% of criterion in the SCD group. September 2018 \| Volume 10 \| Article 264 6 Language After Training in Elders López-Higes et al. TABLE 4 \| Linear regression models for sentences not ﬁtted to canonical word order in Spanish included in ECCO_Senior. How Single Predictors Explain Language performance After CT CI group SCD group Model BIC AIC BF R2 p BIC AIC BF R2 p 0 Intercept 125.65 122.85 128.34 125.75 1 CR 129.00 124.79 2.84 0.00 0.813 127.29 123.40 1.06 0.10 0.037 2 TMT 124.01 119.81 2.20 0.15 0.025 131.42 127.53 3.04 0.00 0.642 3 Inter 128.89 124.69 2.68 0.01 0.692 129.14 125.26 1.22 0.06 0.115 4 DR 127.80 123.59 2.06 0.03 0.262 130.59 126.70 3.02 0.00 0.307 5 CR + TMT 127.32 121.71 1.10 0.16 0.077 130.37 125.19 2.17 0.10 0.102 6 CR + Inter 132.15 126.55 5.52 0.01 0.861 129.80 124.62 1.07 0.17 0.077 7 CR + DR 131.19 125.59 4.04 0.04 0.533 127.87 122.68 1.92 0.11 0.029 8 TMT + Inter 126.55 120.94 1.19 0.18 0.052 132.27 127.09 1.82 0.10 0.265 9 TMT + DR 126.79 121.18 1.09 0.17 0.059 133.60 128.42 6.19 0.01 0.516 10 Inter + DR 130.84 125.24 1.20 0.05 0.447 130.72 125.54 2.84 0.07 0.122 11 CR + TMT + Inter 129.53 122.52 1.56 0.19 0.097 132.91 126.43 2.05 0.18 0.150 12 CR + TMT + DR 130.16 123.15 1.90 0.17 0.128 130.83 124.35 3.79 0.11 0.060 13 TMT + Inter + DR 129.08 122.07 1.35 0.20 0.079 133.78 127.30 3.77 0.10 0.218 14 CR + TMT + Inter + DR 132.21 123.80 2.41 0.21 0.134 133.10 125.33 1.53 0.27 0.077 CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor. Bolded values correspond to models whit better ﬁt to data. TABLE 5 \| Linear regression models for sentences with two propositions included in ECCO_Senior. September 2018 \| Volume 10 \| Article 264 DISCUSSION SCD is the group in which beneﬁts are greater, conﬁrming one of our previous hypothesis. This group considerably improves their results in naming (in which we have observed the largest eﬀect size) and, in to a lesser extent, in sentences not ﬁtted to canonical word order in Spanish at the post-treatment phase. It should be noted that these results are especially favorable for UMAM program, since the older adults with SCD had at baseline greater susceptibility to distraction or lesser eﬃciency in inhibiting irrelevant information (assessed with the Stroop test) than the CI group, as well as worse results in the two aforementioned indicators. In the post-training phase, the two groups showed The present study aims to draw some conclusions regarding UMAM beneﬁts in the language area. Based on our results, we might say in ﬁrst place that the UMAM program produces eﬀects in abilities related to older people’s daily life, that is, in sentence reading comprehension and naming. Considering the percentages of moderate improvement (of greater relevance from a clinical point of view), as well as the resulting diﬀerences between groups in CT outcomes and intra-group diﬀerences between pre- and post-training measures, we can conclude that September 2018 \| Volume 10 \| Article 264 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 7 Language After Training in Elders López-Higes et al. FIGURE 2 \| Cognitive reserve, Trail Making Test ratio (TMT ratio) and digit reordering effects on the difference in sentences with two propositions between the endpoint (Tl) and the baseline (TO) for the control group, according to model 12 (Table 5). Dotted lines delimit 95% conﬁdence interval. FIGURE 2 \| Cognitive reserve, Trail Making Test ratio (TMT ratio) and digit reordering effects on the difference in sentences with two propositions between the endpoint (Tl) and the baseline (TO) for the control group, according to model 12 (Table 5). Dotted lines delimit 95% conﬁdence interval. TABLE 6 \| Linear regression models for the Boston Naming test. TABLE 6 \| Linear regression models for the Boston Naming test. DISCUSSION This would be an adequate explanation given that, as some authors maintain (for example: Lustig et al., 2009), intervention programs that incorporate multiple strategies and a great variety of tasks produce greater (near) transfer eﬀects. It should bear in mind that the UMAM program includes vocabulary exercises, and other requiring reading texts or following instructions, which are all related to written language comprehension. In summary, four main conclusions can be drawn of this study. Firstly, the ECCO_Senior test has been revealed as a screening instrument with enough sensitivity to detect diﬀerences in written language comprehension among adults with and without SCD. Secondly, older adults with SCD obtain greater beneﬁts in the language domain after training in comparison to CI older adults. However, near transfer eﬀects to language in the elderly group SCD cannot With respect to the main objective of the study, that is, to explore the role of CR and executive functions as factors which could modulate or predict the eﬃcacy of the UMAM program in language (complex sentence comprehension and naming), there is a clear diﬀerence between the two groups. With respect to non-canonical sentence comprehension the results for the CI group emphasize that high TMT ratios (meaning low cognitive ﬂexibility) are signiﬁcantly related to high CT beneﬁts (since t statistical test value was positive), and that this variable explains 15% of the total criterion variance. However, in the SCD group although we found a model including CR and digit reordering which explained 11% of the total variance, none of these variables reached statistical signiﬁcance. Thus, the results show that CT outcomes regarding sentences not ﬁtted to canonical word order in Spanish were higher for those CI older adults who had lower cognitive ﬂexibility scores at baseline. Furthermore, CI older adults who had higher TMT ratio scores (low cognitive ﬂexibility) and lower digit reordering scores at baseline, also showed a signiﬁcant improvement in their performance after CT in sentences with two propositions. These two factors explain 34% of the total variance (reaching 39% if CR is added), so their weight in sentence comprehension is moderately high. This result is consistent with that obtained in a good number of studies that show the important role of working memory in understanding complex sentences, especially linked to the number of propositions in the sentence (Waters and Caplan, 2004). DISCUSSION Selected predictors hardly explain SCD older adults’ post-training performance, so a substantial part of its variance remained unexplained, suggesting that other variables not considered in the study may account for it. This pattern of results contradicts, at least in part, our hypothesis regarding the role of executive functions in both groups as factors explaining language performance. Although CT beneﬁted language performance in older adults with SCD more than in CI participants, the chosen factors (executive functions) did not explain these beneﬁts. On the contrary, the same factors predicted the beneﬁts of CT for CI individuals. This is an paradoxical pattern of results that demand an explanation. It has been suggested that CT may cause a decrease in depressive symptomatology, or an improvement in general mood, well-being or motivation in SCD participants’ performance (Bherer, 2015). Therefore, the aforementioned changes, could be responsible for the observed improvement in the area of language in the group of older adults with SCD. Other authors have suggested that a change in lifestyle (variety of cognitive, physical, and social activities) associated to voluntary participation in training program has positive eﬀects in global cognition and memory (Küster et al., 2016). Some studies have also found eﬀects of multitasking training on processes such as perception and attention (Anguera et al., 2013), which are basic for others of higher level, so they could also be considered as factors to be taken into account. A complementary explanation arise when the pattern of diﬀerences across predictors (in isolation) are observed: (a) CR has a marginal weight as a factor explaining language performance in the SCD group (also in the CI group); (b) However, in the group of CI older adults two speciﬁc executive functions, alternation (cognitive ﬂexibility) and updating and monitoring (evaluated by digit reordering), explain a signiﬁcant portion of the variance associated to sentence comprehension. Inhibition eﬃciency, a common part for all the executive functions (following Miyake and Friedman model), explains a substantial portion of naming performance in this later group. Thus, it seems that UMAM program enhances CI older adults’ executive functioning, which beneﬁts, in turn, CI participant’s performance in the linguistic tasks considered here (many studies have pointed out close relationships between executive functions and language, especially in the process of reanalysis of complex sentences; see for example: Caplan and Waters, 2013; Yoon et al., 2015). DISCUSSION CI group SCD group Model BIC AIC BF R2 p BIC AIC BF R2 p 0 Intercept 144.30 141.50 160.20 157.46 1 CR 147.69 143.48 2.90 0.00 0.907 160.10 155.99 1.28 0.07 0.063 2 TMT 147.65 143.45 2.87 0.00 0.821 162.09 157.98 1.23 0.06 0.224 3 Inter 143.11 138.90 2.75 0.17 0.032 163.55 159.45 3.08 0.00 0.916 4 DR 146.45 142.24 1.55 0.05 0.263 163.14 159.04 2.09 0.03 0.514 5 CR + TMT 151.04 145.43 5.97 0.00 0.969 161.30 155.83 1.01 0.16 0.060 6 CR + Inter 146.16 140.56 1.17 0.15 0.085 162.97 157.50 1.61 0.13 0.138 7 CR + DR 149.84 144.24 4.04 0.04 0.532 162.36 156.89 2.79 0.07 0.102 8 TMT + Inter 146.00 140.39 1.28 0.18 0.078 165.40 159.93 3.01 0.06 0.464 9 TMT + DR 149.65 144.04 3.19 0.06 0.483 165.14 159.67 1.45 0.11 0.408 10 Inter + DR 145.77 140.17 1.39 0.19 0.070 166.47 161.01 5.42 0.02 0.795 11 CR + TMT + Inter 148.96 141.96 1.99 0.17 0.136 164.29 157.45 1.71 0.19 0.111 12 CR + TMT + DR 153.03 146.03 7.08 0.05 0.689 163.71 156.88 1.97 0.17 0.086 13 TMT + Inter + DR 148.42 141.41 1.20 0.21 0.107 168.42 161.58 4.30 0.09 0.597 14 CR + TMT + Inter + DR 151.11 142.70 2.58 0.20 0.147 166.74 158.53 2.43 0.21 0.140 CR, cognitive reserve; TMT, Trail Making Test; Inter, Stroop’s interference; DR, digit reordering; BIC, Bayesian Information Criterion; AIC, Akaike Information Criterion; BF, Bayes Factor. Bolded values correspond to models whit better ﬁt to data. September 2018 \| Volume 10 \| Article 264 8 Frontiers in Aging Neuroscience \| www.frontiersin.org Language After Training in Elders López-Higes et al. an equivalent performance. Some studies have reported similar beneﬁts in language comprehension, but after a verbal WM training program which was applied to CI older adults aged 65–75 years (Carretti et al., 2013). Those results are also in line with that obtained in the study conducted by Payne and Stine-Morrow (2017) using a novel home-based computerized CT program focused on verbal WM, and comparing CI older adults with a control group. The UMAM program includes vocabulary exercises, and other requiring reading texts or following instructions, which are all related to written language comprehension. These features can lead to positive training outcomes associated with sentence comprehension skills and naming, and could be considered as near transfer eﬀects. older adults in the CI group. Frontiers in Aging Neuroscience \| www.frontiersin.org September 2018 \| Volume 10 \| Article 264 DISCUSSION In the SCD group, CR almost reached statistical signiﬁcance for this type of sentences, presenting a slight tendency implying better training outcomes for participants with lower CR at baseline. Regarding participants’ performance in naming, the results pointed out that CI older adults with lower inhibition eﬃciency at baseline had better results in BNT after CT, and that this factor explains 17% of the total variance associated with criterion. However, in the SCD group none of the models computed for this dependent measure reached statistical signiﬁcance. These results enable drawing another conclusion: executive functions at baseline (Ranganath et al., 2011) play a role as predictors of language performance after training, but only in CI older adults. The association between executive functions at the baseline and CT beneﬁts is especially important in complex sentence comprehension (exemplars with two verbs or propositions) and, to a lesser extent, in naming, but restricted to September 2018 \| Volume 10 \| Article 264 9 Language After Training in Elders López-Higes et al. be explained adequately with the predictors selected in this study. Thirdly, the UMAM program seems to enhance CI older adults’ executive functioning, which beneﬁts, in turn, CI participants’ performance in sentence comprehension and naming. Finally, as we expected, CR has a marginal role as predictor of participants’ linguistic performance. other hand, the results found in the group of participants with SCD, which reveal important diﬀerences with CI older adults in relation to the mechanisms that underlie the beneﬁts produced by the UMAM program in a speciﬁc area such as language, serve as an incentive to deepen the study of other predictors that provide an adequate explanation. Taken as a whole, the results of this study could be indicating the need to adapt the UMAM program in a ﬂexible way to the characteristics of the users as a consequence of its location in the continuum that goes from normality to mild cognitive deterioration, and to the speciﬁc functions that are intended to maintain or improve. Since we have obtained results that contrast with other studies which have also considered cognitive functioning at baseline (Guye et al., 2017), future research should address if these diﬀerences might emerge as a function of the population considered, the type of training, or other factors such as motivation, etc. REFERENCES Chafetz, M. D., and Matthews, L. H. (2004). A new interference score for the Stroop test. Arch. Clin. Neuropsychol. 19, 555–567. doi: 10.1016/j.acn.2003. 08.004 Anguera, J. A., Boccanfuso, J., Rintoul, J. L., Al-Hashimi, O., and Faraji, F. (2013). Video game training enhances cognitive control in older adults. Nature 501, 97–101. doi: 10.1038/nature12486 Clark, D. O., Xu, H., Unverzagt, F. W., and Hendrie, H. (2016). Does targeted cognitive training reduce educational disparities in cognitive function among cognitively normal older adults? Int. J. Geriatr. Psychiatry 31, 809–817. doi: 10.1002/gps.4395 Arbuthnott, K., and Frank, J. (2000). 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Neuropsychology 30, 742–755. doi: 10.1037/neu0000235 Au, J., Sheehan, E., Tsai, N., Duncan, G. J., Buschkuehl, M., and Jaeggi, S. M. (2015). Improving ﬂuid intelligence with training on working memory: a meta-analysis. Psychon. Bull. Rev. 22, 366–377. doi: 10.3758/s13423-014-0699-x Bamidis, P. D., Fissler, P., Papageorgiou, S. G., Zilidou, V., Konstantinidis, E. I., Billis, A. S., et al. (2015). Gains in cognition through combined cognitive and physical training: the role of training dosage and severity of neurocognitive disorder. Front. Aging Neurosci. 7:152. doi: 10.3389/fnagi.2015.00152 Gross, A. L., Parisi, J. M., Spira, A. P., Kueider, A. M., Ko, J. Y., Saczynski, J. S., Rebok, G. W. (2012). Memory training interventions for older adults: a meta-analysis. Aging Ment. Health 16, 722–734. doi: 10.1080/13607863.2012. 667783 Guye, S., De Simoni, C., and von Bastian, C. C. (2017). Do individual diﬀerences predict change in cognitive training performance? A latent growth curve modeling approach. J. Cogn. Enhanc. 1, 374–393. Bherer, L. (2015). Cognitive plasticity in older adults: eﬀects of cognitive training and physical exercise. Ann. N. Y. Acad. Sci. 1337, 1–6. DISCUSSION A possible line of future research would involve exploring the role of other factors/predictors such as those that have been suggested to explain the pattern of results obtained in the group of older adults with SCD; it would involve to study the changes that occur in mood, general well-being or daily activity (physical, social, intellectual) in this population for their participation in the UMAM program, as well as the relevance of these variables in the prediction of SCD participants’ performance in diﬀerent cognitive domains, including language. Likewise, it would be interesting to explore the relationships between executive functions and language in cognitively intact older adults, checking if the training program produces a signiﬁcant improvement in the executive functions (especially those which are more related to language), and if this is also related directly with an improvement in language skills. Potential limitations of this study would be that the percentage of females were greater than the corresponding of males (66% vs. 34%). Similarly, the sample size could also be extended in future studies to strengthen conclusions. FUNDING The UMAM program has demonstrated its eﬃcacy improving memory in older adults with and without SCD and in mild cognitive impairment. From a practical point of view the present results are useful since they allow us to know which factors explain to a large extent the beneﬁts that the UMAM program has in the language area in cognitively intact older adults. 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https://openalex.org/W2899249011	https://ntnuopen.ntnu.no/ntnu-xmlui/bitstream/11250/2577233/1/Lu_et_al-2018-European_Transport_Research_Review.pdf	English	null	A new adaptive cruise control strategy and its stabilization effect on traffic flow	European transport research review	2,018	cc-by	8,221	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. ORIGINAL PAPER Open Access Abstract Connected and autonomous vehicle (CAV) technologies are likely to be gradually implemented over time. In this paper, an adaptive cruise control, named Smart Driver Model (SDM), is proposed to describe the autonomous vehicles flow. The stability criteria is proposed for SDM to judge the stability of homogeneous traffic flow. Numerical simulations were conducted to verify the results of the theoretical analysis. Single-lane vehicle dynamics in a traffic stream with connected and autonomous vehicles are simulated by varying model parameters. Simulation results are consistent with the results of linear stability analysis. As a result, a set of parameters is proposed to investigate the stabilization effect of the proposed model on homogeneous traffic flow considering realistic driving cycle and cut-in condition. By simulating a platoon with a lead vehicle which follows the Urban Dynamometer Driving Schedule (UDDS), we find out that the proposed model can stabilize the traffic flow with proposed parameters. The results from simulation and linear stability analysis show that SDM outperforms the IDM-ACC and the ACC proposed by Milanés and Shladover in terms of stabilization effect on homogeneous traffic flow. The simulation result shows that the SDM- equipped vehicles are able to stabilize the homogeneous traffic flow under cut-in condition. Keywords: Autonomous vehicle, Adaptive cruise control, Traffic flow stability (VTH) [8]. CSH is not string stable and not suitable for ACC systems [9–11]. Among these three headway selec- tion policies, the ACCs based on CTH are most closely re- lated to normal manual car-following behavior. Moreover, Swaroop et al. [12] pointed out that the control design and stabilization challenges of the CTH-based car follow- ing criteria are significantly easier for other rule-based car-following models. As a result, in this paper, we focus on rule-based ACCs with constant time headway policies. A new adaptive cruise control strategy and its stabilization effect on traffic flow Chaoru Lu* and Arvid Aakre (2018) 10:49 (2018) 10:49 European Transport Research Review Lu and Aakre European Transport Research Review https://doi.org/10.1186/s12544-018-0321-9 * Correspondence: chaoru.lu@ntnu.no Traffic Engineering Research Center, Department of Civil and Environmental Engineering, Norwegian University of Science and Technology, Høgskoleringen 7 A, 7034 Trondheim, Norway 1 Introduction Connected and autonomous vehicle (CAV) technologies have gained a lot of attention all over the world because of its potential in improving safety and congestion of the road transportation system. In particular, adaptive cruise control (ACC) is one of the most important CAV tech- nologies that can enhance driving comfort, reduce driv- ing errors, improve safety, increase traffic capacity and reduce fuel consumption [1]. Existing ACC methods are categorized into optimization-based ACCs, artificial intelligence technique-based ACCs and rule-based ACCs. Consider- ing the fuel efficiency, signalized intersection, and road geometry, several optimization-based ACCs have been de- veloped [2–6]. Naranjo et al. [7] proposed an ACC based on artificial intelligence techniques. Moreover, existing rule-based ACCs are based on three main headway selec- tion policies: Constant Space-Headway (CSH), Constant Time-Headway (CTH) and Variable Time-Headway Several rule-based ACC methods have been proposed in the literature. Davis [13] proposed an ACC that auto- matically maintained a safe distance and minimized the speed difference between the following vehicle and its immediate preceding vehicle. Kesting et al. [14] pro- posed an ACC based on intelligent driver model (IDM) and inherited its intuitive parameters proposed by Trei- ber et al. [15]. The ACC proposed by Kesting et al. par- tially eliminates the unrealistic behavior of IDM in cut-in situations. However, since the ACC proposed by Kesting et al. inherits the instability of IDM under homogenous traffic flow condition [16]. Moreover, an ACC, which is proprietary to Nissan, was described by Shladover et al. [17]. Since this ACC is simplified Page 2 of 11 Page 2 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review proportion of the direct power cooperation of the preced- ing vehicle. Recently, Wen-Xing and Li-Dong [31] pro- posed two lemmas and one theorem as criteria to judge the stability of homogeneous autonomous vehicles flow. representation for computation efficiency in simulation, it is not able to describe the ACC car-following behavior in the field test. Consequently, Milanés and Shladover [18] developed an ACC system to match the experimen- tal result obtained from the production vehicle road tests. However, their research did not consider the stabil- ity in multi-car following scenarios. 1 Introduction Among existing studies, most of the existing rule- based adaptive cruise control strategies are derived based on the human driver models, such as Intelligent Driver Model [15], Full Velocity Difference Model [32] and Optimal Velocity Model [33]. These adaptive cruise control strategies may inherit the limitation of the hu- man driver models, such as instability [16, 34] and un- realistic behavior [13, 15, 33]. In this paper, we propose a rule-based adaptive cruise control to improve the sta- bility and behavior of Intelligent Driver Model [15]. The stabilization effect of the proposed adaptive cruise con- trol on traffic flow is discussed theoretically. Moreover, in order to estimate the performance of the proposed ACC, the stabilization effect of the proposed adaptive cruise control is compared with existing models. y g By modeling ACC-equipped vehicles’ behavior, the im- pact of ACC on traffic flow is widely studied [10, 19, 20]. Davis showed that the traffic congestions are not formed when the ACC-equipped vehicle reaches 20% [13]. Kest- ing et al. reported that the traffic congestion was com- pletely eliminated when the share of the ACC-equipped vehicle reaches 25% [14]. Jerath and Brennan reported that the highway capacity drastically increases when the percentage of the ACC-equipped vehicle approaches 100% [21]. Moreover, by simulating a mixed traffic con- sisting of ACC-equipped and manually driven vehicles, Jiang et al. found that the introduction of ACC-equipped vehicles would enhance the free flow stability [22]. Yuan et al. investigated the transition probability from the syn- chronized flow to congestion and pointed out that ACC-equipped vehicles enhance the traffic stability of synchronized flow [23]. The rest of the paper is organized as follows: section 2 presents the proposed adaptive cruise control strategy. The linear stability analysis of the proposed model is represented in Section 3. Section 4 provides results from simulating single-lane vehicle dynamics with or without cut-in scenarios. The conclusions and limitations are presented in Section 5. Consequently, scholars paid a lot of attention to the stabilization effect of ACC-equipped vehicles on traffic flow. Darbha and Rajagopal [11] mathematically investi- gated the stability of an intelligent cruise control system with a constant time headway policy and provided a framework for designing cruise control laws. Liang and Peng [10] proposed a framework to analysis string- stability of a string with ACC-equipped vehicles and human-driven vehicles. 1 Introduction Davis [24] investigated the effect of vehicle response time and delay on string stability of adaptive cruise control systems. Later, Davis [25] investi- gated the impact of mechanical response of the dynamics and string stability of a platoon of adaptive cruise control vehicles. Hu et al. [26] derived a stability criteria of a platoon of ACC-equipped vehicles with actuator lag and sensor delay. Considering different models with the technology-appropriate assumption, Talebpour and Mahmassani [16] proposed a framework to investigate the impact of the connected and autonomous vehicle on traf- fic flow stability. Moreover, Davis [25] investigated the im- pact of mechanical response on string stability of a platoon of ACC-equipped vehicles. Considering the delay, Xing et al. [27], Wang et al. [28] and Besselink and Johans- son [29] proposed appropriate control strategies to en- hance string stability of ACC-equipped vehicle platoon. Moreover, Li et al. [30] proposed an extended intelligent driver model and analyzed the stability against a small per- turbation by use of the linear stability method for the pro- posed model on a single lane. They found that the traffic flow stability can be improved by increasing the 2 Smart driver model In the past decades, numbers of car-following models have been introduced to simulate manually driven ve- hicle, such as Multi-anticipative Model [35], Tampere Model [36], Newell Model [37], Gipps Model [38], Full Velocity Difference Model [32] and Optimal Velocity Model [33]. Based on Gipps model, Treiber et al. pro- posed a human driver model named Intelligent Driver Model (IDM) [15]. By capturing different congestion dy- namics, IDM provides greater realism than most of the deterministic acceleration modeling frameworks [16]. The IDM is formulated as follows: an IDM ¼ amax 1−vn v0 δ − s Δx 2 " # ð1Þ s ¼ s0 þ vnT þ vn vn−vn−1 ð Þ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ amaxb p ð2Þ an IDM ¼ amax 1−vn v0 δ − s Δx 2 " # ð1Þ ð1Þ s ¼ s0 þ vnT þ vn vn−vn−1 ð Þ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ amaxb p ð2Þ ð2Þ where, an IDM is the acceleration of the following vehicle based on Intelligent Driver Model (m/s2); an IDM is the acceleration of the following vehicle based on Intelligent Driver Model (m/s2); g δ is the acceleration exponent; δ is the acceleration exponent; δ is the acceleration exponent; s0 is the standstill distance between stopped vehicles (m); amax is the maximum acceleration (m/s2), which is predetermined and less than the Planck acceleration; Page 3 of 11 Page 3 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review Δx is the gap between the leading and the following vehicle (m); amax½1−ðvn v0Þδ is used to offset the acceleration term. Moreover, considering the range policy, it ensures that the real deceleration is equal to or slight larger than the expected deceleration, which is v2 n−v2 n−1 2Δx . Moreover, since exp(∙) > 0 and expð0Þ ¼ 1; 1 expð Δx s0þvnT−1Þ is applied to describe the dependence of decel- eration term on the ratio between actual spacing and de- sired spacing. Therefore. when there is no speed difference between the leading and following vehicle, a SDM-equipped vehicle ‘s acceleration increases with the ra- tio of Δx to the desired spacing. According to the character- istics of the exponential function, the jerk of SDM-equipped vehicle, which represents the changing rate of SDM-equipped vehicle ‘s acceleration, decreases with the ratio of Δx to the desired spacing. Consequently, the vehicle equipped with SDM can achieve smoother acceleration and deceleration which is able to stabilize the traffic flow. T is the desired time gap (s); T is the desired time gap (s); v0 is the maximum speed (m/s); vn is the speed of the following vehicle (m/s); vn −1 is the speed of the leading vehicle (m/s); s∗is the desired gap (m); and. b is the desired deceleration (m/s2) b is the desired deceleration (m/s2). In recent years, car-following models have evolved to describe the behavior of vehicles with advanced cruise controls, which take advantage of the sensing and ve- hicle to vehicle/vehicle to infrastructure (V2V/V2I) com- munication technologies. By using constant-acceleration heuristic (CAH) as an indicator, Kesting et al. proposed a rule-based adaptive cruise control based on IDM [34] (IDM-ACC). The ACC proposed by Kesting et al. par- tially eliminates the unrealistic behavior of IDM in cut-in situations. However, since the ACC proposed by Kesting et al. where, inherits its intuitive behavioral parameters of IDM, it inherits the instability of IDM under homogenous traffic flow condition [16]. Several properties of SDM are discussed as follows, considering special cases: First, when SDM-equipped vehicle is cruising (i.e. an SDM ¼ 0; vn−vn−1 ¼ 0), the speed-dependent spacing Δx between the preceding and the following vehicle is given by A rule-based ACC, named Smart Driver Model (SDM) is proposed to address the instability of IDM under homogenous traffic condition. In this study, a single-lane car-following scenario is considered and the lane changing behavior is ignored. In this paper, we as- sume that on-board sensors measure vehicle speed, gap (relative distance) and relative speed with respect to the preceding vehicle on regular time intervals [28]. ð4Þ Δx ¼ s0 þ vn T ð4Þ The speed-dependent spacing Δx equals to the desired spacing, that is, Δx = s0 + vn × T. The speed-dependent spacing Δx equals to the desired spacing, that is, Δx = s0 + vn × T. The acceleration of the following vehicle equipped with SDM is determined by the following equation: Second, when the traffic density is low (i.e. Δx is much larger than the desired spacing), SDM-equipped vehicles will accelerate to the maximum speed. When the leading vehicle is out of the detection range of SDM-equipped vehicle (Δx →∞), v2 n−v2 n−1 2Δx is close to 0 and eð Δx s0þvnT−1Þ is close to infinity. As a result, the acceleration of SDM is approximately equal to the maximum acceleration, an SDM ≈amax ½1−ðvn v0Þ4. After the speed of the SDM-equipped vehicle reaches the maximum speed, acceleration of SDM is 0. an SDM ¼ amax 1−vn v0 δ " # − amax 1− vn v0 δ þ v2 n−v2 n−1 2Δx exp Δx s0 þ vn T −1 ð3Þ ð3Þ where, 9, we can obtain Forth, when the spacing is much smaller than the de- sired spacing (Δx ≪s0 + v0 × T) and there is no signifi- cant speed differences (vn −vn −1 ≈0), the acceleration is determined as follows: €yn t þ τ ð Þ ¼ €xn t þ τ ð Þ−xn t þ τ ð Þ ð Þ00 ¼ €xn t þ τ ð Þ ð12Þ By substituting Eq. 7 into Eq. 10, we have ð12Þ By substituting Eq. 7 into Eq. 10, we have an SDM ≈amax 1− 1 e Δx s0þvnT−1 0 B @ 1 C A ð7Þ €yn t þ τ ð Þ ¼ f vn tð Þ; sn tð Þ; Δvn tð Þ ð Þ ð13Þ By linearizing Eq. 11, we get the following equation an SDM ≈amax 1− 1 e Δx s0þvnT−1 0 B @ 1 C A ð7Þ €yn t þ τ ð Þ ¼ f vn tð Þ ð By linearizing Eq. 11 an SDM ≈amax 1− 1 e Δx s0þvnT−1 0 B @ 1 C A ð7Þ €yn t þ τ ð Þ ¼ f vn tð Þ; sn tð Þ; Δvn tð Þ ð Þ ð13Þ ð13Þ ð7Þ By linearizing Eq. 11, we get the following equation Especially, when Δx →0, Eq. 5 reduces to Especially, when Δx →0, Eq. 5 reduces to €yn t þ τ ð Þ ¼ f v_yn tð Þ þ f s yn tð Þ−yn−1 tð Þ ð Þ þ f Δv _yn tð Þ−_yn−1 tð Þ ð Þ ð14Þ Where f v ¼ ∂f ∂vn jðve;se;0Þ ≤0, f Δv ¼ ∂f ∂Δvn jðve;se;0Þ ≤0, and f s ¼ ∂f ∂sn jðve;se;0Þ ≥0. ð14Þ an SDM ≈amax 1−e ð Þ ð8Þ an SDM ≈amax 1−e ð Þ ð8Þ ¼ ∂f ∂sn jðve;se;0Þ ≥0. where, where, Third, when SDM-equipped vehicle is following a slower vehicle or approaching a stopped vehicle (i.e. vn −vn −1> 0) with the limited spacing (Δx →s0 + v0 × T), the acceleration equation of SDM is given by an SDM is the acceleration of the following vehicle that is equipped with SDM (m/s2); an SDM is the acceleration of the following vehicle that is equipped with SDM (m/s2); According to Eq. 1, SDM inherits the acceleration maneuver from Intelligent Driver Model [15]. As a re- sult, the acceleration exponent is set as 4 according to existing studies [15, 34, 39]. Therefore, the vehicles equipped with SDM tend to accelerate with amax½1−ðvn v0Þ4 when Δx is large. An SDM-equipped vehicle will brake while the speed of the SDM-equipped vehicle is greater than the leading vehicle speed and Δx is less than the an SDM→− v2 n−v2 n−1 2 s0 þ vn T ð Þ ð5Þ ð5Þ ð5Þ Especially, when a SDM-equipped vehicle with the maximum speed approaches a stopped vehicle (i.e. vn = v0, vn −1 = 0), the maximum kinematic deceleration is ap- plied to avoid a collision, as follows. desired spacing. amax½1−ðvn v0Þδþ v2n−v2 n−1 2Δx expð Δx s0þvnT−1Þ is used to describe the deceleration maneuver. In the deceleration term, desired spacing. amax½1−ðvn v0Þδþ v2n−v2 n−1 2Δx expð Δx s0þvnT−1Þ is used to describe the deceleration maneuver. In the deceleration term, Lu and Aakre European Transport Research Review (2018) 10:49 Page 4 of 11 Review (2018) 10:49 Page 4 of 11 (2018) 10:49 Lu and Aakre European Transport Research Review (2018) 10:49 Page 4 of 11 an SDM ¼ − v2 0 2 s0 þ v0 T ð Þ ð6Þ yn tð Þ ¼ xn tð Þ−xn tð Þ; yn tð Þ→0 ð11Þ yn tð Þ ¼ xn tð Þ−xn tð Þ; yn tð Þ→0 ð11Þ an SDM ¼ − v2 0 2 s0 þ v0 T ð Þ ð6Þ ð11Þ ð6Þ By taking the second derivative of Eq. 3 Linear stability analysis ð e; e; Þ We rewrite Eq. 12 to obtain the difference equation ð e; e; Þ We rewrite Eq. 12 to obtain the difference equation Linear stability method is widely applied to analyze the stabilization performance of car-following models [40– 47]. In this section, we apply the linear stability method for the Smart Driver Model described in the last section. The general form of time-continuous car-following models is yn t þ 2τ ð Þ−_yn t þ τ ð Þ ¼ τf s yn tð Þ−yn−1 tð Þ ð Þ þ f v yn t þ τ ð Þ−yn tð Þ ð Þ þf Δv yn t þ τ ð Þ−yn tð Þ−yn−1 t þ τ ð Þ þ yn−1 tð Þ ð Þ ð15Þ By substituting ynðtÞ ¼ ceiaknþzt into Eq. 13, we have yn t þ 2τ ð Þ−_yn t þ τ ð Þ ¼ τf s yn tð Þ−yn−1 tð Þ ð Þ þ f v yn t þ τ ð Þ−yn tð Þ ð Þ þf Δv yn t þ τ ð Þ−yn tð Þ−yn−1 t þ τ ð Þ þ yn−1 tð Þ ð Þ yn t þ 2τ ð Þ−_yn t þ τ ð Þ ¼ τf s yn tð Þ−yn−1 tð Þ ð Þ þ f v yn t þ τ ð Þ−yn tð Þ ð Þ þf Δv yn t þ τ ð Þ−yn tð Þ−yn−1 t þ τ ð Þ þ yn−1 tð Þ ð Þ ð15Þ ð15Þ By substituting ynðtÞ ¼ ceiaknþzt into Eq. 13, we have By substituting ynðtÞ ¼ ceiaknþzt into Eq. 13, we have eτz−1 ð Þ eτzz−f v−f Δv 1−e−2iak ¼ τf s e−iak−1 ð16Þ Where c is constant, ak = 2πk/N(k = 0, 1, …, N −1). By expanding z = z1(iak) + z2(iak)2 + …, eτz ¼ 1 þ τz þ τ2z2 2 þ ∙∙∙; and inserting it into Eq. 13, then the first order and second order terms of coefficients of the ex- pression of z are described as follows eτz−1 ð Þ eτzz−f v−f Δv 1−e−2iak ¼ τf s e−iak−1 ð16Þ Where c is constant, ak = 2πk/N(k = 0, 1, …, N −1). By expanding z = z1(iak) + z2(iak)2 + …, eτz ¼ 1 þ τz þ τ2z2 2 þ ∙∙∙; and inserting it into Eq. 3 Linear stability analysis Page 5 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review In order to investigate the stabilization effect of the SDM model on homogeneous traffic flow, we have a = 1.4 m/s2, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m. The traffic flow is stable with uniform velocity steady state above the neutral sta- bility line; conversely, the traffic is unstable and the traf- fic congestion emerges. From Fig. 1, one can observe that the neutral stability line moves up with the increase of time delay. The effect of the stability of traffic flow by considering the time delay can be further illustrated in Fig. 2, where the average speed ve = 4 m/s, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m. Same as in the study conducted by Van Arem et al. [49], the maximum comfortable acceleration is 2 m/s2. From Fig. 2, one can see that by increasing the maximum ac- celeration a, the neutral stability curve changes down, which means that traffic flow will become more and more stable if the maximum acceleration of SDM-equipped vehicle increases, i.e., the traffic stability can be improved by raising the maximum acceleration. However, by increasing the maximum acceleration may make driving less comfortable. The improvement in traf- fic stability by decreasing the time delay also can be found here. an SDM ¼ amax 1−vn v0 δ " # − amax 1− vn v0 δ þ v2 n−v2 n−1 2Δx exp Δx s0 þ vn T −1 0 n f s ¼ amax 1− ve v0 4 s0 þ ve T ð Þ ð20Þ f Δv ¼ − ve s0 þ ve T ð Þ ð21Þ f v ¼ − amaxT s0 þ ve T ð Þ 1−ve v0 4 " # −8amaxv3 e v4 0 ð22Þ ð20Þ ð22Þ By substituting Eqs. 18–20 into Eq. 17, the stable con- dition is derived as follows τ 2 < 1 2 amaxT s0 þ ve T ð Þ 1−ve v0 4 " # þ 8av3 e v4 0 ! 3 Linear stability analysis 13, then the first order and second order terms of coefficients of the ex- pression of z are described as follows eτz−1 ð Þ eτzz−f v−f Δv 1−e−2iak ¼ τf s e−iak−1 ð16Þ ð16Þ €xn t þ τ ð Þ ¼ f vn tð Þ; sn tð Þ; Δvn tð Þ ð Þ ð9Þ ð9Þ Where, þ τ2z2 2 þ ∙∙∙; and inserting it into Eq. 13, then the first order and second order terms of coefficients of the ex- pression of z are described as follows f is a general nonlinear function; €xnðtÞ is the acceleration; τ is the time delay; τ is the time delay; vn(t) is the speed of vehicle n; vn(t) is the speed of vehicle n; z1 ¼ f s f v ð17Þ z2 ¼ 1−τ 2 f v z2 1−f Δvz1−1 2 f s f v ð18Þ sn(t) is the spacing between vehicles n and n + 1; and. sn(t) is the spacing between vehicles n and n + 1; and. Δvn(t) is the relative velocity between vehicles n and n + 1. ð17Þ Δvn(t) is the relative velocity between vehicles n and n + 1. Moreover, the steady state of Eq. 1 is set as ð18Þ f v xn tð Þ ¼ N−n ð Þse þ vet; n ¼ 1; 2; 3; …; N ð10Þ ð10Þ If z2 < 0, the homogenous traffic flow is unstable. Al- ternatively, If z2 > 0, the homogenous traffic flow is stable. Therefore, the stability condition is described as follows Where, se is the spacing of adjacent vehicles in homogenous flow; N is the total number of vehicles in homogenous flow; ve is the speed of vehicles in homogenous flow; and. Stability ¼ 1 2 f 2 v−f s þ f v f Δv þ τ 2 f vf s > 0 ð19Þ ð19Þ xnðtÞ is the location of vehicle n at time t. Moreover, yn(t) is introduced as a small perturbation from the steady state solution of the vehicle n at time t with the linear Fourier-mode expanding: This stability condition is consistent with existing studies [30, 48]. 3 Linear stability analysis s0 þ ve T ð Þ amax 1− ve v0 4 − 1 amaxT s0 þ ve T ð Þ 1−ve v0 4 " # þ 8amaxv3 e v4 0 ! þ ve amax 1− ve v0 4 ð23Þ According to Eq. 19, the stability of SDM is compared with IDM-ACC and the ACC proposed by Milanés and Shladover [18] by using the typical parameters from lit- erature [18, 34]. Since IDM-ACC only eliminates the un- realistic behavior of IDM in cut-in situations, the linear stability of IDM-ACC is the same as the linear stability of IDM. As shown in Fig. 3, the ACC proposed by Milanés and Shladover is unstable with the proposed pa- rameters. Same as the result in [16], the platoon of vehi- cles equipped with IDM-ACC is stable for speeds below 3.5 m/s and unstable for speed above 3.5 m/s. Moreover, the stability curve of SDM is above 0. When the speed is ð23Þ According to Eq. 23, the phase transition curves are derived. Figure 1 shows the neutral stability curves for different time delays, where the maximum acceleration Fig. 1 Phase diagram in the velocity-desired time gap for different time delay, where the maximum acceleration a = 1.4 m/s2, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m Fig. 1 Phase diagram in the velocity-desired time gap for different time delay, where the maximum acceleration a = 1.4 m/s2, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m Fig. 1 Phase diagram in the velocity-desired time gap for different time delay, where the maximum acceleration a = 1.4 m/s2, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m Lu and Aakre European Transport Research Review (2018) 10:49 Page 6 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review Page 6 of 11 (2018) 10:49 Fig. 2 Phase diagram in the Maximum acceleration-desired time gap for different time delay, where the average speed ve = 4 m/s, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m Fig. 4.1.1 Case 1 larger than 3.5 m/s, SDM outperforms IDM-ACC in terms of stability. A simple case is applied to investigate the impacts of de- sired time gap and maximum acceleration on the stabilization performance of the proposed model. Ini- tially, the speed vinitial=10 m/s, stand still distance s0= 1.5 m and the spacing sinitial=s0 + vinitial × T. The initial steady state will be broken when the leading vehicle brakes urgently from 10 to 4 m/s during the time period 10 s ≤t ≤13 s, followed by the following vehicle moving according to Eq. (4). As shown in Fig. 4(a), the traffic flow is unstable when the desired time gap is less than 1.6, where the maximum acceleration is 1.4 m/s2. More- over, while the desired time gap is 1.6, the traffic flow is unstable when the maximum acceleration is 0.8 m/s2. 3 Linear stability analysis 2 Phase diagram in the Maximum acceleration-desired time gap for different time delay, where the average speed ve = 4 m/s, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m 4.1 Platoon without cut-in To validate the results of our theoretical stability analysis, a platoon with 100 vehicles are simulated on a one-lane highway (no lane-changing) with an infinite length. Moreover, the time delay of ACC- equipped vehicle are ignored to investigate the stabilization performance of the proposed model under ideal conditions. Two cases are conducted in this section. Fig. 3 Stability of vehicle with different ACCs, which is using the typical parameters from literature [18, 34] Fig. 3 Stability of vehicle with different ACCs, which is using the typical parameters from literature [18, 34] Fig. 3 Stability of vehicle with different ACCs, which is using the typical parameters from literature [18, 34] Lu and Aakre European Transport Research Review (2018) 10:49 Page 7 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Page 7 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Th lt i t t ith th lt d i d i l t Fi 5 M th i iti l i d ti h d (a) (b) (c) (d) (e) Fig. 4 Acceleration and speed profiles of 1st, 25th, 50th, and 100th vehicle for different maximum acceleration and desired time gap (a) ( ) (b) (b) (b) (c) (c) (d) (d) (e) (e) ration and speed profiles of 1st, 25th, 50th, and 100th vehicle for different maximum acceleration and desired time gap Fig. 4 Acceleration and speed profiles of 1st, 25th, 50th, and 100th vehicle for different maximum acceleration a Fig. 5. Moreover, the initial spacing and time headway are the desired spacing and desired time gap, respect- ively. According to the result of case 1, we set the max- imum acceleration a = 1.4 m/s2, desired time gap T = 1.6 s, maximum speed v0 = 30 m/s, and the standstill distance s0 = 1.5 m. The parameters from case 1 is simi- lar to the parameters of IDM-ACC proposed by Kesting et al. [34]. Moreover, the ACC proposed by Milanés and The results are consistent with the results derived in last section. The results are consistent with the results derived in last section. 4.1.2 Case 2 In order to investigate the stabilization effect of the pro- posed model considering the realistic driving cycle, we assume the lead vehicle of platoon follows the Urban Dynamometer Driving Schedule (UDDS), as shown in Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review (2018) 10:49 Page 8 of 11 0 5 10 15 20 25 30 0 200 400 600 800 1000 1200 Speed (m/s) Time (s) Fig. 5 Urban Dynamometer Driving Schedule (UDDS) [52] parameters, the proposed model can stabilize the homo- geneous traffic flow considering a realistic driving cycle. Moreover, SDM outperforms the IDM-ACC and Milanés and Shladover Model in terms of stabilization effect on homogeneous traffic flow. Shladover [18] is also simulated to compare with the IDM-ACC and SDM. In order to avoid the instability of the ACC proposed by Milanés and Shladover [18], we have k1 = 0.49 and k2 = 0.07, where the stability calcu- lated based on Eq. 19 is 0.02. Therefore, the parameters from case 1 is used in the simulation for both IDM-ACC and SDM. As shown in Fig. 6, the acceleration variance is significantly decreasing for the SDM-equipped vehicle towards the end of the platoon. With before mentioned 4.2 Platoon with cut-in Based on the simulation conducted by [50], a platoon with 16 vehicles, when a cut-in occurs between the (a) (b) (c) (d) Fig. 6 Acceleration profiles of 1st, 25th, 50th, and 100th vehicle equipped with Milanés and Shladover Model, IDM-ACC and SDM (b) (a) (b) (a) (d) (c) Fig. 6 Acceleration profiles of 1st, 25th, 50th, and 100th vehicle equipped with Milanés and Shladover Model, IDM-ACC and SDM Page 9 of 11 Lu and Aakre European Transport Research Review (2018) 10:49 Lu and Aakre European Transport Research Review (2018) 10:49 fourth and fifth positions, is simulated to investigate the stability of SDM under cut-in condition. The cut-in rules and parameters are based on the study conducted by Davis [51]. The simulation starts with the 16 SDM-equipped vehicles. The leading vehicle is manually driven and its speed profile is generated using real data from experimental tests. The other vehicles follow the leading vehicle. One can appreciate how the speed changes are not amplified upstream. Then a vehicle cuts in between the fourth and fifth vehicles. For the sake of clarity in the figure, the speeds of the first four vehicles have been removed from the plots of the results for the rest of the simulation. From second 340, the cut-in ve- hicle depicted in the graph with the red solid lane splits the string in two. This vehicle is computer generated and it is assumed that it is manually driven with small fourth and fifth positions, is simulated to investigate the stability of SDM under cut-in condition. The cut-in rules and parameters are based on the study conducted by Davis [51]. The simulation starts with the 16 SDM-equipped vehicles. The leading vehicle is manually driven and its speed profile is generated using real data from experimental tests. The other vehicles follow the leading vehicle. One can appreciate how the speed changes are not amplified upstream. Then a vehicle cuts in between the fourth and fifth vehicles. For the sake of clarity in the figure, the speeds of the first four vehicles have been removed from the plots of the results for the rest of the simulation. From second 340, the cut-in ve- hicle depicted in the graph with the red solid lane splits the string in two. This vehicle is computer generated and it is assumed that it is manually driven with small speed oscillations around 25 m/s. 4.2 Platoon with cut-in As shown in Fig. 7, the oscillations from the cut-in vehicle are reduced by the 5th to the 16th vehicles. With the proposed parame- ters, the proposed model can stabilize the homogeneous traffic flow under cut-in condition. 5 Conclusion and future work In this paper, an adaptive cruise control, named Smart Driver Model (SDM), is proposed to describe the au- tonomous vehicles flow. Meanwhile, the stability criteria is proposed for SDM to keep the homogeneous traffic flow stable. Numerical simulations were conducted to verify the results of the theoretical analysis. We find that simulation results are consistent with the results of lin- ear stability analysis. Considering a platoon without cut-in, the simulation result shows that the proposed (a) (b) Fig. 7 Simulation results from a vehicle performing a cut-in in the fourth position of 16 ACC vehicles (a) ( ) (b) Fig. 7 Simulation results from a vehicle performing a cut-in in the fourth position of 16 ACC vehicles Fig. 7 Simulation results from a vehicle performing a cut-in in the fourth position of 16 ACC vehicles Lu and Aakre European Transport Research Review (2018) 10:49 Page 10 of 11 Page 10 of 11 Lu and Aakre European Transport Research Review model can stabilize the homogeneous traffic flow consid- ering a realistic driving cycle. Moreover, the results from simulation and linear stability analysis show that SDM outperforms the IDM-ACC and the ACC proposed by Milanés and Shladover in terms of stabilization effect on homogeneous traffic flow. Considering a platoon with cut-in, a platoon with 16 vehicles, when a cut-in occurs between the fourth and fifth positions, is simulated to investigate the stability of SDM under cut-in condition. The simulation result shows that the SDM-equipped ve- hicles are able to stabilize the homogeneous traffic flow under cut-in condition. In the future, considering the in- formation from multiple preceding vehicles and the surrounding traffic, SDM can be extended to energy- efficient control strategies and personalized information systems. 3. Vajedi M, Azad NL (2016) Ecological Adaptive Cruise Controller for Hybrid Electric Vehicles Using Nonlinear Model Predictive Control. IEEE Trans Intell Transp Syst 17:113–122. https://doi.org/10.1109/TITS.2015.2462843. 3. 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JSME Int J Ser C Mech Syst Mach Elem Manuf 13:671–677. y The present paper has the following limitations. First, the stability analysis is based on the theoretical model and simulation. Second, the lane-changing behavior is ignored to investigate the stability of the proposed model. Third, heterogeneous traffic conditions with ACC-equipped vehicles and human-driven vehicles is not considered in this paper. The fourth limitation comes from the same model parameter setting within a vehicle string in the simulation experiments. The im- pacts of different driver characteristics, such as reaction time, acceleration capabilities, and desired time gaps, within a string can be found by a sensitive analysis in subsequent simulations. In the future, the data collected from SDM-equipped vehicles should be used to validate the theoretical results. The proposed model will be im- plemented in an advanced and sophisticated traffic simulation model to discover the traffic impact of ACC vehicles on heterogeneous traffic flow. Moreover, specific lane-change maneuver needs to be designed for SDM in order to improve the stabilization effect of autonomous vehicles on the road transportation system. 11. Darbha S, Rajagopal KR (1999) Intelligent cruise control systems and traffic flow stability. Transp Res Part C 7:329–352. 11. Darbha S, Rajagopal KR (1999) Intelligent cruise control systems and traffic flow stability. Transp Res Part C 7:329–352. 12. Authors’ contributions CL d l d h ACC Authors’ contributions CL developed the ACC and drafted the paper. AA participated in the design of the study. Both authors read and approved the final manuscript. 20. Fancher P, Peng H, Bareket Z et al (2002) Evaluating the influences of adaptive cruise control systems on the longitudinal dynamics of strings of highway vehicles. 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https://openalex.org/W2937017956	https://research.utwente.nl/files/105397166/sensors_19_01730.pdf	English	null	Possibilities for Groundwater Flow Sensing with Fiber Bragg Grating Sensors	Sensors	2,019	cc-by	9,507	Received: 15 February 2019; Accepted: 8 April 2019; Published: 11 April 2019 Abstract: An understanding of groundwater ﬂow near drinking water extraction wells is crucial when it comes to avoiding well clogging and pollution. A promising new approach to groundwater ﬂow monitoring is the deployment of a network of optical ﬁbers with ﬁber Bragg grating (FBG) sensors. In preparation for a ﬁeld experiment, a laboratory scale aquifer was constructed to investigate the feasibility of FBG sensors for this application. Multiparameter FBG sensors were able to detect changes in temperature, pressure, and ﬁber shape with sensitivities inﬂuenced by the packaging. The ﬁrst results showed that, in a simulated environment with a ﬂow velocity of 2.9 m/d, FBG strain effects were more pronounced than initially expected. FBG sensors of a pressure-induced strain implemented in a spatial array could form a multiplexed sensor for the groundwater ﬂow direction and magnitude. Within the scope of this research, key technical speciﬁcations of FBG interrogators for groundwater ﬂow sensing were also identiﬁed. Keywords: ﬁber Bragg grating; aquifer simulator; thermal tracer; FBG interrogators; multiplexed temperature sensing sensors sensors sensors Possibilities for Groundwater Flow Sensing with Fiber Bragg Grating Sensors Sandra Drusová 1,2,∗,† , Wiecher Bakx 1,3 , Adam D. Wexler 1 and Herman L. Offerha 1 Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands; wiecher.bakx@arcadis.com (W.B.); adam.wexler@wetsus.nl (A.D.W.) 1 Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands; wiecher.bakx@arcadis.com (W.B.); adam.wexler@wetsus.nl (A.D.W.) 2 Optical Sciences, University of Twente, Hallenweg 23, 7522 NH Enschede, The Netherlands; h.l.offerhaus@utwente.nl 2 Optical Sciences, University of Twente, Hallenweg 23, 7522 NH Enschede, The Netherlands; h.l.offerhaus@utwente.nl 3 Arcadis Nederland B.V., Beaulieustraat 22, 6814 DV Arnhem, The Netherlands * Correspondence: sandra.drusova@wetsus.nl † Current address: Wetsus, European centre of excellence for sustainable water technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands. † Current address: Wetsus, European centre of excellence for sustainable water technology, Oostergoweg 9, 8911 MA Leeuwarden, The Netherlands. 1. Introduction The majority of groundwater reserves are stored within porous sedimentary structures and fractured rocks [1]. Groundwater is often extracted from underground aquifers via drilled wells and globally provides almost 50% of all drinking water and 43% of all consumptive use in agriculture [2]. The careful assessment of local hydrogeological conditions is necessary when drilling, installing, and operating a groundwater extraction well. Understanding groundwater ﬂow near wells helps to identify the mechanisms of groundwater recharge and to estimate water resources for extraction. Accurate ﬂow information helps to prevent well clogging, salt water intrusion in coastal areas, or contamination of the drinking water. Natural groundwater velocities typically do not exceed a few meters per day [3]. Therefore, groundwater-related problems are slow to develop and may require months or years to detect. Once identiﬁed, the mitigation of the problem can take years and is usually accompanied by high costs [4,5]. This is why all groundwater sources should be monitored and protected. Groundwater ﬂow velocities are a fundamental input to ﬂow models and are typically measured using in situ methods. Two of the most common methods are discrete ﬂow meters and tracer dilution Sensors 2019, 19, 1730; doi:10.3390/s19071730 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 15 Sensors 2019, 19, 1730 tests. Regardless of the method chosen, boreholes must be drilled in the area of study and can potentially disrupt the natural ﬂow of groundwater. Flow logging using discrete ﬂow meters placed in a borehole is the current industry standard [6]. These devices use a variety of operating principles including electromagnetic, acoustic, impeller, or heat pulses to measure velocity [7]. The selection of a suitable tool for ﬂow monitoring relies on a priori information about hydrogeological conditions, which is not always available [8]. Tracer tests work by adding a concentrated tracer at a constant rate for a predetermined period of time into a borehole upstream, and ﬂow velocity is determined as a function of a tracer arrival time in downstream boreholes. The velocity can be also determined with a single borehole measurement by monitoring the concentration of a tracer as a function of time due to dilution [9]. Tracer tests are a powerful technique for identifying ﬂow patterns and the hydraulic conductivity of aquifers, as they not only provide velocities but can also identify preferential ﬂow paths in the area [6]. However, especially at low groundwater ﬂow rates, completing tracer tests can be time-consuming. 1. Introduction Heat is becoming more utilized as a tracer for three primary reasons: speed, cost, and environmental friendliness [10]. Measuring the temperature is fast and less costly than measuring the solute concentration. Moreover, it is an environmental tracer—variations in groundwater temperature already occur naturally due to the surface water or fracture inﬂows [11]. Active heat tracing experiments involve either heating a discrete volume of groundwater or injecting hot/cold water into an aquifer [12,13]. While this approach is generally considered to be environmentally safe, high temperature changes may have an effect on the chemical properties and microbiological stability of aquifers [14]. When relatively large temperature differences are created in the subsurface, the resulting changes in density and viscosity modify the ﬂow regime [15]. The collection of spatial temperature data from the ﬁeld has, until recently, been a limitation to heat tracing methods. Innovations in optical ﬁber sensing technology have opened new possibilities for this method by improving spatial and temporal resolutions. Optical fibers have quickly gained acceptance as temperature sensors due to their high resolution. Their main advantage is their serial multiplexing capability—a single fiber can provide a large number of highly accurate temperature measurements along the entire length, reducing the costs of the deployment. Their small size and durability open up the possibilities for embedding them inside materials. One optical fiber-based technology that is becoming more widely implemented in hydrogeological studies is distributed temperature sensing (DTS). In DTS, the temperature is calculated from the intensity of inelastically backscattered light propagating in an optical fiber [16]. The temperature readings are spatial averages from discrete sections in the fiber. DTS in combination with hot water as a tracer was used to characterize groundwater-surface inflows [17], vertical groundwater flows in boreholes [18,19], and horizontal groundwater flow in an unconsolidated aquifer [20]. Another ﬁber-optic technology with a potential use for groundwater ﬂow monitoring is ﬁber Bragg grating (FBG) sensors [21,22]. FBG sensors already have a broad application range in aerospace (load monitoring and shape sensing [23–25]), in civil engineering (structural health monitoring [26–28]), and in the oil and gas industry (temperature and pressure monitoring [29]). An FBG sensor is a periodic variation of the refractive index within a ﬁber core which acts like a wavelength-speciﬁc mirror. 1. Introduction When an FBG sensor is illuminated by a broadband light source, part of the light is reﬂected when it satisﬁes the Bragg condition [30]: λB = 2 · ne f f · ΛG (1) (1) where λB is a reﬂected Bragg wavelength, ne f f is an effective refractive index for the propagating light, and ΛG is a grating period. Changes in the temperature or mechanical strain affect both the refractive index of the ﬁber and the grating period, causing a shift ∆λ in the initial Bragg wavelength λB0 (Figure 1). Multiple physical quantities can be measured with FBG sensors when their inﬂuence is translated into strain or temperature variations, e.g., pressure, vibrations, and curvature of the ﬁber [31,32]. FBG sensors provide instantaneous and point-wise measurements. 3 of 15 Sensors 2019, 19, 1730 FBG Transmitted signal Temperature Strain λ +∆λ B0 Input signal Reﬂected signal Figure 1. The basic sensing principle of an ﬁber Bragg grating (FBG) sensor. Temperature Strain Input signal Transmitted signal Reﬂected signal Figure 1. The basic sensing principle of an ﬁber Bragg grating (FBG) sensor. A choice between FBG and DTS sensing depends on the intended application. DTS ﬁbers are usually standard single-mode or multimode ﬁbers used in the telecom industry, which makes them affordable in large lengths. FBG sensors can be written in standard and specialty optical ﬁbers, and the ﬁber price increases per number of sensors. Fiber interrogators for collecting the DTS sensing data still remain around ﬁve times more expensive than FBG units. Current DTS systems provide continuous measurements along the ﬁber length with intervals of 12.5 cm, a spatial resolution of 30 cm, an accuracy of 1 ◦C, and a temperature resolution of 0.01 ◦C [33–35]. The temperature resolution of an FBG sensor is typically determined by the resolution of the interrogator and the sensitivity of a sensor to temperature changes. The current state-of-the art interrogators provide around 1 pm spectral resolution [36] corresponding to a 0.1 ◦C change in bare ﬁbers. The temperature sensitivity of an FBG sensor can be improved through packaging [37–39]. The location of FBG sensors in a ﬁber can be tailored by the user to a submillimeter spatial resolution. The temperature calibration of both DTS and FBG sensors needs to be performed to acquire the value of temperature sensitivity coefﬁcients for a given packaging. 2.1. Aquifer Simulator and FBG Fibers 2.1. Aquifer Simulator and FBG Fibers Aquifer simulators are commonly used to validate groundwater sensors under realistic conditions on a small scale. An aquifer simulator consists of a watertight container ﬁlled with a porous sediment saturated with water. Controlled groundwater ﬂow conditions have previously been used to study the migration of a contaminant plume [41,42] and the temperature distribution in a dike [43] or to evaluate the accuracy of ﬂowmeters measuring the horizontal groundwater velocity [8]. The AS used in this study was constructed from a metal frame with glass walls measuring 2 × 1 × 1 m (length × width × height) which was ﬁlled with sand of 0.1–0.25 mm grain sizes, a porosity of 0.41, and a hydraulic conductivity of 9 m/d. The sand layer was deposited by hand in order to reduce the variation in sediment structure and was subsequently sealed with a 4-cm-thick clay layer, forcing the water to ﬂow through the sand rather than over the top. The base was ﬁlled with a coarser sand and covered with a plastic sheet. A water ﬂow through the sand was created by a hydraulic head gradient between the inﬂow and outﬂow reservoirs. Water in the inﬂow reservoir was pumped from a feed reservoir and had an overﬂow at a controlled height, keeping the inﬂow water level stable. An even distribution of the ﬂow into the sand both at the inﬂow and outﬂow sides of the system was created using six vertically placed perforated tubes with vertical slots of a 0.5-mm width. Hydraulic head differences were checked by two piezometer tubes near inﬂow and outﬂow inside the AS. The ﬂow in the simulator was expected to be laminar with some local variations due to differences in the way the sediment settled and due to an obstruction by the frame. The mass discharge ˙m of water with density ρ through an AS with a cross-sectional area A was measured regularly to determine the ﬂow velocities v: v = ˙m ρ · A (2) v = ˙m ρ · A (2) FBG ﬁbers in the sediment were attached to a frame of polyvinyl chloride (PVC) tubes to ensure the placement of the sensor strings with respect to each other (Figure 2). The FBG interrogator used for the experiment was capable of simultaneously interrogating 8 sensors. 1. Introduction The groundwater temperature is stable, and variations smaller than 0.1 ◦C are insigniﬁcant for heat tracing experiments. Differential temperature measurements are more important than absolute values due to small variations in background groundwater temperature. Active heat tracing experiments require heating the injected water between 10 and 80 ◦C in order to compensate for the heat losses by heat diffusion in the sediment [40]. In order to determine whether a sensor can be used for a particular application, one must consider the following parameters: resolution, dynamic range, accuracy, and stability. Standard FBG sensors already have a sufﬁcient temperature resolution to measure groundwater temperature changes without additional packaging. The required dynamic range is determined by the expected temperature difference between the heat pulse magnitude and aquifer temperature. The measurement accuracy is affected by the temperature calibration process and FBG interrogator characteristics. Generally, a temperature-stabilized FBG interrogator is required for groundwater ﬂow measurements, and the long-term wavelength stability needs to be evaluated under ﬁeld conditions. Current ﬂow models are limited by an insufﬁcient spatial sampling of the heterogeneous subsurface. In order to better manage an increased aquifer use, e.g., extraction and thermal energy storage systems, more precise ﬂow sensing techniques need to be deployed. An ideal groundwater ﬂow monitoring system should provide a reliable long-term operation and give real-time warnings for abrupt changes. These conditions could be fulﬁlled by a distributed network of FBG sensors producing a 3-D map of the groundwater ﬂow ﬁeld. FBG temperature and strain sensing capabilities were validated during a heat tracing experiment, which was performed in a laboratory scale aquifer. The accuracy and suitability of FBG sensors for groundwater ﬂow monitoring has been evaluated in this paper. 4 of 15 Sensors 2019, 19, 1730 2. Materials and Methods An aquifer simulator (AS) was used in the laboratories of Deltares, Delft, the Netherlands to investigate the feasibility of FBG sensors for groundwater ﬂow monitoring applications. This section describes the experimental setup, the calculation of temperature from FBG data, and the temperature calibration process. 2.1. Aquifer Simulator and FBG Fibers BL BM BH 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 EL EM EH 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 0 40 80 B C D E F Z (height) [cm] AL AM AH 1 2 3 4 5 6 7 8 A 0 30 60 90 105 150 X (ﬂow direction) [cm] Inﬂow Outﬂow PT100 FBG PVC coating FBG teﬂon coating Figure 3. The position of all FBG and PT100 sensors in the aquifer simulator (AS): The side view. 2.2. FBG Data Acquisition and Temperature Calculation BL BM BH 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 EL EM EH 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 0 40 80 B C D E F Z (height) [cm] AL AM AH 1 2 3 4 5 6 7 8 A 0 30 60 90 105 150 X (ﬂow direction) [cm] Inﬂow Outﬂow PT100 FBG PVC coating FBG teﬂon coating Figure 3. The position of all FBG and PT100 sensors in the aquifer simulator (AS): The side view. 2.2. FBG Data Acquisition and Temperature Calculation PT100 FBG PVC coating FBG teﬂon coating Figure 3. The position of all FBG and PT100 sensors in the aquifer simulator (AS): The side view. Figure 3. The position of all FBG and PT100 sensors in the aquifer simulator (AS): The side view. 2.2. FBG Data Acquisition and Temperature Calculation The data from the FBG sensors was collected using an FBG interrogator (Gator, Technobis, Alkmaar, The Netherlands). The interrogation was performed by a photonic integrated circuit with an arrayed waveguide grating. The interrogator emited broadband light in the range of 1516–1584 nm into the ﬁber. The reﬂected signal was directed by an optical circulator to an arrayed waveguide grating serving as a demultiplexer for up to 8 Bragg wavelengths. A proprietary center-of-gravity algorithm calculated the peak Bragg wavelengths from the measured photodiode current. A 1 × 16 ﬁber optic switch (eol 1 × 16, LASER COMPONENTS GmbH, Olching, Germany) was used to extend the number of interrogated ﬁbers in the system. Both the switch and the interrogator were supervised by a microcontroller (Raspberry Pi Model 3B, Raspberry Pi Foundation, Cambridge, UK) using a Python serial library. 2.1. Aquifer Simulator and FBG Fibers For this reason, all FBG ﬁbers had 8 FBG sensors each, with Bragg wavelengths of 1518 nm, 1527 nm, 1536 nm, 1545 nm, 1554 nm, 1563 nm, 1572 nm, and 1581 nm. The FBG sensors were inscribed in a standard single-mode Corning SMF28 ﬁber with an acrylate coating using an ultraviolet femtosecond laser (Loptek GmbH & Co., KG, Berlin, Germany). Two types of FBG ﬁbers were used in the AS: 3 ﬁbers (B, C, and D) had a sensor spacing of 15 cm and a PVC buffer of 3 mm in diameter, and 3 ﬁbers (A, E, and F) had a 10-cm spacing of the sensors and a 1-mm-thick teﬂon tube of 3 mm diameter in the sensor section. Nine resistive platinum PT100 temperature sensors were placed in the AS as an additional temperature reference plus one PT100 sensor in the inﬂow reservoir (FS400P, Conrad). The data from the PT100 sensors was acquired by an Ecograph RSG30 (Endress + Hauser, Reinach, Switzerland). q y g p The distribution of the FBG ﬁbers in the X (ﬂow) direction was almost equal, with a spacing of 30 cm (see Figure 2). The only exception was row E. This row was shifted 15 cm towards the inﬂow to create more space between rows E and F for the experiments with a ﬁber placement in sand as a preparation for future ﬁeld studies. One of the experimental tools for the placement of the ﬁbers was a hollow push rod with a cone-shaped driving point. The nonuniform distribution of FBG sensors in the Z direction (height) was a result of the ﬁber mounting procedure (see Figure 3). The ﬁbers were taped on the two ends to the PVC frame to allow a certain degree of freedom to prevent ﬁber damage 5 of 15 Sensors 2019, 19, 1730 with the heavy sand load. The ﬁbers slipped up to 5 cm compared to the initial location during the attachment and stretching (all selected FBG sensors for the data were within 5 cm distance from each other). The taped ﬁbers also adapted their shape as the container was ﬁlled with sand; this was why the displayed results were from a 10-cm interval in the middle of the setup, as it was not possible to guarantee an equal distribution in the aquifer simulator. 2.1. Aquifer Simulator and FBG Fibers 0 30 60 90 105 150 A B C D E Inﬂow Outﬂow X (ﬂow direction) [cm] 0 20 40 60 85 Y (width) [cm] F PT100 FBG PVC coating FBG teﬂon coating Figure 2. The position of the FBG ﬁbers and PT100 sensors on the PVC frame: The top view. The data from the highlighted section are displayed in the results. BL BM BH 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 EL EM EH 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 0 40 80 B C D E F Z (height) [cm] AL AM AH 1 2 3 4 5 6 7 8 A 0 30 60 90 105 150 X (ﬂow direction) [cm] Inﬂow Outﬂow PT100 FBG PVC coating FBG teﬂon coating Figure 3. The position of all FBG and PT100 sensors in the aquifer simulator (AS): The side view. 2. FBG Data Acquisition and Temperature Calculation 0 30 60 90 105 150 A B C D E Inﬂow Outﬂow X (ﬂow direction) [cm] 0 20 40 60 85 Y (width) [cm] F PT100 FBG PVC coating FBG teﬂon coating Figure 2 The position of the FBG ﬁbers and PT100 sensors on the PVC frame: The top view The data 0 30 60 90 105 150 A B C D E Inﬂow Outﬂow X (ﬂow direction) [cm] 0 20 40 60 85 Y (width) [cm] F PT100 FBG PVC coating FBG teﬂon coating Figure 2. The position of the FBG ﬁbers and PT100 sensors on the PVC frame: The top view. The data from the highlighted section are displayed in the results. PT100 FBG PVC coating FBG teﬂon coating Figure 2. The position of the FBG ﬁbers and PT100 sensors on the PVC frame: The top view. The data from the highlighted section are displayed in the results. 2.1. Aquifer Simulator and FBG Fibers A sampling period of 10 s was chosen to yield enough sampling points to capture the heat processes in the sediment in great detail. The interrogator continuously sent the reﬂected wavelengths information via USB at a frequency of 1 kHz, overwriting previous data within the device buffer. 6 of 15 Sensors 2019, 19, 1730 The 1-kHz sampling interval was too high for the desired application, but this factory setting could not be changed. Fifty datasets were acquired every 10 s and averaged to increase the accuracy. The groundwater temperature was very stable and rarely ﬂuctuated 0.1 ◦C in an hour; thus, a long-term stability of the interrogator light source was required for the heat-tracing experiments. According to the published interrogator speciﬁcations, the wavelength stability was 5 pm in a steady-state environment [44]. However, a measurement with the FBG ﬁbers in a stable laboratory environment showed a wavelength variation up to 20 pm, which translated to a drift of 2 ◦C. The measured drift did not originate in the ﬁber but was rather due to the thermal instabilities affecting the interrogator photonic integrated circuits. At the time of this study, the interrogator had no built-in wavelength referencing system so the manufacturer provided an additional external reference. This external reference was an FBG sensor with a Bragg wavelength of 1550 nm surrounded by thermoelectric elements controlled by a thermoelectric cooling (TEC) controller (ITC4005, Thorlabs, Newton, MA, USA). With this FBG reference sensor embedded, immobilized, and temperature-stabilized, the reﬂected wavelength could be used to quantify the drift of the system at 1550 nm directly (see Figure 4). The drift was approximately 70 pm during the initial 12-h operational period and, afterwards, ﬂuctuated in the range of 20 pm. Thus, the interrogator could be used for the heat-tracing experiments only in combination with the external reference sensor since a drift correction needed to be performed. Δλ Time [d] [pm] ref 0 1 2 3 -20 20 60 Figure 4. The long-term wavelength drift of the FBG interrogator at 1550 nm as measured with a temperature-controlled FBG sensor. Δλ Time [d] [pm] ref 0 1 2 3 -20 20 60 Figure 4. The long-term wavelength drift of the FBG interrogator at 1550 nm as measured with a temperature-controlled FBG sensor. Since the FBG sensors had Bragg wavelengths different from the reference, the wavelength-dependent drift could not be directly subtracted from the measured data. 2.3. Thermal Calibration of FBG Sensors The sum of the coefﬁcients αe + αn was obtained experimentally during additional calibration measurements. FBG ﬁbers with two different coatings (in rows A and B) were placed in a calibration water bath with a controlled temperature, together with a reference PT100 sensor (TSP01, Thorlabs, Newton, MA, USA). The water in the bath was heated to +25 ◦C corresponding to the temperature of the hot inﬂow water entering the AS in the heat-tracing experiment. The data from the FBG sensors was collected using a different interrogator with a built-in wavelength reference for a more precise calibration (Hyperion si155, Micron optics, Atlanta, GA, USA). The interrogator had a swept laser in the range of 1500–1600 nm scanning the entire spectrum with a 1-kHz frequency. A full optical spectrum was digitized, and the peak wavelengths were calculated with a centre-of-gravity algorithm. With this technology, the number of interrogated FBG sensors is limited by the desired dynamic range. The device has an internal wavelength referencing system consisting of a Fabry–Perot cavity and a gas cell [45]. The measured drift for this interrogator was less than 1 pm. The temperature sensitivity coefﬁcient α can be calculated from Equation (6), while neglecting the strain and reference term, as ∆λ α = αe + αn = ∆λ λB0∆T (7) (7) The α values for each coating in Table 1 are an average of 8 FBG sensors within each ﬁber from a 15-min calibration interval when the water temperature was held stable (Figure 5). The accuracy of the PT100 calibration sensor determined the conﬁdence bounds of the α values. In the interval of 20–45 ◦C, the accuracy of the differential temperature measurement with the reference sensor was ±0.8 ◦C. The temperature sensitivity of the FBG sensors, i.e., the wavelength shift per 1 ◦C, could be calculated from Equation (7). The values for a 1550-nm FBG sensor are presented in Table 1. The temperature resolution of the FBG sensors depends on the resolution of the spectrum analyzer in the interrogator, in this case, 1 pm, and is calculated from the sensitivity S as 1 (pm)/S (pm/◦C). Time [min] calibration interval A FBG 1 B FBG 1 PT100 ref 0 0 0 100 10 10 200 20 20 300 30 30 [°C ] [pm] Δλ ΔT Figure 5. An example of the calibration curves for two FBG sensors: Each sensor is inscribed in a ﬁber with a different coating. 2.1. Aquifer Simulator and FBG Fibers An additional correction or gain factor, G, needed to be determined, which scaled the measured FBG wavelengths to match the reference: λ G = λB λre f (3) (3) where λB is the Bragg wavelength from an FBG sensor and λre f is the Bragg wavelength of the reference FBG sensor. The wavelength dependency of G was investigated using the AS under the following conditions: the AS was off, the temperature inside was stable, and there were no factors that could introduce strain effects in the sediment. In this case, the data collected from the FBG sensors directly represented the system drift at those corresponding Bragg wavelengths. The experiment showed that G = G(λ) is linear; therefore, the gain value for any FBG sensor can be simply calculated as a constant: G = λB0 λre f0 (4) (4) where λB0 is the initial Bragg wavelength of an FBG sensor and λre f0 is the initial Bragg wavelength of the reference sensor. The Bragg wavelength shift ∆λ with respect to a change of the external temperature ∆T and the strain ∆ϵ is described by the following equation [30], with the reference t bt t d where λB0 is the initial Bragg wavelength of an FBG sensor and λre f0 is the initial Bragg wavelength of the reference sensor. The Bragg wavelength shift ∆λ with respect to a change of the external temperature ∆T and the strain ∆ϵ is described by the following equation [30], with the reference term subtracted: λ ∆λ ∆λ λB0 −G ∆λre f λre f0 = (1 −p)∆ϵ + (αe + αn)∆T (5) (5) 7 of 15 Sensors 2019, 19, 1730 where p is a strain-optic coefﬁcient, αe is a thermal expansion coefﬁcient (describes changes in the grating length due to temperature), and αn is a thermo-optic coefﬁcient (describes changes in effective refractive index due to temperature). If the ﬁbers are ﬁrmly held in the sediment, then the strain effects can be neglected. Relative temperature changes in the AS can be determined from Equation (5) as: ∆T = 1 αe + αn · ∆λ λB0 −G ∆λre f λre f0 ! (6) (6) 2.3. Thermal Calibration of FBG Sensors The sensor A FBG 1 has a teﬂon coating; the sensor B FBG 1 has a PVC coating. Time [min] calibration interval A FBG 1 B FBG 1 PT100 ref 0 0 0 100 10 10 200 20 20 300 30 30 [°C ] [pm] Δλ ΔT Figure 5. An example of the calibration curves for two FBG sensors: Each sensor is inscribed in a ﬁber with a different coating. The sensor A FBG 1 has a teﬂon coating; the sensor B FBG 1 has a PVC coating. 8 of 15 Sensors 2019, 19, 1730 Table 1. The results of the thermal calibration: The thermal sensitivities were calculated for two types of FBG coating (packaging) material used in the AS. FBG Coating α ± ∆α (1/◦C) Sensitivity at 1550 nm (pm/◦C) T Resolution (◦C) Teﬂon (7.43 ± 0.26) × 10−6 11.51 0.09 PVC (6.91 ± 0.24) × 10−6 10.71 0.09 The conﬁdence interval for α can be used to calculate the accuracy of the differential temperature measurements with the FBG sensors in the AS. If α changes by ∆α, ∆T will change as ∆T′ = ∆λ λB0(α + ∆α) (8) (8) The accuracy can, thus, be calculated in comparison to the original ∆T as ∆T′ ∆T = α α + ∆α (9) (9) A varied temperature sensitivity is caused by differences in the thermal expansion properties of the coating material. The thermal expansion coefﬁcient of teﬂon is slightly larger than that of PVC, (112 −135) × 10−6 (1/◦C) for teﬂon and (54 −110) × 10−6 (1/◦C) for PVC. 3.1. Temperature Effects Experimental stages: I—hot inﬂow, II—cold inﬂow, and III—no inﬂow. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 -5 0 5 10 15 20 25 30 [°C] A Inflow B E I II III FBG PT100 Δ T Figure 6. A comparison of the FBG data translated into temperatures with the PT100 temperature proﬁles: The displayed data are from the sensors highlighted in Figure 3. Identical colors indicate sensors in the same row on the frame (the same distance from the inﬂow system). Experimental stages: I—hot inﬂow, II—cold inﬂow, and III—no inﬂow. Figure 6. A comparison of the FBG data translated into temperatures with the PT100 temperature proﬁles: The displayed data are from the sensors highlighted in Figure 3. Identical colors indicate sensors in the same row on the frame (the same distance from the inﬂow system). Experimental stages: I—hot inﬂow, II—cold inﬂow, and III—no inﬂow. 2.4. Heat-Tracing Experiments • 2-D temperature maps—the time development of a temperature from all sensors along a vertical slice through the AS (all sensors in Figure 3). 2.4. Heat-Tracing Experiments 2.4. Heat-Tracing Experiments FBG technology has already been successfully deployed in subsurface environments, but not yet for the purpose of monitoring groundwater ﬂow. One way to achieve this would be to use an array of FBG sensors for localized temperature measurements in heat-tracing experiments. Therefore, a heat-tracing experiment was performed in an idealized version of an aquifer. FBG ﬁbers with two different thermal sensitivities were buried in the AS with a constant ﬂow velocity of 2.9 m/d and varying inﬂow conditions: I. Hot inﬂow. Time t = 0 h. Hot water (48 ◦C) from a feed reservoir was pumped into the inﬂow reservoir where it slowly mixed with the room temperature (19 ◦C) water before ﬂowing through the inﬂow pipes and into the AS. II. Cold inﬂow. Time t = 5.5 h. In this stage, the feed reservoir was ﬁlled with cold tap water (19 ◦C) and pumped into the inﬂow reservoir. ﬂ In this stage, the feed reservoir was ﬁlled with cold tap water (19 ◦C) and pumped into the inﬂow reservoir. III. No inﬂow. Time t = 7.5 h. III. No inﬂow. Time t = 7.5 h. The feed pump to the inﬂow reservoir was stopped; both the inﬂow and outﬂow systems were closed. Prior to making measurements, the inﬂow system was open for two hours to allow the ﬂow to reach a steady-state condition. The ﬂow velocity was chosen to correspond to that of the natural groundwater ﬂow, and we assumed that, for this low velocity, the strain effects would be negligible compared to the temperature effects. Surprisingly, some strain effects were observed and will be discussed below. The temperature and strain cross-sensitivity of the FBG sensors was separated by a comparison with the PT100 sensors. The wavelength shifts measured by the FBG sensors were translated into temperature, and the results are displayed as • 1-D temperature proﬁles—the time development of a temperature from all sensors along a line in the ﬂow direction (highlighted in Figure 3). A comparison between the PT100 and FBG sensors also allowed the identiﬁcation of the strain effects. 9 of 15 Sensors 2019, 19, 1730 • 2-D temperature maps—the time development of a temperature from all sensors along a vertical slice through the AS (all sensors in Figure 3). 3.1. Temperature Effects The majority of FBG signals consists of temperature contributions. Generally, temperature curves from both type of sensors, FBG and PT100, have similar shapes (see Figure 6). The differences are within the accuracy range of the sensors (Table 2). Moreover, the compared sensors were not at identical locations and had different physical dimensions. The PT100 sensors, for example, provided a temperature spatially averaged over 6 cm. Heat transport by convection was a dominant process in stages I and II. Thermal diffusion took place not only in the pore ﬂuid but also in the sand particles. As a result, the advancing thermal tracer front was not a sharp boundary. Diffusion was dominant in stage III; heat transport by diffusion was much slower than convection in this case. A heat exchange between the inﬂow water, the sand particles, and the AS walls was apparent from the gradual decrease in the maximal temperature as water progressed through the AS. Table 2. A comparison of the temperature accuracy of the FBG and PT100 sensors used in the heat-tracing experiment. heat-tracing experiment. FBG Coating ∆T Accuracy (%) ∆T = 25◦C Accuracy (◦C) Teﬂon ±3.38 ±0.85 PVC ±3.36 ±0.84 PT100 ±1.1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time [h] -5 0 5 10 15 20 25 30 [°C] A Inflow B E I II III FBG PT100 Δ T Figure 6. A comparison of the FBG data translated into temperatures with the PT100 temperature proﬁles: The displayed data are from the sensors highlighted in Figure 3. Identical colors indicate sensors in the same row on the frame (the same distance from the inﬂow system). Experimental stages: I—hot inﬂow, II—cold inﬂow, and III—no inﬂow. FBG Coating ∆T Accuracy (%) ∆T = 25◦C Accuracy (◦C) FBG Coating ∆T Accuracy (%) ∆T = 25◦C Accuracy (◦C) Teﬂon ±3.38 ±0.85 PVC ±3.36 ±0.84 PT100 ±1.1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time [h] -5 0 5 10 15 20 25 30 [°C] A Inflow B E I II III FBG PT100 Δ T Figure 6. A comparison of the FBG data translated into temperatures with the PT100 temperature proﬁles: The displayed data are from the sensors highlighted in Figure 3. Identical colors indicate sensors in the same row on the frame (the same distance from the inﬂow system). 3.2. Thermal Plume Propagation The temperature of the inﬂow water was altered in the experiment, and the FBG sensors were deployed to visualize the propagation of a thermal plume across the AS (see the plume shape in Figure 7). A majority of the strain effects were a fast perturbation in a slow temperature dynamics; therefore, it was possible to choose FBG data not affected by the strain to visualize the thermal plume propagation. The thermal plume developed asymmetrically over time in the vertical direction and exhibited a quasi-parabolic proﬁle consistent with laminar ﬂow. Initially, the hot plume was centered in the upper 10 of 15 Sensors 2019, 19, 1730 section of the AS due to buoyancy effects (see Figure 7a). The hot plume propagated downwards during stages I and II. The heat propagation direction did not coincide with the direction of the ﬂow, possibly due to the asymmetric thermal properties of the upper and lower sediment boundaries. The top of the AS had wet saturated clay as a sealant, and the base was ﬁlled with a coarse sand covered with a plastic sheet. The buoyancy effects were expected to prevail during the entire experiment, but this was not the case. This unexpected heat propagation in the sand was captured by the FBG sensors with a high resolution of 0.09 ◦C. 3 h A B C D E F (a) 5 h A B C D E F (b) 0 5 10 15 20 25 [° C] ΔT 7 h A B C D E F (c) 14 h A B C D E F (d) 0 5 10 15 20 25 [° C] ΔT Figure 7. The time evolution of the thermal plume in the AS at four different moments: (a) t = 3 h; (b) t = 5 h; (c) t = 7 h; (d) t = 14 h from the start of the experiment. The black circles indicate the calculated temperature from the FBG sensors. The displayed temperature map was generated by a linear interpolation from the FBG data. 3.3. Strain Effects Prior to this study, it was assumed that the wavelength shifts from FBG sensors in an AS would be caused exclusively by temperature changes. However, as these experiments showed, this was not valid for all FBG sensors. In the case of the FBG sensors in rows A and C, the strain effects were non-negligible (Figure 8). Possible explanations for the observed strain events could be 1. Setup adjustments, e.g., inﬂow connection check 1. Setup adjustments, e.g., inﬂow connection check 1. Setup adjustments, e.g., inﬂow connection check 2. Local ﬂow changes, e.g., preferential ﬂow paths around the sensors or settling of the s 2. Local ﬂow changes, e.g., preferential ﬂow paths around the sensors or settling of the sedime 3 Global ﬂow changes e g hydraulic head changes 2. Local ﬂow changes, e.g., preferential ﬂow paths around the sensors or settling of the sediment 3. Global ﬂow changes, e.g., hydraulic head changes Local ﬂow changes, e.g., preferential ﬂow paths around the sensors or settling of the sediment Global ﬂow changes, e.g., hydraulic head changes 2. Local ﬂow changes, e.g., preferential ﬂow paths around the sensors or settling of the sediment 3. Global ﬂow changes, e.g., hydraulic head changes 3. Global ﬂow changes, e.g., hydraulic head changes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time [h] -50 0 50 100 150 200 250 300 [pm] A C I II III Δλ Figure 8. The wavelength shifts of the FBG sensors in rows A and C: The red arrows indicate strain events resulting from (1) setup adjustments, (2) local ﬂow, or (3) global ﬂow changes. The Roman numerals demarcate the different experimental stages consistent with Figure 6. Figure 8. The wavelength shifts of the FBG sensors in rows A and C: The red arrows indicate strain events resulting from (1) setup adjustments, (2) local ﬂow, or (3) global ﬂow changes. The Roman numerals demarcate the different experimental stages consistent with Figure 6. The step strain response at t = 0.2 h (arrow 1) is common for the FBG sensors from rows A and C. It is the result of physical adjustments made to the inﬂow system. The connection between the inﬂow reservoir and the inﬂow tubes is made of several plastic hoses. At the start of the experiment, these connections were checked and tightened. 3.2. Thermal Plume Propagation 5 h A B C D E F (b) 0 5 10 15 20 25 [° C] ΔT 3 h A B C D E F (a) 3 h 5 h (b) (a) 14 h A B C D E F (d) 0 5 10 15 20 25 [° C] ΔT 7 h A B C D E F (c) A (d) (c) Figure 7. The time evolution of the thermal plume in the AS at four different moments: (a) t = 3 h; (b) t = 5 h; (c) t = 7 h; (d) t = 14 h from the start of the experiment. The black circles indicate the calculated temperature from the FBG sensors. The displayed temperature map was generated by a linear interpolation from the FBG data. Predicting the propagation of a thermal plume was not a trivial task even in a designed subsurface environment with a quasi-uniform particle distribution and laminar ﬂow. In our case, the heat transport was affected by boundary conditions with different thermal conductivities. The same situation occurred during the heat-tracing tests in the ﬁeld. A heterogeneity of the subsurface complicated the interpretation of heat-tracing results, as heat does not always propagate in the same direction as the ﬂow. An array of temperature sensors could, then, help to visualize the heat transport in porous sediment structures. The FBG sensors could be used in the heat-tracing tests if they were combined with a certain packaging to eliminate the strain effects (e.g., rigid metal tubing). However, in terms of the cost and resolution, DTS was still a more suitable option for temperature sensing in the subsurface. The use of active heat-tracing methods had been successfully applied to the characterization of groundwater ﬂows in diverse geological formations [12,46]. However, due to the need for a constant power input to run the heating equipment, such an approach is not feasible for long-term ﬂow monitoring. In this 11 of 15 Sensors 2019, 19, 1730 regard, FBG ﬂow sensors present an interesting alternative as they also exhibit a sensitivity to strain which would not require power for heating. regard, FBG ﬂow sensors present an interesting alternative as they also exhibit a sensitivity to strain which would not require power for heating. 3.3. Strain Effects Such manipulations could result in either a physical strain due to the small movements of parts in the AS or changes in the hydraulic conveyance efﬁciency of the system. The FGB sensors in rows A and C had the largest strain response at t = 7.5 h (stage III) when the ﬂow in the AS stops—it was a fast step response followed by a slow relaxation with amplitudes up to 50 pm. In the case of ﬁber A, the slow relaxation lasted for 6.5 h. It is likely that those FBG sensors measured the local pressure (hydraulic head) changes. The strain effect at the start of stage II was caused by a small hydraulic head change when the feed reservoir was drained from hot water. The hydraulic head differential in the AS at the start of stage III was 22 cm and decreased slowly to zero after many hours. Hydraulic head changes near drinking water wells can start from a few tens of cm and reach to several meters depending on how far from the well one measures and the extraction rate. Hence, the strain effects measured by FBG sensors near groundwater extraction wells are expected to be even larger than those found in this study. FBG sensors are sensitive to pressure if they are translated to axial strain through suitable packaging. The most common design of an FBG pressure sensor uses a perforated metal cylinder to surround the grating which is embedded in an elastic polymer [47]. When both ﬁber ends are ﬁxed, an increasing pressure compresses the ﬁber and decreases the grating period, thus causing a negative 12 of 15 Sensors 2019, 19, 1730 wavelength shift. FBG sensors in the AS, attached to the PVC frame, are similar to pressure sensors with unequal sensitivities. The FBG sensors in row A were more sensitive than the sensors in row C, while the sensors in the other rows showed no such response. The pressure sensitivity was presumably linked to the amount of pre-strain in the ﬁber. When the ﬂow in the AS suddenly stopped, the pressure also suddenly increased, causing a drop in the Bragg wavelength (as can be observed in Figure 8). The slow relaxation response (arrows labeled 3) could describe the pressure equalization or elastic response of the ﬁber material. 3.3. Strain Effects Apart from global pressure changes throughout the system, the FBG sensors from rows A and C exhibited abrupt changes with short relaxation times that were localized to one or a few sensors (arrows labeled 2). The amplitude was typically less than 5 pm. These responses could be associated with changes in the local ﬂow due to the nonuniformity of the sand distribution around the ﬁbers or possibly the settling effects. The latter would cause changes in the local radius of curvature as the ﬁber adapts its shape to the change in sediment distribution. The authors intend to further investigate the sources of the strain in FBG sensors buried in aquifers. The fact that strain effects are visible for ﬂow velocities as low as 2.9 m/d indicates that pressure–strain-sensitized FBG sensors could be used near drinking water wells with higher ﬂow velocities. Since pressure differences are the driving force for the ﬂow, pressure-induced strains in FBG sensors hold information directly related to groundwater ﬂow. The pressure in the subsurface is not uniform due to a variability in the sediment distribution, and this is where distributed pressure sensors can provide more insight than simple water table measurements. When connected into a network of point sensors, FBG sensors can further offer a possibility to estimate ﬂow magnitude and direction. References 1. Todd, D. Groundwater Hydrology; Wiley: Hoboken, NJ, USA, 1980. 1. Todd, D. Groundwater Hydrology; Wiley: Hoboken, NJ, USA, 1980. . Smith, M.; Cross, K.; Paden, M.; Laban, P. Managing Groundwater Sustainably; IUCN: Gland, Switzerland, 2 2. Smith, M.; Cross, K.; Paden, M.; Laban, P. Managing Groundwater Sustainably; IUCN: Gland, Switze 3. Heath, R.C. Basic Ground-Water Hydrology; US Department of the Interior, US Geological Survey: Reston, VA, USA, 1983. doi:10.1614/0890-037X(2000)014[0451:]2.0.CO;2. 3. Heath, R.C. Basic Ground-Water Hydrology; US Department of the Interior, US Geological Survey: Reston, VA, USA, 1983. doi:10.1614/0890-037X(2000)014[0451:]2.0.CO;2. 4. Rinaudo, J.D.; Arnal, C.; Blanchin, R.; Elsass, P.; Meilhac, A.; Loubier, S. Assessing the cost of groundwater pollution: The case of diffuse agricultural pollution in the Upper Rhine valley aquifer. Water Sci. Technol. 2005. 52, 153–162. [CrossRef] [PubMed] 4. Rinaudo, J.D.; Arnal, C.; Blanchin, R.; Elsass, P.; Meilhac, A.; Loubier, S. Assessing the cost of groundwater pollution: The case of diffuse agricultural pollution in the Upper Rhine valley aquifer. Water Sci. Technol. 2005. 52, 153–162. [CrossRef] [PubMed] 5. Van Beek, K.; Breedveld, R.; Stuyfzand, P. Preventing two types of well clogging. 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Author Contributions: Conceptualization, S.D., A.D.W. and H.L.O.; methodology, S.D.; software, S.D.; validation, S.D. and W.B.; formal analysis, S.D.; investigation, S.D. and W.B.; resources, S.D.; data curation, S.D.; writing—original draft preparation, S.D.; writing—review and editing, S.D., W.B., A.D.W. and H.L.O.; visualization, S.D.; supervision, A.D.W. and H.L.O. Funding: This work was performed in the cooperation framework of Wetsus, European Centre of Excellence for Sustainable Water Technology (www.wetsus.eu). Wetsus is cofunded by the Dutch Ministry of Economic Affairs and Ministry of Infrastructure and Environment, the European Union Regional Development Fund, the Province of Fryslân, and the Northern Netherlands Provinces. This research has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 665874. Acknowledgments: We are grateful to the participants of the research theme “Groundwater technology” for the fruitful discussions and ﬁnancial support. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had a role in the decision to publish the results. Abbreviations The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: FBG Fiber Bragg grating DTS Distributed temperature sensing AS Aquifer simulator TEC thermoelectric cooling PVC polyvinyl chloride FBG Fiber Bragg grating DTS Distributed temperature sensing AS Aquifer simulator TEC thermoelectric cooling PVC polyvinyl chloride FBG Fiber Bragg grating DTS Distributed temperature sensing 4. Conclusions An idealized lab-scale aquifer simulator was constructed to evaluate the suitability of FBG sensors for groundwater ﬂow sensing. The heat-tracing experiment demonstrated that FBG sensors can measure the relative temperature with an accuracy of 0.85 ◦C for the differential measurements. The FBG point sensors used in these experiments provided localized temperature data with a spatial distribution of 10 and 15 cm. Since smooth temperature changes were expected during the heat-tracing experiment, the spatial FBG dataset was linearly interpolated. As a result, the thermal plume propagation in the aquifer could be visualized. An FBG system, as used for these experiments, is capable of a multiple-sensor interrogation with the possibility of an expansion into a larger distributed sensing network. With these FBG sensors, it is not possible to decouple wavelength shifts caused by changes in the ambient temperature from the ﬁber strain. If the application requires only the temperature information in an unconsolidated subsurface environment, the FBG sensors should be packaged to remain strain-free and to increase their temperature sensitivity. A different packaging can increase both the temperature and strain sensitivity and/or to decouple both effects. As shown in the results, the strain effects caused by hydraulic head changes are the most signiﬁcant strain contributions to measure the FBG signal. These effects will be examined and quantiﬁed in our future research towards a pressure-based groundwater ﬂow sensor. A suitable FBG interrogator for groundwater ﬂow monitoring needs to have a low noise level for maximum temperature and strain sensitivity. Currently available interrogators with noise levels under 1 pm are already sufﬁcient for this application. Furthermore, the long-term wavelength stability of a light source in the interrogator needs to be guaranteed. This is only possible when the interrogator is combined with a wavelength reference. As a ﬁrst step, the results demonstrated that FBG sensors can be used as temperature sensors in the subsurface environment, but in this implementation, they did not present an advantage over the existing ﬁber sensing technologies. However, multiplexed FBG pressure or strain sensors have a potential in groundwater ﬂow monitoring. This research will be followed by a ﬁeld validation and derivation of groundwater ﬂow vectors. 13 of 15 13 of 15 Sensors 2019, 19, 1730 Author Contributions: Conceptualization, S.D., A.D.W. and H.L.O.; methodology, S.D.; software, S.D.; validation, S.D. and W.B.; formal analysis, S.D.; investigation, S.D. References Heat as a tracer for understanding transport processes in fracturedmedia: Theory and ﬁeld assessment from multiscale thermal push-pull tracer tests. Water Resour. Res. 2016, 52, 5442–5357. [CrossRef] 14 of 15 14 of 15 Sensors 2019, 19, 1730 14. 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https://openalex.org/W4281567365	https://bmcmededuc.biomedcentral.com/counter/pdf/10.1186/s12909-022-03481-w	English	null	Mind your language: the importance of english language skills in an International Medical Programme (IMP)	BMC medical education	2,022	cc-by	6,045	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mind your language: the importance of english language skills in an International Medical Programme (IMP) Sharon Min Hui Chan1 , Norul Hidayah Mamat2* and Vishna Devi Nadarajah3 Abstract Background: Language proficiency is crucial for doctors as they communicate with patients, peers and other health- care professionals. Although proficiency in English is part of admission requirements, there is a gap in the knowledge of medical students’ perception of factors enhancing English language (EL) skills during training in international medical programmes (IMPs). The gap prevents educators and policy makers from helping students who struggle with communication skills during medical training. This study therefore explores the importance of English language skills from medical students’ perspectives. Methods: Six focus group interviews with 24 medical students were conducted in an IMP. Data were analysed using Braun and Clarke’s framework of thematic analysis. Results: Results established three themes that constitute the importance of EL skills, namely the use of EL in medical training and practice, influence of university culture in EL mastery and individual EL proficiency as perceived by medi- cal students. Conclusions: Findings of this study demonstrate how students perceived the importance of EL skills as a profes- sional and social requirement during medical training and for future practice. It also informs that setting English language admission pre requisites needs to be complemented with opportunities to practice context specific com- munication skills. Thus, international medical programmes should embed diverse and inclusive strategies to support and develop medical students’ English language skills. Keywords: English Language skills, Medical education, Medical students, International medical prog Background perspectives of health professions education offering IMPs [2]. With IMPs, the curriculum, teaching, learn- ing and assessment activities are conducted in English [3]. EL serves as prerequisite for enrolment into IMPs which include assessments such as International English Language Testing System (IELTS) or its equivalent, Test of English as a Foreign Language (TOEFL). Originally, EL assessments were meant to assess the language ability of candidates who need to study or work where English is used as the language of communication. Nonetheless, higher education institutions in Anglophone countries English language and medical educationh English language and medical educationh The importance of English language (EL) skills in medi- cal education is demonstrated when students from mul- tilingual backgrounds enrol into international medical programmes (IMPs) [1]. The past three decades have seen an increasing number of universities with global *Correspondence: NorulHidayah@imu.edu.my 2 Centre for Education, International Medical University, No 126 Jalan Jalil Perkasa 19, Bukit Jalil , 57000 Kuala Lumpur, Malaysia Full list of author information is available at the end of the article Chan et al. BMC Medical Education (2022) 22:405 https://doi.org/10.1186/s12909-022-03481-w Open Access Data collection I f d Informed consent was obtained from participants prior to each focus group interview. The interview was audio recorded to facilitate transcription. The interview ques- tions were categorised in three areas. The first area, English language usage in medical education, consisted of two questions; ‘How is English language used in med- ical studies?’ and ‘In what ways did you see the usage of English Language in school compared to university?’. In the second area, the impact of English language profi- ciency on medical students, the interview question was; ‘Can you explain instances where learning was impeded by English language?’. Likewise, in the third area, the influence of university culture in English language mas- tery, the interview question was; ‘In what way has the university learning environment enhanced your mas- tery of English language?’. Each question was probed to gain in depth perspective of the importance of Eng- lish language skills. At the end of the interview, par- ticipants were given the opportunity to share additional Sampling context A total of 24 medical students from Year 1, 2 and 3 of a five-year IMP [2] participated in six focus group inter- views. The students have fulfilled a minimum IELTS requirement of an overall band score between 6.5 to 7.0 for all sections (reading, writing, speaking and listen- ing) upon entry to the IMP. We used maximum diverse sampling method to recruit participants based on the inclusion criteria of medical students who were in Year 1 to Year 3. The selection of participants from Year 1 to 3 provided the range of pre-clinical and clinical phases to ensure that data could be obtained from participants at different stages of the IMP. This context highlights the need for studies to iden- tify factors that enable enhancement of English language skills from the perspectives of medical students. The current knowledge gap prevents educators and policy makers from helping students who struggle with com- munication skills during medical training [11]. Findings from this study will be useful in determining learning and support strategies for students in both multilingual set- tings and IMPs. Chan et al. BMC Medical Education (2022) 22:405 Page 2 of 7 seem to have broadened the use of these assessments to gauge students’ academic performance [4]. in recognizing the patterns of learning present in the learning environment [14]. Henceforth, the use of this theory is preferred as it encapsulates learning as a contin- uous process of internalization during training, in which development of EL skills and knowledge are transformed and intertwined between the social and cognitive planes. English is an international language of medicine. It is also expanding as an international communication and educational tool [5]. The literature emphasises the importance of communication skills among medical practitioners as “effective doctor-patient communication is a crucial aspect of quality patient care” [6]. However it is scarce in relation to the perception of medical students towards factors enhancing English language and com- munication skills. This particularly applies in multilingual environments and IMPs [7]. Studies on medical students’ English language needs in Taiwanese higher education institutions were mostly concerning English language teaching [8]. The study highlighted that although stu- dents value English proficiency in their training as medical doctors, universities failed to satisfy students’ expectations and facilitate attainment of language profi- ciency [8]. In these environments, students are faced with double cognitive task [9]; studying English as the medium of their chosen studies while trying to improve their Eng- lish proficiencies [10]. Similarly, in a study from a Gulf University, students acknowledged and identified weak- ness in English language as a hindrance to their commu- nication and information handling skills [10].h When medical students participate in conversations and activities in a multicultural group, they will be able to connect with their peers, and assimilate the experience, which includes use of EL in medical training [11]. There- fore, students engage in a wide range of joint activities and subsequently, acquire new strategies and knowledge of the culture in which they are placed [15]. In summary, the gaps in literature and the broad con- textual needs and similarities of IMPs led to this study exploring medical students’ perspectives on the impor- tance of EL skills in medical training. Data analysis Th d The data were analysed using the Braun and Clarke’s framework of thematic analysis [16]. The framework includes six steps: 1) Data familiarization, 2) Iden- tification of codes, 3) Finding themes, 4) Reviewing of themes, 5) Definition and naming of themes and 6) Report production. It provides a clear and usable framework, distinguished at two levels; semantic and latent. At the semantic level, the explicit or surface meanings of the data, based on surfaced themes are analysed, while at the latent level, the analysis looks beyond what was said during the focus group discus- sions, giving it more depth in analysis [16]. In Step 1, the data from the transcription was repeatedly read to familiarize transcript and identify the codes. Samples of codes from transcription data are shown in Additional file 1: Table S1. In Step 2, the coding process started with initial codes generated on students’ perception of English language, the availability of university support on mastery of English language and the impact of Eng- lish language on medical students, as shown in Addi- tional file 1: Table S1. In Step 3, the subsequent coding process involved examinining the identified codes and narrowed it into more specific codes. The codes iden- tified in Step 1 and Step 2 were further reviewed and led to emergence of preliminary themes such as ‘English language as a learning tool’, ‘university support in Eng- lish language mastery’ and ‘English language for aca- demic progression’. In Step 4, the preliminary themes identified through data coding that represented the context of the importance of English language skills were reviewed and refined. In Step 5, a total of three themes were defined and established. Finally in Step 6, results based on the established themes were finalised. In the medical programme, learning activities include lectures, problem-based learning sessions, clinical skills and simulation sessions, hospital and bedside teaching. It is evident that students perceived EL as a learning tool in these activities as they cited that activities were in English. “If we’re in class, sure, English only speaking, if we’re at Clinical Skills & Simulation Centre sure, English only speaking.” Furthermore, students also perceived EL to be an important learning tool as medical learning resources were mainly in English. “Reference or the books that we use are mostly used in English. So, English is very important actually in our studies of our medical programme”. Results perspectives and contribute further. The interviews were transcribed and coded to identify similarities and differences of perspectives. The saturation was deemed to be achieved when similar and redundant perspec- tives shared by the participants and no new informa- tion were obtained across the focus groups. A total of three themes were established, namely, ‘EL usage in medical training and practice’, ‘the influence of university culture and environment in EL mastery’ and ‘individual EL proficiency’. Data analysis Th d English language is perceived to be equally important as a professional language. Medical students communi- cate in English with patients, peers and lecturers and par- ticipate in external events such as community support, conferences and extra curricular activities (ECAs). Stu- dents shared that speaking in English made them appear more professional, particularly when they represented the university in external events. “It’s also something to do with the image because like if we do if we are representing IMU and going to a different public university in Malaysia for example. So if we since we are carrying the name of IMU, if we converse in English, I would say that it will bring a better image to IMU”. Medical students communicated with peers formally and informally in the learning setting and during social interactions. Furthermore, when students were from multilingual backgrounds and nationalities, English lan- guage was also the main communication tool. Vygotsky’s sociocultural theory Given the context which relates to the importance of the English language and the gaps in medical education literature on factors enhancing EL skills, the theoreti- cal framework adopted for this study is the sociocul- tural theory by Vygotsky (1978). This framework posits that an individual’s learning is linked to cultural, institu- tional and historical contexts [12]. The four core tenets of Vygotsky’s sociocultural theory which are (i) learning precedes development, (ii) language is the main vehicle (tool) of thought, (iii) mediation is central to learning and (iv) social interaction is the basis of learning and devel- opment [13]. The tenets can be adapted to the study learning environment ranging from curricula outcomes to teaching and learning methods through social inter- actions. The tenets which initially focused on children’s learning have been extended to include adult learning. The application of this theory to adult learning guides Chan et al. BMC Medical Education (2022) 22:405 Chan et al. BMC Medical Education (2022) 22:405 Chan et al. BMC Medical Education (2022) 22:405 Page 3 of 7 perspectives and contribute further. The interviews were transcribed and coded to identify similarities and differences of perspectives. The saturation was deemed to be achieved when similar and redundant perspec- tives shared by the participants and no new informa- tion were obtained across the focus groups. Research team and reflexivity Th fi h The first research team member is a language teacher who has a background in linguistics. The second mem- ber is trained in educational psychology and teaches in the Health Professions Education Programme. The third team member is a biochemist and medical educationist by training. Throughout the study, the team was actively involved in the research process while at the same time aware of subjectivity due to their different backgrounds. “We have people from various cultural backgrounds and different countries, even we have international students here. So, it’s logical if we speak in English and we converse with each other in English since that that will make the international students feel more comfortable as they will be able to understand what we’re talking.” Page 4 of 7 Chan et al. BMC Medical Education (2022) 22:405 Chan et al. BMC Medical Education (2022) 22:405 Page 4 of 7 “Our lecturer actually encouraged him to speak Eng- lish more.” “Our lecturer actually encouraged him to speak Eng- lish more.” Lack of English proficiency impeded students’ day to day learning. Some students were afraid to ask their lec- turers when unsure of the content taught or participated less in problem based learning discussions. Nonetheless some students faced difficulties learning and communicating in an English speaking environment. “At first I was quite struggling to adapt with English speaking environment.” “It’s hard when you are among with your peers because you need to let them to understand and also the facilitator to understand. Some they don’t know certain my language.” Students from multilingual and multicultural back- grounds with lesser opportunities to communicate socially in English expressed that the English speaking environment came as a shock, as some of them did not have experience of speaking with different ethnicities or nationalities. Therefore, in order to improve their English proficiency and cope with learning, students mentioned that they took initiatives on their own and approached peers for support. Students also perceived that proficiency in EL had an impact on patient communication. Lack of English proficiency may cause the interactions to sound rude, awkward and lead to misunderstanding and errors in instructions given to patients. For sensitive issues such as sexual history, medical students explained that profi- ciency in English is needed when exploring details with- out causing patients’ uneasiness. Most importantly, they felt proficiency helped gain a patient’s confidence when discussing such matters. “If they really don’t know the language, they will come to us and ask us what does it mean… We have to translate it to them, either using their own language, or we use really simple terms to explain it.” “I think communication with patients, that will also be a problem because some people I heard my friends say, they directly translate it from Manda- rin to English. And then sometimes, it might sound wrong, it might sound rude in English.” Besides that, students watched and listened to Eng- lish reading and entertainment materials, online learn- ing tools and registered for English classes outside the university.“I tried to improve myself with a lot of things like, I watch English movies, I listened to English songs. Theme 3 – Individual EL proficiency Students felt that limitations in EL impacted their learn- ing as they needed more time and effort to comprehend the resources. As students were multilingual with varied cultural back- grounds and nationalities, it was necessary to explore university support in mastering the English language. Students felt that the university learning environment with English as a medium of instruction and extra cur- ricular activities helped in strengthening their command of English. There was obvious improvement in English language proficiency for peers who had initially strug- gled with English language after the first semester of the medical programme. “They might actually be speed lagging behind learn- ing like learning the syllabus, I mean, they have to be/ they have to put like extra effort to learn the same thing that we learned/ we have to learn because maybe it’s not in the language that they are comfortable in.” Furthermore, assessments such as assignments, pres- entations, reports and the Objective Structured Clinical Examinations were carried out in English. Hence, having poor command of English had an impact on their assess- ments when examiners were not clear about what was presented. “But what I noticed from them is that after the six months of our semester one their English skills like improve tremendously. And when we asked the person about it, what they said was that it was the talking with all the peers was the one that helped them.” “If you are not so proficient in the language, you will have difficulty trying to structure your points according to paragraphs and making the flow look nice. And because of the inability to do that, then the lecturer, the person marking the paper might feel like, this person is all over the place, he doesn’t really know what he’s writing about.” To support improvement in EL communication skills, students mentioned that lecturers equally encouraged them to communicate in English. Discussionh Additionally, students observed that in both learning and healthcare environments, EL is used in a professional setting amongst healthcare professionals and for patient care. Vygotsky proposed that mediation (a tool) is often used by learners to resolve a problem or achieve a tar- get, and hence language is considered a significant tool for learners to develop the knowledge that they require [17]. In this context, medical students reminded educa- tors that the EL, as a communication tool, was crucial for person-centred care whereby communication skills is crucial for patient engagement and safety of care [21]. This importance as a communication tool is expanded to peers and other health professionals and helps them develop personally and professionally, opening opportu- nities to further their medical education abroad, special- ise or conduct research. The aim of this study was to explore the importance of English language skills in an IMP from medical students’ perspectives. Evidence obtained from the perspectives resonates with Vygotsky’s sociocultural theory, its ten- ets and model that learning is a continuous process and involves multiple factors in promoting the language usage and its importance within the context of medical education and multilingual settings.h The bidirectional elements of culture, thought and lan- guage showed that the importance of EL skills are multi faceted, aiding in developing contextual social and cog- nitive skills in multilingual healthcare settings. This is demonstrated through the emphasis on usage of English during medical training and practice, the influence of university culture and environment on EL mastery and students’ individual EL proficiency.i However, students also identified the challenges faced in acquiring EL skills especially in a multilingual environ- ment and the subsequent impact that may occur from the lack of it. Therefore, they employed various strategies such as getting peer assistance, enrolling in external lan- guage courses to cope with these challenges. Additionally, efforts can be made in the curriculum to support medi- cal students with English learning. At our institution, this was done through the introduction of English Literature and Medicine module as an optional elective for medical students. Whilst learning takes places informally through observations of doctors’ and patients’ interactions and i Medical students identified that they learnt EL skills through participation in facilitated teaching and learn- ing activities such as problem-based learning sessions and simulation and clinical skills training. “Our lecturer actually encouraged him to speak Eng- lish more.” And of course, I tried to gain the courage to speak in Eng- lish with my friends.” During their studies, students were involved in medi- cal research, met with visiting professors from other uni- versities, attended conferences and received training at Page 5 of 7 Page 5 of 7 Chan et al. BMC Medical Education (2022) 22:405 Chan et al. BMC Medical Education (2022) 22:405 medical students to enhance their EL skills. Evidence showed that lecturers emphasised the importance of communication skills and linked it to accuracy in the EL vocabulary. health clinics and wrote case reports. These encounters, which were mainly in English, contributed to the per- sonal and professional development of a future doctor. Students explained that EL is important to review litera- ture, conduct and communicate research. This resulted in opportunities to interact professionally in academia or healthcare at the workplace or when collaborating with international colleagues. Besides that, students acknowledged that they improved their language skills through peer interac- tion during extra curricular activities. Vygotsky empha- sized the dominant effect of social experience on human development was that thoughts developed through social interaction will be internalized through the cultural con- text in which the learner is placed [18]. Therefore, stu- dents will develop thought and knowledge within the community of practice where learning in a second lan- guage context takes place [19]. To this effect, evidence showed that multilingual students who acquired Eng- lish language skills experienced the assimilation pro- cess through peer learning. This correlates with a study conducted among university students from a multicul- tural background in Canada and Russia on the perceived impact of ECA on foreign language learning. Participants of the study indicated that they had more opportunities to practice speaking in English language, learnt more, strengthened their comprehension and developed com- munication skills when participating in extra curricular activities [20]. “Our lecturer actually encouraged him to speak Eng- lish more.” “…eventually when you go out into the working world, when you do literature reviews, when you review other people’s research, when you do your own research, eventually you’re going to have to reach that level of like academic, that sort of aca- demic standard that is required of the community when they do research projects, and you do litera- ture reviews.” “For me usage of English might not be very impor- tant in a community level, but for professional level like from colleagues to colleagues, we still need to use English or professionalism and to discuss any- thing about medical, I think it’s very important to speak in English.” Discussionh These learning activities provide an opportunity for students to commu- nicate with peers, simulated and real patients. Vygotsky puts forward the concept of Zone Proximal Development (ZPD) in which a learner’s level of potential develop- ment is determined through adult guidance or collabora- tive work with more capable peers [17]. Through guided participation, learning and development take place in a social context between learner and teacher [12] allowing Chan et al. BMC Medical Education (2022) 22:405 Chan et al. BMC Medical Education (2022) 22:405 Page 6 of 7 bilingual and monolingual settings can also be investi- gated and compared to identify similarities and differ- ences in medical students’ experiences. discussions of patients’ cases, these interactions are often neglected in clinical reports [22]. Hence in foster- ing medical students’ doctor-patient communication, the module was designed to awaken students’ emotions and awareness toward empathetic behaviours through Eng- lish literary texts. More recently another elective mod- ule for stories and perspective taking was introduced to understand patient care. In a study to explore the effec- tiveness of this newly introduced module for dental stu- dents, the findings showed that it facilitates empathy in students, stimulates their self-awareness and motivates students to be perceptive communicators [23]. In conclusion, various factors come into play during the students’ journey to enhance their EL proficiency. As seen in the results, for the medical students, uni- versity culture and institutional support both for- mally and informally matters as much as students’ self-initiatives and peer support. The context of medi- cal training which includes patient communication, peer interactions and future practice in healthcare also increased the awareness and interest to be pro- ficient in English amongst medical students. Hence, it is imperative that IMPs continue to invest in and broaden approaches both within the formal cur- riculum and extracurricular activities to support and develop medical students’ EL skills. The study findings highlighted the need to continously make available EL enhancement programmes and support structures for medical students. Whilst IMPs already have requirements of English competence prior to entry [24], generic tools such as IELTS or TOEFL, may be insuffi- cient for some students to navigate through medical train- ing. Even at our institution, where EL support is given for students identified through mentor feedback, more can still be done to support students. IMU Joint Committee on Research and Ethics (IMUJC) Project ID No IMU 452/2019. IMU Joint Committee on Research and Ethics (IMUJC) Project ID No IMU 452/2019. This study was limited by the perceptions of medical students of an IMP in a single institution. Furthermore, as it was challenging to encourage students to participate in focus groups, students who participated in the focus groups may not fully represent all IMP students. With the increasing globalisation of higher education and English as the common language of higher education, medical students of multi demographic settings may have similar or different perception of the importance of EL skills. IMU funded the study in terms of its design, the collection, analysis and interpretation of data, the writing of manuscript along with publication fee and study materials (e.g. token of appreciation for participants). The funding approval was obtained during the IMU Joint Committee on Research and Eth- ics (IMUJC) dated 25 July 2019. Availability of data and materials Data were obtained through focus group interviews, provided in Additional file 1: Table S1 and the datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgements g The authors would like to express their heartfelt gratitude to the participants of this study for their valuable input. We would also like to thank Prof Ian Wilson for proof reading the manuscript. Discussionh There are students, as indicated in this study, who would like to gain more social and academic experience in EL to thrive in medical school. One initiative that has been gaining ground is the introduction of humanities to enhance inter disciplinary collaboration, patient communication and care [22]. A study conducted among medical and healthcare students in Taiwan concluded that narrative medicine has positive effects on the students whereby it helped with interdisci- plinary collaboration and enabled them to listen to illness narratives from multiple perspectives [25]. Recommen- dations would be to invest and increase access to these types of programmes, diversify the narratives to be inclu- sive of contemporary culture and arts, and to intergrate these programmes with clinically related modules in the medical curriculum, rather than offer them as stand alone modules. Concordantly, education of medical and nursing students is suggested to include interdisciplinary experi- ences in humanities and its’ relevance to the practice of healthcare providers [26]. Authors’ contributions The research team consisted of education researchers from an international medical institution who were varied in terms disciplinary perspectives and backgrounds. VD contributed to the conception of ideas. NH worked on the qualitative method, SC collected and analyzed the data. VD, NH and SC wrote and edited the manuscript. AND approved the submitted version (and any substantially modified version that involves the author’s contribution to the study); AND agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not person- ally involved, are appropriately investigated, resolved, and the resolution documented in the literature. AND all authors have read and approved the manuscript for submission. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12909-022-03481-w. Additional file 1: Table S1. Sample of Codes. Additional file 1: Table S1. Sample of Codes. 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W2770262852.txt	https://sciforum.net/manuscripts/4684/slides.pdf	en		<strong>Synthesis of Novel <em>N</em>-{[4-(1,2,3-Triazol)pyridin-3-yl]sulfonyl}urea Derivatives With Potential Anticancer Activity</strong>	null	2,017	cc-by	677	Synthesis of Novel N-{[4-(1,2,3-Triazol)pyridin-3-yl]sulfonyl}urea Derivatives With Potential Anticancer Activity Krzysztof Szafrański A, Jarosław A Sławiński , Anna B Kawiak A Department of Organic Chemistry, Medical University of Gdańsk, BDepartment of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk Introduction Diarylsulfonylureas (DSUs) are a group of compounds which, unlike the N-alkyl sulfonylurea derivatives with hypoglycaemic activity, exhibit high potential as antineoplastic compounds [1]. The discovery of the compound LY181984, 181984, followed by the clinical trials of the compound LY186641 (sulofenur), initiated a series of studies on the antitumor activity of sulfonylurea derivatives (Figure 1).. Figure 1. Sulfonylureas with anticancer activity Continuing our previous studies, in which we have shown a significant potency for antitumor activity of 1-(4-substituted pyridine-3-sulfonyl)-3-phenylureas [2], ], we have undertaken the synthesis and evaluation of cytostatic activity of novel series of N-{[4-(1,2,3-triazol)pyridin-3-yl]sulfonyl}urea derivatives. The presence of the 1,2,3-triazole linker at the position 4 of the pyridine ring, which has a significant influence on the antitumor activity [2,3], allows easy functionalization of the basic scaffold by the copper-catalyzed alkyne-azide cycloaddition reaction (CuAAC (CuAAC). Figure 2. Most active previous N-(pyridin-3-yl)sulfonylurea derivative and structure of the target compounds 8-28 Synthesis X N N S R1 R2 compd Ph 4-ClPh 4-BrPh 4-FPh 4-CH3Ph 4-CF3Ph cyclohexane Ph 4-ClPh 4-BrPh 4-FPh 4-CH3Ph 4-CF3Ph cyclohexane Ph 4-ClPh 4-BrPh 4-FPh 4-CH3Ph 4-CF3Ph cyclohexane 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 yield Molecular formula M.W. m.p. [°C] logP* 88% 85% 91% 86% 85% 78% 76% 77% 67% 68% 84% 91% 67% 82% 83% 81% 67% 55% 79% 72% 82% C21H18ClN7O3S C21H17Cl2N7O3S C21H17BrClN7O3S C21H17ClFN7O3S C22H20ClN7O3S C22H17ClF3N7O3S C21H24ClN7O3S C22H21N7O3S C22H20ClN7O3S C22H20BrN7O3S C22H20FN7O3S C23H23N7O3S C23H20F3N7O3S C22H27N7O3S C22H20N6O3S2 C22H19ClN6O3S2 C22H19BrN6O3S2 C22H19FN6O3S2 C23H22N6O3S2 C23H19F3N6O3S2 C22H26N6O3S2 483,93 518,37 562,83 501,92 497,96 551,93 489,98 463,52 497,96 542,41 481,51 477,54 531,51 469,56 480,56 515,00 559,46 498,55 494,59 548,56 486,61 160-162 161-163 174-175 186-188 172-174 164-166 188-190 164-166 167-169 166-168 164-166 134-137 167-169 179-181 167-170 158-162 146-148 164-167 190-192 183-184 167-170 2,32 2,93 3,09 2,46 2,84 3,20 2,29 1,78 2,39 2,55 1,93 2,30 2,66 1,75 3,15 3,76 3,92 3,29 3,67 4,03 2,93 Starting from 4-chloropyridine-3-sulfonamide 1 in the first step, 4- (2-propyn-1yl yl-thio/ amino) pyridine-3-sulfonamides were obtained. Either by previously reported method (a and b on scheme) or by the simple aromatic nucleophilic substitution (method c). Subsequent reaction of alkynes 3 and 4 with 4chloroazide benzene or azidophenylmethane was carried out in standard CuAAC conditions in DMSO and water using copper (II) sulphate and sodium ascorbate to generate Cu+ ions, giving compounds 5-7. The desired, sulfonylurea derivatives 8-28 were obtained by treatment of primary sulfonamides 5-7 with the appropriate isocyanates in dry acetone at r.t. t. in the presence of anhydrous potassium carbonate. The initially formed intermediate potassium salts of type A was acidified with diluted hydrochloric acid to pH 2, to afford products in good yields (55-91%). *Calculated using JChem for Office 17.14.0, 2017, ChemAxon (http://www.chemaxon.com) Biological studies Compounds 8-28 were submitted to test towards their effect on growth of human cancer cell lines: colon cancer HCT-116, breast cancer MCF-7 and cervical cancer HeLa. Cell viability was measured using MTT assay after 72 h of incubation with tested compound in concentration 1 – 100 μM. Preliminary results for compounds 15-18 and 22-25 show their moderate activity (IC50> 100 μM). Literature: [1] Pasello G, et al. Oncologist 2013; 18: 1118–1125. [2]Szafrański K, Sławiński J. Molecules. 2015; 20; 12029–12044. [3]Sławiński J, et al. Eur J Med Chem. 2013;69:701–710. [4]V.R. Avupati et al. 2012, 22, 1031-1035 [5]M.A. El-Sherbeny, et al. Eur. J. Med. Chem., 2010, 45, 689-697. [6]C. Kharbanda et al. Bioorg. Med. Chem. Lett., 2014,24, 5298-5303.; [7]I.M. El-Deeb et al. Eur. J. Med. Chem., 2010, 45, 2516-2530 Table. Percent of growth inhibition (GI%) of human cancer cell lines incubated with tested compounds 1518 and 22-25 in concentration 10 µM. compd. HCT-116 HeLa MCF-7 15 28 34 13 16 20 23 9 17 17 24 11 18 18 21 15 22 17 12 40 23 6 4 21 24 5 9 3 25 1 0 12
https://openalex.org/W2589274676	https://ro.ecu.edu.au/cgi/viewcontent.cgi?article=3617&context=ecuworkspost2013	English	null	Comparison of the HUI3 and the EQ-5D-3L in a nursing home setting	PloS one	2,017	cc-by	5,616	10.1371/journal.pone.0172796 Lung, T., Howard, K., Etherton-Beer, C., Sim, M., Lewin, G., & Arendts, G. (2017). Comparison of the HUI3 and the EQ-5D-3L in a nursing home setting. PloS One, 12(2), Article e0172796. https://doi.org/10.1371/journal.pone.0172796 This Journal Article is posted at Research Online. https://ro.ecu.edu.au/ecuworkspost2013/2611 Follow this and additional works at: https://ro.ecu.edu.au/ecuworkspost2013 Edith Cowan University Edith Cowan University Research Online Research Online Edith Cowan University Edith Cowan University Research Online Research Online Edith Cowan University Edith Cowan University Research Online Research Online Comparison of the HUI3 and the EQ-5D-3L in a nursing home Comparison of the HUI3 and the EQ-5D-3L in a nursing home setting setting See next page for additional authors Follow this and additional works at: https://ro.ecu.edu.au/ecuworkspost2013 Part of the Geriatrics Commons 10.1371/journal.pone.0172796 Lung, T., Howard, K., Etherton-Beer, C., Sim, M., Lewin, G., & Arendts, G. (2017). Comparison EQ-5D-3L in a nursing home setting. PloS One, 12(2), Article e0172796. https://doi.org/10.1371/journal.pone.0172796 This Journal Article is posted at Research Online. https://ro.ecu.edu.au/ecuworkspost2013/2611 Follow this and additional works at: https://ro.ecu.edu.au/ecuworkspost2013 Part of the Geriatrics Commons Part of the Geriatrics Commons Part of the Geriatrics Commons Authors Authors Tom Lung, Kirsten Howard, Christopher Etherton-Beer, Moira Sim, Gill Lewin, and Glenn Arendts m Lung, Kirsten Howard, Christopher Etherton-Beer, Moira Sim, Gill Lewin, and Glenn Arendt Authors Authors Tom Lung, Kirsten Howard, Christopher Etherton-Beer, Moira Sim, Gill Lewin, and Glenn Arendts This journal article is available at Research Online: https://ro.ecu.edu.au/ecuworkspost2013/2611 Authors Authors This journal article is available at Research Online: https://ro.ecu.edu.au/ecuworkspost2013/2611 RESEARCH ARTICLE Results Both measures were completed for 199 residents at baseline and 177 at 6-month follow-up. Mean baseline utility scores for EQ-5D-3L (0.45; 95% CI 0.41–0.49) and HUI3 (0.15; 95% CI 0.10–0.20) were significantly different (Wilcoxon signed rank test, p<0.01) and agree- ment was poor to moderate between absolute scores from each instrument (intra-class cor- relation coefficient = 0.63). The EQ-5D-3L appeared more sensitive to change over the 6- month period. Data Availability Statement: Due to ethical restrictions, the data used for this study is only available upon request. Currently, ethics approval with the University of Western Australia Human Research Ethics Committee (RA/4/1/5123) does not allow any form of data as part of this study to be released into the public domain. Data are available from the University of Western Australia Human Research Ethics Committee (RA/4/1/5123) for researchers who meet the criteria for access to confidential data. Readers with questions about these restrictions or who wish to access the data Tom Lung1,2☯, Kirsten Howard2☯, Christopher Etherton-Beer3☯, Moira Sim4☯, Gill Lewin5☯, Glenn Arendts6☯ Tom Lung1,2☯, Kirsten Howard2☯, Christopher Etherton-Beer3☯, Moira Sim4☯, Gill Lewin5☯, Glenn Arendts6☯ 1 The George Institute for Global Health, University of Sydney, Sydney, New South Wales, Australia, 2 Sydney Medical School, School of Public Health, University of Sydney, Sydney, New South Wales, Australia, 3 School of Medicine and Pharmacology Royal Perth Hospital Unit, The University of Western Australia, Crawley, Western Australia, Australia, 4 School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia, 5 School of Nursing, Midwifery and Paramedicine, Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia, 6 Emergency Medicine, School of Primary, Aboriginal and Rural Health Care, The University of Western Australia, Crawley, Western Australia, Australia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. * tlung@georgeinstitute.org.au ☯These authors contributed equally to this work. * tlung@georgeinstitute.org.au * tlung@georgeinstitute.org.au Editor: Nan Luo, National University of Singapore, SINGAPORE Editor: Nan Luo, National University of Singapore, SINGAPORE Methods EQ-5D-3L and HUI3 scores were measured as part of a cluster randomised controlled trial of nurse led care coordination in a nursing home population in Perth, Western Australia at baseline and 6-month follow up. Copyright: © 2017 Lung 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. Background Citation: Lung T, Howard K, Etherton-Beer C, Sim M, Lewin G, Arendts G (2017) Comparison of the HUI3 and the EQ-5D-3L in a nursing home setting. PLoS ONE 12(2): e0172796. doi:10.1371/journal. pone.0172796 Accurately assessing changes in the quality of life of older people living permanently in nurs- ing homes is important. The multi-attribute utility instrument most commonly used and rec- ommended to assess health-related quality of life in the nursing home population is the three-level EuroQol EQ-5D-3L. To date, there have been no studies using the Health Utili- ties Index Mark III (HUI3). The purpose of this study was to compare the level of agreement and sensitivity to change of the EQ-5D-3L and HUI3 in a nursing home population. ☯These authors contributed equally to this work. * tlung@georgeinstitute.org.au Introduction The growth in the proportion of older people [1] and those who live permanently in a nurs- ing home requiring complex care has implications both for health service delivery and the increase in demand of expenditure in order to meet this need [2]. Health-related quality-of- life (HRQoL), measured using a multi-attribute utility instrument (MAUI), provides a sum- mary score that estimates quality of life (QoL). These scores, in combination with survival estimates, are typically used in health economic evaluations of different interventions to esti- mate quality adjusted life years (QALYs) [3]. Two widely used and accepted MAUIs in economic evaluations are the Health Utilities Index Mark 3 (HUI3) and the EuroQol EQ-5D-3L [4, 5], both of which describe health states in terms of health attributes or health dimensions. A recent systematic review of instruments for measuring outcomes in economic evaluation identified a limited number of studies using MAUIs in an aged care setting [6]. All 7 studies identified used the self-reported EQ-5D-3L instrument and no studies used the HUI3 questionnaire. Four further studies used three mea- sures which encapsulated dimensions outside of health (The Adult Social Care Outcome Toolkit (ASCOT); the ICEpop CAPability measure for Older people (ICECAP-O); and the World Health Organization Quality of Life old (WHOQOL-Old)). Currently, the EQ-5D-3L is the recommended MAUI to use when conducting economic evaluations for health technology assessments in the UK [7] whilst for older people, in addition to the EQ-5D-3L the use of either the ICECAP-O or ASCOT is recommended in order to facil- itate the measurement and valuation of both QALYs and broader quality of life dimensions of importance to the specific population [6, 8, 9]. However, there is no strong rationale for choos- ing the EQ-5D-3L over other MAUIs; indeed there has been concern about the EQ-5D-3L having significant limitations in older people whereby small differences in health status can result in very large changes in QOL scores [10]. Differences between the EQ-5D-3L and the HUI3 have been noted in other settings [11–13], but not in a nursing home setting. To appropriately interpret summary HRQoL scores based on the HUI3 or EQ-5D-3L in a nursing home population, a comparison between the values and properties of these instru- ments is needed. Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting considering the implications of the cost-effectiveness of particular interventions and we con- clude that the HUI3 is no better suited to measuring health-related quality of life in a nursing home population when compared to the EQ-5D-3L. considering the implications of the cost-effectiveness of particular interventions and we con- clude that the HUI3 is no better suited to measuring health-related quality of life in a nursing home population when compared to the EQ-5D-3L. may contact Mr. Peter Carl Miller (Human Ethics Officer) Email: peter.miller@uwa.edu.a. Funding: This work was funded by the JO and JR Wicking Trust to GA. TL receives salary support as the HCF Research Foundation Postdoctoral Research Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Introduction In this study, we use a nursing home population from a cluster controlled clinical trial in Australian nursing homes to assess HRQOL and compare agreement and sensi- tivity to change between the HUI3 and EQ-5D-3L. Materials and methods Health-related quality of life measures Conclusion Our findings show that the EQ-5D-3L and HUI3 estimate different utility scores among nurs- ing home residents. These differences should be taken into account, particularly when 1 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Health-related quality of life measures The EQ-5D-3L questionnaire consists of a Visual Analog Scale (VAS) and five dimensions of health: mobility, self-care, daily activities, pain/discomfort and anxiety/depression [4, 5]. Each dimension has three response levels, yielding a possible 243 unique health states. These prefer- ences are summarised into a utility score that ranges from -0.59 to 1.0 using the time trade-off method and we use Australian derived EQ-5D-3L weights in this study [14]. 2 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting The HUI3 has eight dimensions of health (vision, hearing, speech, ambulation, dexterity, emotion, cognition, and pain/complaints) and each dimension has five or six response levels, resulting in 972,000 possible unique health states. Preference-based scoring algorithms con- verted the descriptive health classifications into values for each health dimension [15, 16]. The health dimensions are designed to be structurally independent, and a multi-attribute model using the standard gamble method derives a utility score that ranges from -0.36 to 1.0 [16]. Study population and data collection Nursing home residents were recruited to a multicentre open label cluster controlled trial of nurse led care coordination in Perth, Western Australia (Australian and New Zealand Clinical Trials Registry number 12611000933954). In brief, individuals were eligible for the study if they were 65 year or older, eligible for Medicare and a permanent high level care resident in an accredited facility [17]. The EQ-5D-3L and HUI3 questionnaires were administered at baseline and 6-month follow-up, and co-morbidities were measured using the Charlson co-morbidity index [18]. Ethics approval was obtained from the University of Western Australia Human Research Ethics Committee (RA/4/1/5123). Written informed consent was obtained from all individual participants and for cognitively impaired individuals incapable of providing informed consent, provision for a written waiver of consent was granted with next of kin acknowledgement. This was directed by the supervising ethics committee and the national statement on Ethical Conduct in Human Research (‘‘National Statement on Ethical Conduct in Human Research”, 2007). Cognitive status and disability of the resident (assessed as part of the study) determined whether they were able to self-populate the questionnaires. For individuals cognitively capable but unable (due to disability) to populate the questionnaires, a recruiter recorded resident responses at the bedside. Cognitively unable residents had a nominated proxy medical decision maker complete an informant questionnaire. Questionnaires were administered at enrolment (baseline) and at six and twelve-month follow-up. This study analyses only fully completed questionnaires at baseline and after 6 months. For the purposes of this study, we combined both intervention and control groups in the analysis to conduct a cohort analysis of the utility scores. All analysis was conducted using Stata SE version 14.0 (StataCorp LP, College Station, TX, USA). PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Results For the 200 residents in the study, 199 EQ-5D-3L and HUI3 questionnaires were completed at baseline. Residents had a mean age of 85 years and 25% were men. By six months’ follow-up, 22 residents had died and were excluded from the analysis as they did not complete a MAUI questionnaire, leaving 177. Characteristics of the study population at baseline and 6-month follow-up are reported in Table 1. Sensitivity to change The sensitivity of an instrument to changes in health was examined by calculating effect sizes. Effect size is classified as the difference between mean scores at baseline and 6-month follow- up divided by the standard deviation of baseline values. Effect sizes were defined as: small (0.20), moderate (0.50) and large (>0.80) [19]. Paired t-tests were conducted for baseline and 6-month follow-up utility values for EQ-5D-3L and HUI3, respectively to determine whether utility values changed over the observation period. Agreement between instruments Comparisons were made at baseline between mean HUI3 and EQ-5D-3L scores by sex, num- ber of co-morbidities, age, treatment group and method of completing the questionnaires (self-completed versus informant-completed) using the Mann-Whitney U test. The distribu- tions of the absolute values of the two instruments were tested individually for normality (Shapiro-Wilk test) and whether the two distributions were equal using the two-sample Kol- mogorov-Smirnov test. To assess agreement between the utility values from the two instru- ments, a Wilcoxon signed rank test was used for all sub-groups. Kendall’s tau was computed to determine whether there was correlation between the dimensions of the EQ-5D-3L and HUI3: mobility (EQ-5D-3L) and ambulation (HUI3); pain/ complaints (EQ-5D-3L) and pain (HUI3); and anxiety/depression (EQ-5D-3L) and emotion (HUI3). Correlation scores were classified into three groups: 0.1–0.3 was considered small, 0.3–0.5 as medium and >0.5 interpreted as a large correlation [19]. Intra-class correlation (ICC) estimates the proportion of variance from both instruments’ scores attributable to differences between individuals, as opposed to differences between the two instruments [20, 21]. A two-way fixed mixed effect model was computed, where the PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 3 / 10 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting instrument effect was fixed and the individual effect was random. ICC values close to 1 sug- gests that the instruments are similar in terms of agreement [22]. We used Bland-Altman [23, 24] plots to graph the difference between an individual’s EQ- 5D-3L and HUI3 value against the mean of the two scores, which summarises the level of agreement between the two methods. PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting Table 2. Mean utility scores and standard deviations of the study population at baseline (n = 199) and 6-month (n = 177) follow-up for EQ-5D-3L and Health Utilities Index Mark 3 (HUI3). Characteristics Mean baseline scores (SD) Mean 6 month scores (SD) EQ-5D-3L HUI3 EQ-5D-3L HUI3 Overall 0.45 (0.02) 0.15 (0.02) 0.44 (0.02) 0.16 (0.03) Age<85 years 0.44 (0.03) 0.15 (0.04) 0.41 (0.03) 0.15 (0.04) Age85 years 0.46 (0.03) 0.15 (0.03) 0.46 (0.03) 0.17 (0.03) Male 0.45 (0.05) 0.17 (0.05) 0.44 (0.05) 0.18 (0.05) Female 0.45 (0.03) 0.14 (0.03) 0.44 (0.03) 0.15 (0.03) Charlson Comorbidity Index = 0 0.45 (0.04) 0.14 (0.04) 0.45 (0.04) 0.15 (0.04) Charlson Comorbidity Index>0 0.45 (0.03) 0.16 (0.03) 0.43 (0.03) 0.17 (0.03) Control Group 0.44 (0.03) 0.09* (0.03) 0.44 (0.03) 0.10* (0.03) Intervention 0.46 (0.03) 0.21* (0.04) 0.44 (0.04) 0.21* (0.04) Informant-completed 0.28* (0.03) -0.05* (0.03) 0.25* (0.04) -0.04* (0.03) Self-completed 0.53* (0.03) 0.25(0.03) 0.53 (0.03) 0.25* (0.03) Represents a signiﬁcant (p<0.05) Mann-Whitney test which rejects the null hypothesis that mean utility scores of sub-groups are from populations with the same distribution. able 2. Mean utility scores and standard deviations of the study population at baseline (n = 199) and 6-month (n = 177) follow-up for EQ-5D-3L nd Health Utilities Index Mark 3 (HUI3). ndard deviations of the study population at baseline (n = 199) and 6-month (n = 177) follow-up for EQ-5D-3L UI3) Table 2. Mean utility scores and standard deviations of the study population at baseline (n = 199) and 6-mon and Health Utilities Index Mark 3 (HUI3). Represents a signiﬁcant (p<0.05) Mann-Whitney test which rejects the null hypothesis that mean utility scores of sub-groups are from populations with the same distribution. Represents a signiﬁcant (p<0.05) Mann-Whitney test which rejects the null hypothesis that mean utility scores of s same distribution. test) was strongly rejected (p<0.01). The distribution of scores at 6 months are similar to those at baseline (S2 Fig). Wilcoxon signed rank tests strongly rejected the null hypothesis of equality between individual’s EQ-5D-3L and HUI3 utility scores at both baseline and 6 months’ follow- up in all sub-groups listed in Table 1. At baseline, the majority of HUI3 scores (87%) were lower than the corresponding EQ-5D-3L score. Large positive correlations (S1 Table) were found between the dimensions Pain/Complaints (EQ-5D-3L) and Pain (HUI3) (Kendall’s tau = 0.51), moderately positive correlation between mobility (EQ-5D-3L) and ambulation (HUI3) (Kendall’s tau = 0.40) and a small positive corre- lation was found between anxiety/depression (EQ-5D-3L) and Emotion (HUI3). All correla- tions were strongly statistically significant (p<0.01). The ICC coefficient was 0.63 (95% CI 0.54–0.71), implying a poor to moderate level of agreement between the two instruments (S2 Table). Fig 1 depicts a Bland-Altman plot at base- line, showing a mean difference of 0.3 between EQ-5D-3L and HUI3 scores. The scatterplot shows that as mean utility values increase, the difference of values between the two instruments decreases. Sensitivity to change Effect sizes between baseline and 6-month follow-up for EQ-5D-3L (0.19) and HUI3 (0.02) suggested that the EQ-5D-3L was more sensitive than HUI3 to changes over a 6-month fol- low-up. Paired t-tests between baseline and 6 months’ follow-up strongly rejected the null hypothesis that the mean difference between EQ-5D-3L scores was greater than zero (p<0.02), suggesting a deterioration of EQ-5D-3L utility values over the observation period. No statisti- cally significant differences were found between baseline and 6-month follow-up HUI3 values. Agreement between instruments The mean utility scores from both instruments measured at both baseline and 6-month fol- low-up are reported in Table 2. Overall, at baseline the mean scores for EQ-5D-3L and HUI3 were estimated at 0.45 (range -0.24 to 1) and 0.15 (range -0.37 to 1), respectively. Significant differences in mean utility scores were found between individuals who self-completed and those who required an informant to complete their questionnaire for both EQ-5D-3L and HUI3 scores at baseline (p<0.01) and 6 months (p<0.01). At both baseline (p<0.03) and 6-month follow-up (p<0.05), significant differences were found between the intervention and control groups for the HUI3 but not EQ-5D-3L scores. At baseline, the EQ-5D-3L distribution shows positive skewness whereas the HUI3 distri- bution shows negative skewness (S1 Fig). The assumption of normality for both utility scores (Shapiro-Wilk test) and equality of the two distributions (Two-sample Kolmogorov-Smirnov Table 1. Study characteristics of the population at baseline and 6-month follow-up. Table 1. Study characteristics of the population at baseline and 6-month follow-up. Characteristics Baseline (n = 199) 6-month follow-up (n = 177) Age (SD) 85.1 (8.9) 84.6 (9.0) Male (%) 49 (24.6%) 40 (22.6%) Female (%) 150 (75.4%) 137 (77.4%) Charlson Comorbidity Index = 0 (%) 71 (35.7%) 63 (35.6%) Charlson Comorbidity Index1 (%) 128 (64.3%) 114 (64.4%) Control Group (%) 99 (49.7%) 90 (50.9%) Intervention (%) 100 (50.3%) 87 (49.2%) Informant-completed (%) 66 (33.2%) 56 (31.6%) Self-completed (%) 133 (66.8%) 121 (68.4%) *SD: Standard Deviation doi:10.1371/journal.pone.0172796.t001 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 4 / 10 Discussion This is the first study that has compared two commonly used MAUIs, the EQ-5D-3L and HUI3, in a nursing home setting. We investigated agreement and sensitivity to change for util- ity scores based on the EQ-5D-3L and HUI3, providing insight into the differences between these two instruments for people living in a nursing home. 5 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting Fig 1. Bland-Altman plot of mean EQ-5D-3L versus HUI3 utility values and the difference between the two utility values at baseline (n = 199). doi:10.1371/journal.pone.0172796.g001 Fig 1. Bland-Altman plot of mean EQ-5D-3L versus HUI3 utility values and the difference between the two utility values at baseline (n = 199). doi:10.1371/journal.pone.0172796.g001 doi:10.1371/journal.pone.0172796.g001 doi:10.1371/journal.pone.0172796.g001 At the individual level, the intra-class correlation coefficients and the Bland-Altman plot reveals a poor to moderate level of agreement between the two instruments, however moderate and small positive correlations exist between some health dimensions. EQ-5D-3L utility scores were considerably higher than corresponding HUI3 scores, which corroborates with non- nursing home population literature [11–13]. The multiplicative scoring function of the HUI3 assumes that all dimensions are dependent on one another, a possible explanation of the lower utility values estimated by the HUI3 in this nursing home cohort where polymorbidity is common. Only EQ-5D-3L scores showed a statistically significant difference between baseline and follow-up. One reason for the larger deterioration in utility values when using the EQ-5D-3L is the smaller number of health states available, so a slight change in one health dimension from no or moderate impairment at baseline to severe impairment at follow-up could result in a relatively large decrement in health utility values. The nursing home population poses considerable challenges for researchers using MAUIs. The two main areas of difficulty are a necessity to use proxy/informant measures of QoL in a large proportion of the population and the applicability of instruments for a population that, by definition, has lost the capacity to live independently in the community. When compared to self-completed scores, informant ratings of QoL are consistently lower in nursing home [25] and elderly community dwellings [26, 27], findings replicated in this study for both MAUIs. It is important to stress that broader elements of QoL (i.e. PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Discussion privacy, dignity, autonomy, etc.) [28, 29] as defined by elderly nursing home residents should be measured and valued alongside a MAUI in order to capture all potential benefits of an intervention. Compared to the seven studies that used EQ-5D-3L scores in an aged care setting [6], we found a similar baseline mean EQ-5D-3L score of 0.45 (SD = 0.32) and within the 95% confidence interval of all but one of the reported studies [30–33]. As this is the first study that used the HUI3 in a nursing home setting, future comparison and validation must be conducted to determine whether similar utility values are estimated. 6 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting A strength of this study was the 100% completion rate for those alive at 6-month follow- up for both MAUIs, which enabled an unbiased examination of change in HRQoL scores and their change over time. A limitation of the present study was our inability to examine whether there were differences in utility values according to the order in which instruments were administered. In all subjects, the EQ-5D-3L was administered first, followed by the HUI3. Changes over time in questionnaire administration mode (i.e. self-completion at base- line to proxy/informant at 6-month follow-up or vice versa) were not captured, which may have influenced the variation in mean scores between baseline and 6-month follow-up. We did not measure cognitive burden of the two questionnaires, which can be an important con- sideration in MAUI assessment in older people. The HUI3 contains up to 41 questions, which is substantially longer than the EQ-5D-3L (5 questions). One study comparing the Assessment of Quality of Life (AQoL) (15 questions) to the EQ-5D-3L in an aged population found that participants were more burdened by the AQoL and consequently found it more difficult to complete [10]. Unlike the EQ-5D-3L, the HUI3 is not based upon preferences of the Australian general population, which could account for some differences seen between the instruments in our results. However, the HUI3 has been widely applied in Australian contexts and appears to behave consistently with other multi-attribute utility instruments, despite the non-Australian based scoring system [34–37]. Conclusions In conclusion, there were significant differences when comparing EQ-5D-3L and HUI3 scores in a nursing home population. Over a 6-month period the EQ-5D-3L appears to be more sen- sitive to changes in health status. Overall, the HUI3 appears no better suited than the EQ-5D- 3L in this context. Further studies are required to determine the appropriateness of the HUI3 in this population, particularly given the cognitive burden when compared to the EQ-5D-3L. Discussion Overall, this study has demonstrated that the EQ-5D-3L appears to be more sensitive to changes over a 6-month period compared to the HUI3, yet there appears to be a low level of agreement between the two. Due to the potential cognitive burden of the HUI3, we conclude that the HUI3 appears no better suited to measuring HRQoL in a nursing home setting than the EQ-5D-3L. Given the increasing use of the EQ-5D-5L (with 5 response levels instead of three level in each QOL domain), additional confirmatory studies may be needed. Supporting information S1 Fig. Distribution of EQ-5D-3L and HUI3 scores at baseline. (TIF) S2 Fig. Distribution of EQ-5D-3L and HUI3 scores at 6-month follow-up. (TIF) S2 Fig. Distribution of EQ-5D-3L and HUI3 scores at 6-month follow-up. (TIF) S3 Fig. Bland-Altman plot of mean EQ-5D-3L versus HUI3 utility values and the differ- ence between the two utility values at 6-month follow-up (n = 177). (TIF) S1 Table. Kendall’s tau to determine the agreement between the health state descriptions. (DOCX) S2 Table. Intra-class coefficient two-way fixed effects model estimating the level of consis- tency between the two instruments. (DOCX) 7 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting Writing – review & editing: TL KH CEB MS GL GA. Writing – review & editing: TL KH CEB MS GL GA. Acknowledgments The authors acknowledge the assistance of the Amana Living and Juniper groups where the research was conducted. PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 Funding acquisition: KH CEB MS GL GA. Investigation: TL GA KH. Project administration: CEB MS GL GA. Software: CEB MS GL GA. Supervision: GA KH. Supervision: GA KH. Validation: TL GA KH. Validation: TL GA KH. Visualization: TL GA KH. Visualization: TL GA KH. Writing – original draft: TL. Writing – review & editing: TL KH CEB MS GL GA. References PMID: 16224300 13. Maes IH, Joore MA, Cima RF, Vlaeyen JW, Anteunis LJ. Assessment of health state in patients with tin- nitus: a comparison of the EQ-5D and HUI mark III. 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The agreement between proxy and self-completed EQ-5D for care home residents was better for index scores than individual PLOS ONE \| DOI:10.1371/journal.pone.0172796 February 24, 2017 9 / 10 Comparison of the Health Utilities Index Mark 3 and the EuroQol EQ-5D-3L in a nursing home setting domains. Journal of clinical epidemiology. 2014; 67(9):1035–43. Epub 2014/05/20. doi: 10.1016/j. jclinepi.2014.04.005 PMID: 24837298 domains. Journal of clinical epidemiology. 2014; 67(9):1035–43. Epub 2014/05/20. doi: 10.1016/j. jclinepi.2014.04.005 PMID: 24837298 32. Wolfs CA, Dirksen CD, Kessels A, Willems DC, Verhey FR, Severens JL. Performance of the EQ-5D and the EQ-5D+C in elderly patients with cognitive impairments. Health and quality of life outcomes. 2007; 5:33. Epub 2007/06/16. doi: 10.1186/1477-7525-5-33 PMID: 17570832 33. Makai P, Brouwer WB, Koopmanschap MA, Nieboer AP. 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https://openalex.org/W4206844983	https://hal.univ-grenoble-alpes.fr/hal-01878009/file/Haass2015.pdf	English	null	Silicon etching in a pulsed HBr/O−2 plasma. I. Ion flux and energy analysis.	HAL (Le Centre pour la Communication Scientifique Directe)	2,015	cc-by	8,295	To cite this version: Moritz Haass, Maxime Darnon, Gilles Cunge, Olivier Joubert, David Gahan. Silicon etching in a pulsed HBr/O–2 plasma. I. Ion flux and energy analysis. Journal of Vacuum Science and Technology, 2015, 33 (3), pp.032202. 10.1116/1.4917230. hal-01878009 Distributed under a Creative Commons Attribution 4.0 International License I. INTRODUCTION The advantage of this kind of probe is that it is almost unaf- fected by deposited layers on its surface. The second probe is a retarding ﬁeld analyzer (RFA)18,19 that gives information on the ion ﬂux and the ion velocity distribution function (IVDF). The focus of the experiments lies on the impact of the pulse parameters. The continuous downscaling of microelectronic devices imposes increasing demands on the plasma processes in terms of etch homogeneity, proﬁle tolerance, surface damage, etch materials, throughput and reproducibility. Especially the plasma-induced damage becomes difﬁcult to control since the tolerance range for deformations and surface alterations decreases permanently. Currently, the traditional ways for process optimization to meet the increasing demands reach their limits. New strategies to overcome these limiting factors are needed and innovative approaches are being developed. One of these is the use of pulsed high-density plasmas. In this case, the RF for inductively coupled plasma (ICP) plasma generation and the optional additional RF for ion acceleration (biasing of the substrate) are pulsed in various modes: syn- chronously (source and bias) at various frequencies with ad- justable phase lags and duty cycles, or independently, e.g., with one generator in continuous wave (CW) mode. p p In Paper II,20 the presented results are used to explain the strong impact of plasma pulsing on actual pattern transfer. Although HBr/O2 plasmas are commonly used in the semi- conductor industry, their composition has hardly been studied. Pargon et al.21 investigated the ion composition by mass spec- trometry for an HBr/O2 (70/30 sccm) plasma and found that H3Oþ was the most common ion that was detected. Cunge et al.22 studied the ion ﬂux composition of HBr/O2/Cl2 plas- mas. However, due to the additional Cl2, it is difﬁcult to derive results for a pure HBr/O2 plasma. Furthermore, the impact of plasma pulsing on HBr plasmas (without O2) has been studied, showing that the duty cycle controls the plasma chemistry: as the duty cycle is reduced the densities of molec- ular species (HBr, Br2) rise while the density of atomic radi- cals (Br) drops.23 To our knowledge, the neutral composition of an HBr/O2 plasma has not been studied in detail yet. a)Electronic mail: Maxime.Darnon@USherbrooke.ca I. INTRODUCTION Several authors reported process improvements due to decreased charging effects,1–6 reduced surface damage,7,8 lower ion energies,6,9,10 reduced UV ﬂux,11,12 increased plasma uniformity,13,14 and an improved selectivity.10,15 Although explanations for the individual observations have been proposed, a complete picture of the fundamental processes that lead to the observed results is still missing. The knowledge of the impact of the plasma pulsing parame- ters on the plasma chemistry and ion ﬂux and energy is indis- pensable to fully identify the potential of pulsed plasmas and to transfer the already published results to other plasma chemistries and applications. Silicon etching in a pulsed HBr/O2 plasma. I. Ion flux and energy analysis Moritz Haass, Maxime Darnon,a) Gilles Cunge, and Olivier Joubert LTM (CNRS/UJF-Grenoble1/CEA), 17 Avenue des Martyrs, 38054 Grenoble Cede David Gahan Impedans Ltd., Unit 8 Woodford Court, Woodford Business Park, Santry, Dublin 17, Ireland (Received 29 September 2014; accepted 17 March 2015; published 9 April 2015) (Received 29 September 2014; accepted 17 March 2015; published 9 April 2015) The ion ﬂux and ion velocity distribution function are studied using a capacitively coupled radio frequency ion ﬂux probe and a multigrid retarding ﬁeld analyzer in an HBr/O2 pulsed plasma process, dedicated to silicon etching in gate or shallow trench isolation applications. A decrease of the duty cycle of the pulsed plasma etch process affects strongly these parameters: the mean ion ﬂux decreases almost by the square of the duty cycle. Furthermore, the contribution of high energy ions from the on-time of the plasma is strongly reduced while their energy is slightly increased. In average, this leads to a signiﬁcantly reduced ion energy and a reduced ion ﬂux in the pulsed plasma compared to the continuous wave plasma. V C 2015 American Vacuum Society. [http://dx.doi.org/10.1116/1.4917230] HAL Id: hal-01878009 https://hal.univ-grenoble-alpes.fr/hal-01878009v1 Submitted on 1 Sep 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 032202-1 J. Vac. Sci. Technol. B 33(3), May/Jun 2015 2166-2746/2015/33(3)/032202/8/$30.00 V C 201 032202-1 J. Vac. Sci. Technol. B 33(3), May/Jun 2015 2166-2746/2015/33(3)/032202/8/$30.00 V C 2015 American Vacuum Society 032202-1 2166-2746/2015/33(3)/032202/8/$30.00 V C 2015 American Vacuum Society 032202-1 II. EXPERIMENTAL SETUP In this paper, we investigate HBr and HBr/O2 plasmas used for silicon etching in continuous wave and pulsed condi- tions. A baseline process with 200 sccm HBr, 750 W of source power, and a pressure of 20 mT is chosen. This corresponds to typical process conditions for silicon etching in the semicon- ductor industry. For the analysis of HBr/O2 plasma, 5 sccm of O2 is added. When silicon is etched, an additional 200 W of bias power are used. In pulsed conditions, both source and bias power supplies are pulsed simultaneously. Standard con- dition is 1 kHz for the pulsing frequency while the duty cycles varies between 10% and 75%. In a series of two articles, we investigate the impact of plasma pulsing on an HBr/O2 silicon etch process, dedicated to gate, ﬁn or shallow trench etching. In this paper, the ion ﬂux and energy is studied by using two different probes. The ﬁrst one is a capacitively coupled RF probe16,17 that measures the ion ﬂux at the reactor walls. In the ﬁrst part of the paper, we analyze the ion ﬂux with- out bias power in CW plasma or in pulsed conditions with a pulsing frequency of 1 kHz and a duty cycle varying between 032202-1 J. Vac. Sci. Technol. B 33(3), May/Jun 2015 2166-2746/2015/33(3)/032202/8/$30.0 ol. B 33(3), May/Jun 2015 2166-2746/2015/33(3)/032202/8/$30.00 V C 2015 American Vacuum Society 032202- 032202-1 J. Vac. Sci. Technol. B 33(3), May/Jun 2015 2166-2746/2015/33(3)/032202/8/$30.00 V C 2015 American Vacuum Society 032202-1 2166-2746/2015/33(3)/032202/8/$30.00 032202-2 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma 032202-2 032202-2 sufﬁciently negative with respect to the plasma potential (several 10 V), the electron ﬂux Ce will be negligible com- pared to the ion ﬂux Ci on the probe surface and the blocking capacitor is therefore going to discharge by collecting the ion Bohm ﬂux from the plasma. 10% and 75%. Then, we analyze the impact of the bias power on the ion ﬂux and energy in continuous wave plas- mas. Ion ﬂux and ion energy are then analyzed in pulsed plasma for a constant pulsing frequency of 1 kHz and vari- ous duty cycles, and with constant duty cycles of 20%, 50%, or 75% and changing frequencies. A. Ion flux probe The ion ﬂux is a crucial parameter for etching processes. In this study, a planar ion ﬂux probe (IFP), developed by Braithwaite et al.16 and Booth et al.17 and recently adapted to pulsed plasma measurements,25 was used to directly measure the ion ﬂux onto its surface. The probe itself consists of a large area (1cm2) planar single-sided disk, which is positioned in plane to the plasma reactor wall about 5 cm above the wafer support. A guard ring with the same surface is placed around the probe to avoid edge effects of the sheath (see Fig. 1). II. EXPERIMENTAL SETUP For ion energy analysis in pulsed plasmas, the plasma conditions were modiﬁed to maximize the signal to noise ratio and limit the ion energy to less than 400 eV (maximum energy acceptable by our RFA system), i.e., the source power was increased to 1200 W, the pressure decreased to 10 mTorr, and the bias power was lim- ited to 60 W. The ion induced current is measured directly over a resis- tor that is placed in parallel with two head-to-tail diodes in between the blocking capacitor and the RF generator. The potential drop is measured with a high resolution analog- digital converter from National Instruments at a sample rate of 1.2 MHz with a maximum voltage input of 10 V. When the probe pulsed RF power supply is correctly synchronized with the pulsed plasma RF power supply, this technique can be used to study pulsed plasma dis- charges.25,26 In this case, the time during which the probe discharges needs to be at least as long as one entire period of the plasma RF pulse. The full details on the application of the planar probe for pulsed plasmas analysis are explained elsewhere.25 The experiments are conducted in a commercially avail- able 300 mm AdvantEdgeTM DPS etch tool from Applied Materials, Inc. The plasma is sustained by two ICP coils (source), powered at 13.56 MHz, while the chuck is biased via a capacitively coupled RF power source (bias), likewise working at 13.56 MHz. The power supply is modiﬁed with the PulsyncTM system to allow pulsing of the source and bias power with frequencies between 100 and 20 kHz and duty cycles between 10% and 90%. More details about the etch reactor can be found in the literature.24 The error bar for the ion ﬂux measurement is considered to be below 5%. This mostly comes from nonperfect edge effect suppression by the guard ring and RF noise in the measurement system. Additional errors from the electronic circuit may lead to systematic errors of a few percent. However, these errors do not change the relative variations observed on the measurement and are not included in the error bars. B. Retarding field analyzer An alternative diagnostic tool to measure the ion ﬂux, but in addition the IVDF, is a RFA. In this article, several proto- types of the commercially available SEMION probe from Impedans, Ltd., were used.18,19 In Fig. 2(a), the schematic of such a four grid analyzer is shown. A pulsed RF source is coupled to the probe and the guard ring via capacitors. Thanks to this blocking capacitor Cb, a dc self bias (Vbias) can develop on the probe surface when it is fed with RF power. This effect is equivalent to the self- bias effect of the powered electrode in a capacitively coupled plasma. The ﬁrst grid G1 is a ﬂoating grid to simulate the potential of the ﬂoating wall or the wafer. If the RFA is positioned on a biased wafer, the self-bias potential is transmitted via the metallic frame to G1. The second grid G2, called the discrim- inator grid, is biased with a potential ramp to discriminate the ion energy. The third grid G3 functions as a suppression grid to repel electrons from the plasma and to prevent sec- ondary electrons from the ion bombardment of the collector to escape. The fourth grid (or plate) C is the collector. The current from the incoming ion ﬂux is measured at each step of the potential ramp. After the RF voltage is turned off, the accumulated charge on the plasma side of the capacitor remains. If Vbias is FIG. 1. (Color online) Sketch of the ion ﬂux probe with direct current measurement. All grids are connected via high impedance low-pass ﬁl- ters to an external power supply, which is controlled via Universal Serial Bus by a Personal Computer. The ﬂoating potential of G1 is measured externally with an oscilloscope with respect to an external ground. While all grids follow the RF component of the plasma power supply and the conse- quential potential changes of this time scale, only G1 follows also the averaged DC bias/ﬂoating potential of the substrate due to its direct electrical connection. This means that the incoming ions “see” a surface with ﬂoating potential (G1). The nonﬂoating grid potentials at G2, G3, and C are set with respect to the external ground. Hence, the ﬂoating voltage FIG. 1. (Color online) Sketch of the ion ﬂux probe with direct current measurement. J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 J. Vac. Sci. Technol. B. Retarding field analyzer B, Vol. 33, No. 3, May/Jun 2015 032202-3 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma from G1 needs to be subtracted from the discriminator volt- age values to obtain the ion energy with respect to the ﬂoat- ing surface. Under these conditions, the measured current–voltage (I-V) data from C (collector current) and G2 (varied discriminator voltage) represents the total ﬂux of ions and can be differentiated to obtain the IVDF. For all experiments presented later, the RFA was placed in the center of a 300mm diameter silicon wafer. For the ﬁrst two secondary electrons in order to obtain a smooth IVDF. From this ﬁt function a derivative can be obtained. Regarding the error of the RFA measurements, two errors need to be distinguished. One is the error of the absolute energy and the other is the error of the ion ﬂux at that energy. FIG. 2. (Color online) (a) Example of an RFA with four grids18 and (b) a photo of the used probe with anodized aluminum cover. FIG. 3. (Color online) (a) Measured I-V curve of the RFA with ﬁt. (b) Direct derivative (IVDF) of raw data and derivative of ﬁt. FIG. 3. (Color online) (a) Measured I-V curve of the RFA with ﬁt. (b) Direct derivative (IVDF) of raw data and derivative of ﬁt. FIG. 2. (Color online) (a) Example of an RFA with four grids18 and (b) a photo of the used probe with anodized aluminum cover. FIG. 2. (Color online) (a) Example of an RFA with four grids18 and (b) a photo of the used probe with anodized aluminum cover. from G1 needs to be subtracted from the discriminator volt- age values to obtain the ion energy with respect to the ﬂoat- ing surface. Under these conditions, the measured current–voltage (I-V) data from C (collector current) and G2 (varied discriminator voltage) represents the total ﬂux of ions and can be differentiated to obtain the IVDF. FIG. 3. (Color online) (a) Measured I-V curve of the RFA with ﬁt. (b) Direct derivative (IVDF) of raw data and derivative of ﬁt. secondary electrons in order to obtain a smooth IVDF. From this ﬁt function a derivative can be obtained. Regarding the error of the RFA measurements, two errors need to be distinguished. One is the error of the absolute energy and the other is the error of the ion ﬂux at that energy. B. Retarding field analyzer Considering the RFA geometry and the electronics, the energy measurement error is considered to be less than 10%. For the ion ﬂux, only ions are measured that arrive at the collector and it is difﬁcult to calculate the original ﬂux arriving at the RFA outer surface, especially if the design changes between experiments. Therefore, only the relative ion ﬂux evolution and its error of less than 5% are shown. The error mainly results from geometric effects and temperature variations. For all experiments presented later, the RFA was placed in the center of a 300mm diameter silicon wafer. For the ﬁrst two measurement sessions, a thin ceramic shield (2 mm) with 0.8mm holes for ion collection was used to prevent the metallic body of the sensor from etching. In a third session, the alumi- num body itself was anodized (same material as reactor walls) and thereby converted to a dielectric shield. Only measure- ments with the same setup are compared since the transmission function might change between different hardware designs. Two examples of the measured I-V curve and the result- ing IVDF are shown in Fig. 3. Under plasma conditions where the ion current is large, the IVDF can be obtained by direct derivation of the I-V characteristic, as shown in Fig. 3(a). However, in some cases (typically in pulsed plasmas at low duty cycle), the I-V measurement is very noisy, so that no information can be obtained from the derivative (IVDF). In this case, the I-V curve can be ﬁtted with a combination of several logistic (Boltzmann) functions and, if necessary, with an additional linear term accounting for the escaping JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena B. Ion flux in pulsed plasmas strong impact on the plasma chemistry in halogen-based plasmas.27–30 Therefore, the ion ﬂux was investigated for CW HBr and HBr/O2 plasmas at 20 mTorr and 750 W source power that are operated either in “seasoned” chamber (SiO2 coating formed on reactor walls prior to etch process) or in “clean walls” reactor conditions (exposure to an SF6/O2 plasma prior to etch process). During the actual etch condi- tions, a layer consisting of Si, Br, O, and H is deposited on the reactor wall. In Fig. 5, the measured ion ﬂux in an HBr and in an HBr/ O2 plasma at 20 mTorr and 750 W source power is shown for different duty cycles at a frequency of 1 kHz. For all pulsed cases, the ion ﬂux increases continuously during the on-time of the source power and never reaches a steady state as it is the case for a CW plasma. While in the pure HBr plasma, the ion ﬂux rises strongly at ﬁrst and then slows down, the ion ﬂux in the HBr/O2 plasma starts out with a slow increase that accelerates later during the on-time of the pulse. The slow decrease in the afterglow on the other hand is comparable in both chemistries. Figure 4 shows the obtained ion ﬂuxes in clean and seas- oned condition. Signiﬁcant differences of the ion ﬂux can be observed. The ion ﬂux results from the ionization of neutral species upon electron impact in the plasma. It is therefore highly de- pendent on the electron density, the electron temperature, and the density and the ionization cross section of each neu- tral species. On the other hand, the neutrals’ composition is strongly dependent on the recombination and sticking coefﬁ- cient of the neutral species at the chamber walls. Therefore, changes in the chamber wall conditions result in changes in the neutrals’ composition and consequently changes in the ion ﬂux. For our speciﬁc conditions, our data indicate that the ion ﬂux is larger for the HBr plasma in seasoned condi- tions than in cleaned conditions, while it is vice versa for the HBr/O2 plasma. This tends to indicate that the density of neutral species with a high ionization cross section is larger in seasoned conditions than in clean conditions for the HBr plasma while it is the contrary for HBr/O2 plasma. Oxygen containing ions such as H3Oþ are probably involved in the differences. B. Ion flux in pulsed plasmas Working in seasoned chamber conditions is preferable since it prevents a signiﬁcant variation of the ion ﬂux along the process that would result from the progressive coating of the chamber walls by SiOx species. ture at the beginning and the end of each pulse since the positive ion ﬂux in an electronegative plasma is essentially described by ni vB ¼ ni ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ kBTe ð1 þ aSÞ=mið1 þ aSgÞ p , where aS ¼ ni;=ne is the ratio of negative ions to electrons and g ¼ Te=Ti; is the ratio of negative ion temperature to electron temperature.31 The slower increase during the on- time is mainly attributed to a change in ion density. We also calculated the ion ﬂux originating only from the on time, and the percentage it represents on the full ion ﬂux. FIG. 4. (Color online) Inﬂuence of the nature of the reactor walls on the ion ﬂux in an HBr and an HBr/O2 CW plasma at 20 mTorr and 750 W source power. The seasoning consists of a thin layer of SiO2 on the walls compared to a “clean” chamber where deposited layers have been removed by an SF6/O2 plasma. FIG. 5. (Color online) Ion ﬂux from capacitive probe (IFP) measurements in pulsed (a) HBr and (b) HBr/O2 plasmas at 20 mTorr, 750 W source power, and a frequency of 1 kHz. No bias power is applied. J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 We also calculated the ion ﬂux originating only from the on time, and the percentage it represents on the full ion ﬂux. FIG. 5. (Color online) Ion ﬂux from capacitive probe (IFP) measurements in pulsed (a) HBr and (b) HBr/O2 plasmas at 20 mTorr, 750 W source power, and a frequency of 1 kHz. No bias power is applied. FIG. 4. (Color online) Inﬂuence of the nature of the reactor walls on the ion ﬂux in an HBr and an HBr/O2 CW plasma at 20 mTorr and 750 W source power. The seasoning consists of a thin layer of SiO2 on the walls compared to a “clean” chamber where deposited layers have been removed by an SF6/O2 plasma. FIG. 4. (Color online) Inﬂuence of the nature of the reactor walls on the ion ﬂux in an HBr and an HBr/O2 CW plasma at 20 mTorr and 750 W source power. A. Necessity of reactor coating for reproducible results Since the IFP is unaffected by a thin deposited layer on its surface, it is well suited to investigate the inﬂuence of dif- ferent reactor wall surface conditions on the ion ﬂux. Indeed, it is well known that the chemical nature of the reactor has a JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena 032202-4 032202-4 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma B. Ion flux in pulsed plasmas The exact source for the difference between both plasma conditions is not fully explained here and would require a complete chemical analysis of the plasma species, which is out of the scope of this paper. These data illustrate though the necessity of controlled chamber wall conditions prior to the experiments to ensure reproducible results. Working in seasoned chamber conditions is preferable since it prevents a signiﬁcant variation of the ion ﬂux along the process that would result from the progressive coating of the chamber walls by SiOx species. Interestingly, both plasmas show an additional fast vari- ation of the ion ﬂux when the power is switched on or off. This is attributed to a fast change of the electron tempera- ture at the beginning and the end of each pulse since the positive ion ﬂux in an electronegative plasma is essentially described by ni vB ¼ ni ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ kBTe ð1 þ aSÞ=mið1 þ aSgÞ p , where aS ¼ ni;=ne is the ratio of negative ions to electrons and g ¼ Te=Ti; is the ratio of negative ion temperature to electron temperature.31 The slower increase during the on- time is mainly attributed to a change in ion density. tral species. On the other hand, the neutrals composition is strongly dependent on the recombination and sticking coefﬁ- cient of the neutral species at the chamber walls. Therefore, changes in the chamber wall conditions result in changes in the neutrals’ composition and consequently changes in the ion ﬂux. For our speciﬁc conditions, our data indicate that the ion ﬂux is larger for the HBr plasma in seasoned condi- tions than in cleaned conditions, while it is vice versa for the HBr/O2 plasma. This tends to indicate that the density of neutral species with a high ionization cross section is larger in seasoned conditions than in clean conditions for the HBr plasma while it is the contrary for HBr/O2 plasma. Oxygen containing ions such as H3Oþ are probably involved in the differences. The exact source for the difference between both plasma conditions is not fully explained here and would require a complete chemical analysis of the plasma species, which is out of the scope of this paper. These data illustrate though the necessity of controlled chamber wall conditions prior to the experiments to ensure reproducible results. 2. RFA results Using the RFA, the ion ﬂux and the ion energy can be determined and correlated. The dataset presented in Fig. 6 starts with a bias power of 20 W because the ceramic shield that we used to prevent the metallic body from being sput- tered would induce a too large charging effect at lower bias power. Since it is dielectric, it might be charged differently compared to the wafer and the RFA body itself. For low ion energies, this difference might be sufﬁcient to deﬂect ions and to reduce thereby the number of ions that reach the de- tector. Another possible reason is linked to the very thin plasma sheath in the case without bias. If the sheath is thin enough, the plasma might be able to extend to some degree into the holes of the dielectric shield.32 Since the ion trajec- tory runs perpendicular to the sheath, the distortion in the proximity of the holes could deﬂect some ions toward the ce- ramic hole sidewalls so that they do not reach the RFA col- lector. Hence, we do not report the ion ﬂux at 0 W bias here. TABLE I. Measured (rounded) mean ion ﬂuxes in HBr and HBr/O2 plasmas at 20 mTorr, 750 W source power, and without bias at a pulse frequency of 1 kHz. TABLE I. Measured (rounded) mean ion ﬂuxes in HBr and HBr/O2 plasmas at 20 mTorr, 750 W source power, and without bias at a pulse frequency of 1 kHz. Gas Duty cycle % Mean flux lA/cm2 Mean flux % CW Flux on-time % mean flux Flux on-time % mean flux CW 100 137 100 100 100 75 84 61 79 48 HBr 50 32 24 54 13 35 14 10 41 4.2 20 6 4.1 28 1.1 10 3 2.0 18 0.4 100 107 100 100 100 75 45 42 76 32 HBr/O2 50 22 20 54 11 35 13 13 40 5.0 20 5 4.6 29 1.3 10 4 3.3 16 0.5 JVST B - Nanotechnology and Microelectronics: Materials, Process Comparing the RFA data for the ion ﬂux and energy, two features can be observed: the total ion ﬂux is increased when oxygen is added and at the same time, the mean ion energy is decreased. Furthermore, the ion energy increases linearly with the bias power. 1. IFP results The second point is very important since it means that in a pulsed HBr/O2 plasma, the relative amount of high energy (HE) ions can be drastically reduced. This might be a way to reduce surface damage from energetic ions. The evolution of the ion ﬂux with bias power from the IFP data shows a dependence on the used plasma chemistry. For the HBr case, the ion ﬂux decreases slightly with higher bias power at ﬁrst and starts to increase gradually afterwards. In the HBr/O2 case, the ﬂux is constantly increasing. At higher bias power both ﬂuxes approach each other. By combining both observations, we can also state that in pulsed conditions, the ion ﬂux from only the on-time of the plasma (i.e., the energetic ion dose) increases approximately with dc3 compared to CW mode. In both chemistries, the increase of the ion ﬂux with the RF bias power is attributed mostly to the localized ionization produced by the capacitive coupling of the RF bias power to the chuck. This contribution to the total ionization rate in the plasma volume is small in high density plasma, but is never- theless clearly captured by the ion ﬂux probe for both plas- mas and by the RFA probe for the HBr/O2 plasma in Fig. 6. B. Ion flux in pulsed plasmas The seasoning consists of a thin layer of SiO2 on the walls compared to a “clean” chamber where deposited layers have been removed by an SF6/O2 plasma. FIG. 5. (Color online) Ion ﬂux from capacitive probe (IFP) measurements in pulsed (a) HBr and (b) HBr/O2 plasmas at 20 mTorr, 750 W source power, and a frequency of 1 kHz. No bias power is applied. J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 032202-5 032202-5 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma The (mean) ﬂux contribution from the on time is deﬁned by hCiion ¼ ð1=TÞ Ð dcT 0 Cidt with dc the duty cycle, T the puls- ing period and Ui the ion ﬂux. The mean ion ﬂux being deﬁned as hCii ¼ ð1=TÞ Ð T 0 Cidt, the percentage of the ion ﬂux from the on time in comparison to the full ion ﬂux is deﬁned as hCiion=hCii 100. The mean values for the ion ﬂuxes and their relative contributions are given in Table I. They follow similar trends in both plasma conditions: IV. ION FLUX IN BIASED Si ETCH CONDITIONS From theoretical considerations, the ion ﬂux should not change much with different bias conditions since the plasma density is expected to be controlled mainly by the inductively coupled plasma source power which remains constant. However, changes in the plasma composition would lead to changes in the ion ﬂux. With our plasma con- ditions, the silicon is not etched without bias power, while for bias powers larger than few tens of Watts, silicon is etched and the etch rate hardly varies with the bias power. Therefore, we expect a difference in plasma composition and therefore different ion ﬂuxes without bias power and with bias power, when the plasma is fed with silicon etch by-products. It should also be underlined that the absolute value of the ion ﬂux is small since it hardly reaches 0.12 mA/cm2. This is attributed to the highly electronegative character of the HBr and HBr/O2 plasmas. A. CW plasma Figure 6 shows the total ion ﬂux with respect to the bias power for the HBr and HBr/O2 plasma obtained from RFA and IFP measurements. Figure 7 shows the mean ion energy obtained in the same conditions with the RFA. While for the RFA measurements the ion ﬂux of the pure HBr plasma is lower than the ﬂux in the HBr/O2 plasma, the opposite is the case for the IFP measurements. This difference is due to the different positions of the probes (the IFP is situated in plane with the reactor sidewall, the RFA lies on a dummy silicon wafer). Therefore, a direct comparison between both probes cannot be made. (1) The mean ﬂux increases approximately with the square of the duty cycle (dc2). (1) The mean ﬂux increases approximately with the square of the duty cycle (dc2). (2) The percentage of the ﬂux from only the on-time of the plasma (high energy ions) increases almost linearly with the duty cycle. (2) The percentage of the ﬂux from only the on-time of the plasma (high energy ions) increases almost linearly with the duty cycle. JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena B. Pulsed plasma As shown in Sec. III for the case without bias power, the ion ﬂux decreases with lower duty cycle since the ion density in the plasma is reduced. This evolution is unchanged if pulsed bias power is applied synchronously with the source power. During the on-time of the plasma, the instantaneous bias power is the same as for CW, but the ion ﬂux is signiﬁ- cantly lower, which leads to a much higher ion energy in pulsed mode. Figure 8 shows the time averaged IVDF for a pulsed HBr plasma at different duty cycles, as well as the corresponding ion ﬂux. The plasma conditions studied here (10 mTorr, 1200 W source power, 60 W bias power) are signiﬁcantly different from the standard etch process studied previously: the ICP power was increased to obtain a signal which is strong enough to be analyzed while the bias power was reduced to be able to measure the ion energy (our sensor stops at 400 eV). Also, a different dielectric shield for the RFA was used (anodized probe body instead of additional ceramic shield). FIG. 6. (Color online) Total ion ﬂux for different bias powers in CW mode at 20 mTorr and 750 W source power (HBr ﬂow at 200 sccm and O2 ﬂow at 5 sccm): (a) RFA and (b) IFP data. losses (e.g., ionization or charge transfer) and losses in the hardware (e.g., matchbox) are neglected. In other words, the applied bias power is mainly consumed by accelerating the ions. As discussed previously, this is a good approximation in our conditions since only little ionization is resulting from applying RF bias power to the wafer. In this case, the prod- uct of the ion ﬂux and ion energy is proportional to the bias power. If the ion density and consequently also the ion ﬂux increase while the bias power remains constant, the mean ion energy decreases.26 In the case of added oxygen, the total ion ﬂux is increased according to the RFA measurements. Hence, the mean ion energy of the IVDF should decrease (as observed). In CW mode, a single peak IVDF is measured. If the plasma is pulsed, a low energy (LE) peak from the afterglow appears in addition to the HE peak from the active source and bias power period. 2. RFA results In a rough approximation, the bias power Pbias is proportional to the product of the ion ﬂux Ci and the sheath voltage V S (ion energy Ei)26 if collisional JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena 032202-6 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma 032202-6 losses (e g ionization or charge transfer) and losses in the FIG. 6. (Color online) Total ion ﬂux for different bias powers in CW mode at 20 mTorr and 750 W source power (HBr ﬂow at 200 sccm and O2 ﬂow at 5 sccm): (a) RFA and (b) IFP data. g p p losses (e g ionization or charge transfer) and losses in the case a mean energy of approximately 500 an HBr/O2 and the HBr plasma, respectively. are high for an ICP plasma, but they are a dir of the low value of the ion current in HBr-ba B. Pulsed plasma As shown in Sec. III for the case without ion ﬂux decreases with lower duty cycle sinc in the plasma is reduced. This evolution pulsed bias power is applied synchronously power. During the on-time of the plasma, th bias power is the same as for CW, but the io cantly lower, which leads to a much highe pulsed mode. Figure 8 shows the time averaged IVDF f plasma at different duty cycles, as well as th ion ﬂux. The plasma conditions studied h 1200 W source power, 60 W bias power) different from the standard etch process stu FIG. 6. (Color online) Total ion ﬂux for different bias powers in CW mode at 20 mTorr and 750 W source power (HBr ﬂow at 200 sccm and O2 ﬂow at 5 sccm): (a) RFA and (b) IFP data. FIG. 7. (Color online) RFA measurements of the mean 20 mTorr and 750 W source power. FIG. 7. (Color online) RFA measurements of the mean ion energy in CW at 20 mTorr and 750 W source power. FIG. 7. (Color online) RFA measurements of the mean ion energy in CW at 20 mTorr and 750 W source power. case a mean energy of approximately 500 and 750 eV for the HBr/O2 and the HBr plasma, respectively. 2. RFA results These energies are high for an ICP plasma, but they are a direct consequence of the low value of the ion current in HBr-based plasmas. J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 B. Pulsed plasma For the low energy peak, we would expect an energy of just a few eV, corresponding to a vanish- ing sheath voltage in the afterglow. The rather high meas- ured energy and its slow evolution might be linked to the poor measurement accuracy for the RFA ﬂoating potential that is used to calibrate the IVDF and/or to an imperfect ﬁl- tering at 1 kHz. Also, for low energy ions, charge effects that derogate the IVDF measurement might play a role, as dis- cussed for the ion ﬂux measurement in CW conditions with- out bias power. Therefore, the energy value from the LE peak will not be discussed. Nevertheless, the measured ion Furthermore, in both plasma chemistries, the ion ﬂux is almost constant with changing bias power. Hence, an increase in bias power leads to the linear increase in mean ion energy, as observed in Fig. 7. Since the voltages applied to the RFA are limited to a maximum of 400 V, the IVDF for the process plasma with 200 W bias power could not be measured. Based on the lin- ear approximation of the ion energy, we can estimate for this J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 032202-7 032202-7 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma ﬂux of both peaks and the very large energy shift for the HE peak are assumed to be correct. it is difﬁcult to derive a meaningful IVDF from it. However, it is quite straightforward to extract the total ion ﬂux, which is shown in Fig. 9 for several duty cycles and frequencies. For decreasing duty cycles, the high energy peak shifts to very large values due to the decreasing ion ﬂux. The ratio between high and low energy ﬂux is decreasing until the high energy ﬂux becomes almost insigniﬁcant, shown in Fig. 8(b). This is in good agreement with the time-resolved mea- surement of the ion ﬂux in no-bias conditions in Sec. III and might open the possibility to reduce surface damage caused by high energy ions. It is important to underline that although the high energy ﬂux is strongly reduced, for a given RF biasing power the ion energy of this ﬂux is multiplied by more or less a factor of 5 when the plasma is pulsed at 20% duty cycle. V. SUMMARY In the ﬁrst part of the article, the ion ﬂux was studied in an HBr/O2 plasma without applied bias power. With increasing the pulsing frequency the IVDF also changes, but only slightly. Unfortunately, the ion current mea- surement data are very noisy for high frequencies, and hence, changes, but only slightly. Unfortunately, the ion current mea- surement data are very noisy for high frequencies, and hence, Depending on the nature of the reactor wall and the gas composition of the plasma, the ion ﬂux varies signiﬁcantly. This is due to the changed reaction coefﬁcients on the cham- ber walls that affect the plasma composition and therefore the ion ﬂux. When the plasma is pulsed, the time variations of the ion ﬂux during the pulse depend also on the plasma chemistry: while the ﬂux rises strongly at ﬁrst and then slows down in a pure HBr plasma, the opposite is observed for the HBr/O2 plasma. By reducing the duty cycle, the average ion ﬂux decreases by approximately the square of the duty cycle (dc2) and the percentage of the ion ﬂux that originates from the on-time of the plasma almost linearly. Hence, the average ion ﬂux from only the on-time of the plasma decreases by roughly dc3 FIG. 8. (Color online) (a) IVDFs of a pulsed HBr plasma (200 sccm, 10 mTorr, 1200 W source power, 60 W bias power) at 1 kHz with bias. (b) Corresponding total ion ﬂux, high energy ion ﬂux and mean energy of the latter. FIG. 9. (Color online) Ion ﬂux for pulsed HBr plasmas (200 sccm, 10 mTorr, 1200 W source power, and 60 W bias power) from RFA measurements. Depending on the nature of the reactor wall and the gas composition of the plasma, the ion ﬂux varies signiﬁcantly. This is due to the changed reaction coefﬁcients on the cham- ber walls that affect the plasma composition and therefore the ion ﬂux. compositio This is due ber walls the ion ﬂu When th during the ﬂux rises s plasma, the By redu by approx percentage the plasma only the o FIG. 8. (Color online) (a) IVDFs of a pulsed HBr plasma (200 sccm, 10 mTorr, 1200 W source power, 60 W bias power) at 1 kHz with bias. (b) Corresponding total ion ﬂux, high energy ion ﬂux and mean energy of the latter. FIG. 9. B. Pulsed plasma It follows that when the plasma is pulsed under our typical etching conditions at this duty cycle (with 750 W source and 200 W bias), the ion energy is estimated to be about 2.5 keV, leading to very speciﬁc etching conditions compared to what can be obtained in a CW plasma. Indeed, since the wafer is bombarded by a low ﬂux of very energetic ions, we are in a condition that resembles those obtained in purely capacitively coupled reactors, but at lower pressure with collisionless sheaths and with a high ﬂux of low energy ions during the off-time of the plasma pulses. For all duty cycles, the total ion ﬂux decreases if the fre- quency is increased, comparable to a further reduction of the duty cycle. The relative reduction of the ion ﬂux becomes stronger for lower duty cycle. At higher frequencies, the num- ber of transitions per second between on and off increases and the pulse period could become so small that it approaches the characteristic time constants for ionization, dissociation, and recombination, even more at low duty cycles. Furthermore, even if our system is working at constant absorbed power (the reﬂected power is measured and compen- sated), it is difﬁcult to determine how much of this power is really absorbed by the plasma species and how much is dissi- pated in the matching units. In other words, since matching the plasma impedance becomes more and more difﬁcult as the pulsing frequency is increased, it is possible that a slightly lower RF power is delivered to the plasma at high frequency, thus participating to a reduction of the ion ﬂux. V. SUMMARY Mieno, Plasma Sources Sci. Technol. 5, 132 (1996). 7C. Petit-Etienne, M. Darnon, L. Vallier, E. Pargon, G. Cunge, F. Boulard, bert, S. Banna, and T. Lill, J. Vac. Sci. Technol., B 28, 926 (201 In the second part of the article, the ion ﬂux was investi- gated in silicon etch conditions with applied bias power (200 W). The results indicate mean ion energies of approxi- mately 500 and 750 eV for the CW HBr/O2 and the CW HBr plasma, respectively. 8C. Petit-Etienne, E. Pargon, S. David, M. Darnon, L. Vallier, O. Joubert, d S B J V S i T h l B 30 40604 (2012) 8C. Petit-Etienne, E. Pargon, S. David, M. Darnon, L. Vallier, O. Joubert, and S. Banna, J. Vac. Sci. Technol., B 30, 40604 (2012). 9 C. Petit-Etienne, E. Pargon, S. David, M. Darnon, L. Vallier, O. Joubert, and S. Banna, J. Vac. Sci. Technol., B 30, 40604 (2012). 9 9S. A. Voronin, M. R. Alexander, and J. W. Bradley, Meas. Sci. Technol. 16, 2446 (2005). 10H. Ohtake, K. Noguchi, S. Samukawa, H. Iida, A. Sato, and X. Qian, J. Vac. Sci. Technol., B 18, 2495 (2000). 11 The total ion ﬂux increases slightly with higher bias power due to an increase of the density of silicon etch product in the gas phase as well as to a non-negligible local ionization in front of the sheath that is facing the RF biased wafer. Directly on the wafer, the ion ﬂux from an HBr/O2 plasma is higher than in the pure HBr chemistry. The opposite is observed with the IFP at the reactor sidewall. The reason for this difference is not fully understood but attributed to spatial density gra- dients of the charged species in the reactor. 11S. Samukawa, Y. Ishikawa, S. Kumagai, and M. Okigawa, Jpn. J. Appl. Phys. 40, L1346 (2001). 12M. Okigawa, Y. Ishikawa, and S. Samukawa, Jpn. J. Appl. Phys. 42, 2444 (2003). ( ) 13P. Subramonium and M. J. Kushner, J. Appl. Phys. 96, 82 (2004). 14 14G. Cunge, D. Vempaire, and N. Sadeghi, Appl. Phys. Lett. 96, 131501 (2010). 15R. W. Boswell and D. Henry, Appl. Phys. Lett. 47, 1095 (1985 16 16N. St. J. Braithwaite, J. P. Booth, and G. Cunge, Plasma Sources Sci. Technol. 5, 677 (1996). 17J. P. Booth, N. St. J. Braithwaite, A. Goodyear, and P. Barroy, Rev. Sci. Instrum. 71, 2722 (2000). 1T.-H. Ahn, K. Nakamura, and H. Sugai, Plasma Sources Sci. Technol. 5, 139 (1996). 2G. S. Hwang and K. P. Giapis, Jpn. J. Appl. Phys. 37, 2291 (1998). 3J.-H. Kim, C.-J. Kang, T.-H. Ahn, and J.-T. Moon, Thin Solid Films 345, 124 (1999). V. SUMMARY For pulsed mode, the IVDF shows a high and a low energy peak, which correspond to the ions from the on- and the off-time of the plasma, respectively. If the duty cycle is reduced, the total ion ﬂux, measured by the RFA on the wa- fer, decreases linearly and the high energy peak shifts to even higher energies. The relative contribution from the high energy peak to the total ion ﬂux is also reduced with the duty cycle, leading to an overall decreased averaged ion energy. These results are in good agreement with the IFP measure- ments in unbiased conditions. 18D. Gahan, B. Dolinaj, and M. B. Hopkins, Rev. Sci. Instrum. 79, 033502 (2008). 19D. Gahan, S. Daniels, C. Hayden, D. O’Sullivan, and M. B. Hopkins, Plasma Sources Sci. Technol. 21, 015002 (2012). 20 20M. Haass, M. Darnon, G. Cunge, and O. Joubert, J. Vac. Sci. Technol., B 33, 032203 (2015). 21 21E. Pargon, O. Joubert, T. Chevolleau, G. Cunge, S. Xu, and T. Lill, J. Vac. Sci. Technol., B 23, 103 (2005). 22 22G. Cunge, R. L. Inglebert, O. Joubert, L. Vallier, and N. Sadeghi, J. Vac. Sci. Technol., B 20, 2137 (2002). 23P. Bodart, M. Brihoum, G. Cunge, O. Joubert, and N. Sadeghi, J. Appl. Phys. 110, 113302 (2011). Similar to a decrease in duty cycle, also an increase in fre- quency, especially for low duty cycles, results in a further reduction of the total ion ﬂux. 24S. Banna et al., IEEE Trans. Plasma Sci. 37, 1730 (2009). 25 25M. Darnon, G. Cunge, and N. St. J. Braithwaite, Plasma Sources Sci. Technol. 23, 025002 (2014). 26 26M. Brihoum, G. Cunge, M. Darnon, D. Gahan, O. Joubert, and N. St. J. Braithwaite, J. Vac. Sci. Technol., A 31, 020604 (2013). J. Vac. Sci. Technol. B, Vol. 33, No. 3, May/Jun 2015 V. SUMMARY (Colo 1200 W sour When the plasma is pulsed, the time variations of the ion ﬂux during the pulse depend also on the plasma chemistry: while the ﬂux rises strongly at ﬁrst and then slows down in a pure HBr plasma, the opposite is observed for the HBr/O2 plasma. By reducing the duty cycle, the average ion ﬂux decreases by approximately the square of the duty cycle (dc2) and the percentage of the ion ﬂux that originates from the on-time of the plasma almost linearly. Hence, the average ion ﬂux from only the on-time of the plasma decreases by roughly dc3 FIG. 9. (Color online) Ion ﬂux for pulsed HBr plasmas (200 sccm, 10 mTorr, 1200 W source power, and 60 W bias power) from RFA measurements. FIG. 8. (Color online) (a) IVDFs of a pulsed HBr plasma (200 sccm, 10 mTorr, 1200 W source power, 60 W bias power) at 1 kHz with bias. (b) Corresponding total ion ﬂux, high energy ion ﬂux and mean energy of the latter. FIG. 9. (Color online) Ion ﬂux for pulsed HBr plasmas (200 sccm, 10 mTorr, 1200 W source power, and 60 W bias power) from RFA measurements. JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena FIG. 8. (Color online) (a) IVDFs of a pulsed HBr plasma (200 sccm, 10 mTorr, 1200 W source power, 60 W bias power) at 1 kHz with bias. (b) Corresponding total ion ﬂux, high energy ion ﬂux and mean energy of the latter. FIG. 9. (Color online) Ion ﬂux for pulsed HBr plasmas (200 sccm, 10 mTorr, 1200 W source power, and 60 W bias power) from RFA measurements. FIG. 9. (Color online) Ion ﬂux for pulsed HBr plasmas (200 sccm, 10 mTorr, 1200 W source power, and 60 W bias power) from RFA measurements. JVST B - Nanotechnology and Microelectronics: Materials, Processing, Measurement, and Phenomena ogy and Microelectronics: Materials, Processing, Measurement, and Phenomena 032202-8 Haass et al.: Silicon etching in a pulsed HBr/O2 plasma 032202-8 4S. Samukawa and K. Terada, J. Vac. Sci. Technol., B 12, 3300 (1994). 5 compared to the CW case. The reduction of the relative con- tribution of high energy ions to the total ﬂux might be useful to reduce damages from energetic ions although their ion energy itself increases. 5S. Samukawa, K. Noguchi, J. I. Colonell, K. H. A. Bogart, M. V. 6S. Samukawa and T. ACKNOWLEDGMENT 27G. Cunge, M. Kogelschatz, and N. Sadeghi, Plasma Sources Sci. Technol. 13, 522 (2004). 28 This work was partly supported by the French RENATECH network. 28G. Cunge, M. Kogelschatz, O. Joubert, and N. Sadeghi, Plasma Sources Sci. Technol. 14, S42 (2005). 29G. Cunge, N. Sadeghi, and R. Ramos, J. Appl. Phys. 102, 093305 (2007). 30G. Cunge, D. Vempaire, R. Ramos, M. Touzeau, O. Joubert, P. Bodard, d N. Sadeghi, Plasma Sources Sci. Technol. 19, 034017 (2010). 31M. A. Lieberman and A. J. Lichtenberg, Principles of Plasma Discharges and Materials Processing (Wiley-Interscience, New York, 1994). 32C. B€ohm and J. Perrin, Rev. Sci. Instrum. 64, 31 (1993). 32C. B€ohm and J. Perrin, Rev. Sci. Instrum. 64, 31 (1993).
https://openalex.org/W2747713746	https://europepmc.org/articles/pmc5592016?pdf=render	English	null	Cholinergic Potentiation of Restoration of Visual Function after Optic Nerve Damage in Rats	Neural plasticity	2,017	cc-by	9,582	Hindawi Neural Plasticity Volume 2017, Article ID 6928489, 10 pages https://doi.org/10.1155/2017/6928489 Hindawi Neural Plasticity Volume 2017, Article ID 6928489, 10 pages https://doi.org/10.1155/2017/6928489 Hindawi Neural Plasticity Volume 2017, Article ID 6928489, 10 pages https://doi.org/10.1155/2017/6928489 Research Article Cholinergic Potentiation of Restoration of Visual Function after Optic Nerve Damage in Rats Mira Chamoun,1 Elena G. Sergeeva,2 Petra Henrich-Noack,2 Shaobo Jia,2 Lisa Grigartzik,2 Jing Ma,2 Qing You,2 Frédéric Huppé-Gourgues,1 Bernhard A. Sabel,2 and Elvire Vaucher1 1Laboratoire de Neurobiologie de la Cognition Visuelle, École d'optométrie, Université de Montréal, Montréal, QC, Canada 2Institute of Medical Psychology, Otto von Guericke University, Magdeburg, Germany Correspondence should be addressed to Bernhard A. Sabel; bernhard.sabel@med.ovgu.de and Elvire Vaucher; elvire.vaucher@umontreal.ca Received 7 March 2017; Revised 26 May 2017; Accepted 4 June 2017; Published 27 August 2017 Academic Editor: Preston E. Garraghty Copyright © 2017 Mira Chamoun 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. Enhancing cortical plasticity and brain connectivity may improve residual vision following a visual impairment. Since acetylcholine plays an important role in attention and neuronal plasticity, we explored whether potentiation of the cholinergic transmission has an eﬀect on the visual function restoration. To this end, we evaluated for 4 weeks the eﬀect of the acetylcholinesterase inhibitor donepezil on brightness discrimination, visually evoked potentials, and visual cortex reactivity after a bilateral and partial optic nerve crush in adult rats. Donepezil administration enhanced brightness discrimination capacity after optic nerve crush compared to nontreated animals. The visually evoked activation of the primary visual cortex was not restored, as measured by evoked potentials, but the cortical neuronal activity measured by thallium autometallography was not signiﬁcantly aﬀected four weeks after the optic nerve crush. Altogether, the results suggest a role of the cholinergic system in postlesion cortical plasticity. This ﬁnding agrees with the view that restoration of visual function may involve mechanisms beyond the area of primary damage and opens a new perspective for improving visual rehabilitation in humans. 1. Introduction (b) When all rats reached an adequate brightness discrimination performance and success rate, electrodes (black dots) were implanted in the visual cortex (V1), the superior colliculus (SC), and in the nose bone (Ref.) in the brain/skull of the rats, together with a headstage to ﬁx visual stimulation goggles (gray circles). A potential evoked by ﬂash displayed through the goggles (VEP, red tracing) was recorded and set as a baseline. (c) Then animals were divided into three experimental groups: sham, ONC/DPZ, and ONC/saline, and bilateral partial optic nerve crush (ONC) or sham surgery was performed. (d) DPZ (1 mg/kg then 0.5 mg/kg) or saline i.p. injections were performed each day for the ﬁrst 2 weeks post-ONC (black arrows) then once a week. VIST testing (3 times a week during 4 consecutive weeks) and the VEP recording (once a week during 4 consecutive weeks, red tracing) were performed. (e) One week after the end of the VIST testing and the jugular vein catheter implantation, the neuronal activity was measured by thallium autometallography (TlAMG). deprived before the start of the behavioural study with lim- ited access to water for 15 minutes per day because VIST learning was based on water reward. The study was per- formed over a period of 11 weeks (Figure 1). After VIST training, the baseline brightness discrimination capacity was acquired and the animals were randomly assigned to 3 groups: sham, n = 7 (sham-ONC and no injection), ONC/ DPZ, n = 6 (ONC and DPZ injection), and ONC/saline, n = 7 (ONC and saline injection). Repetitive behavioural and electrophysiological testing and thallium autometallo- graphy were then performed. Note that some animals were excluded from VEP or TlAMG measurements because they did not ﬁt the experimental criteria. All procedures were in accordance with ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the guidelines of the Canadian Council for the Protection of Animals. All the procedures were accepted by the Ethics Committee of the Université de Montréal number 14-164 and of the IRB of the Landesverwaltungsamt Sachsen-Anhalt according to the German National Act. ergic enhancement to potentiate the improvement of residual vision in visually deﬁcient subjects. y j The present study sought to evaluate whether cholinergic enhancement, by DPZ administration, can facilitate the res- toration of the visual capacities following partial bilateral optic nerve crush (ONC) surgery in rats. 1. Introduction term enhancement of responses in the visual cortex [10–13]. Particularly, acetylcholinesterase inhibitors (AChEIs), which prevent the degradation of the acetylcholine (ACh)and enable the buildup of AChat the synapse,could beused forlong-term enhancement of visual experience. AChEIs, such as donepezil (DPZ), are used for sustaining ACh action and treating the dementia symptoms in mild Alzheimer’s disease [14]. Studies have shown that AChEIs enhance visual and cognitive capac- ities in both animals and humans [11–13, 15–18]. They also improve performance inbehaviouraltaskssuchasradialwater maze,spatialrecognition,andcontrastsensitivitydetectionfor healthy and hypocholinergic rats [18–21]. Moreover, combin- ing cholinergic enhancement via DPZ administration with repetitive visual training increases the visual cortical respon- siveness in healthy rats [13] and the learning in a perceptual- cognitive task in healthy young humans [17]. Therefore, administration of DPZ could be an eﬀective method of cholin- The visual system has a substantial plasticity and a potential of partial spontaneous visual recovery both in animals [1–3] and in patients with visual system damage [4–6]. Moreover, clinical studies have shown that the residual vision subse- quent to neural trauma or neuronal degeneration can be improved by training [7–9]. Several approaches have tried to enhance this visual recovery by potentiating cortical plas- ticity and sensory functioning. In this regard, stimulating central neuromodulatory systems that facilitate sensory responses and cortical plasticity is an interesting avenue. There is growing evidence that the cholinergic system is involved in attention, cortical and synaptic plasticity, and potentiation of the cortical responses to visual stimulation. In rats, electrical or pharmacological stimulation of the choliner- gic system when coupled with visual training enables long- 2 Neural Plasticity VIST training (a) V1 SC Ref. VEP recording (b) ONC (c) 1 mg/kg Week 1 Visual stimulation test (3 sessions/week) Donepezil or saline i.p. injections 0.5 mg/kg Week 2 Week 3 Week 4 (d) TIAMG (e) Figure 1: Experimental design. The study was performed over a period of 11 weeks: 5 weeks for the learning of the behavioural task training—VIST training, double-end arrow (a), 1 week for the baseline VEP recording (b) and the crush surgery (c), and 4 weeks for the repetitive behavioural and electrophysiological testing (d). Thallium autometallography (TlAMG) (e) was done one week after the last testing session. (a) The VIST test consisted of the detection of a bright stimulus (light gray square) among 6 openings (5 black and 1 gray square) on a touch screen. 1. Introduction (b) When all rats reached an adequate brightness discrimination performance and success rate, electrodes (black dots) were implanted in the visual cortex (V1), the superior colliculus (SC), and in the nose bone (Ref.) in the brain/skull of the rats, together with a headstage to ﬁx visual stimulation goggles (gray circles). A potential evoked by ﬂash displayed through the goggles (VEP, red tracing) was recorded and set as a baseline. (c) Then animals were divided into three experimental groups: sham, ONC/DPZ, and ONC/saline, and bilateral partial optic nerve crush (ONC) or sham surgery was performed. (d) DPZ (1 mg/kg then 0.5 mg/kg) or saline i.p. injections were performed each day for the ﬁrst 2 weeks post-ONC (black arrows) then once a week. VIST testing (3 times a week during 4 consecutive weeks) and the VEP recording (once a week during 4 consecutive weeks, red tracing) were performed. (e) One week after the end of the VIST testing and the jugular vein catheter implantation, the neuronal activity was measured by thallium autometallography (TlAMG). VIST training (a) V1 SC Ref. VEP recording (b) ONC (c) 1 mg/kg Week 1 Visual stimulation test (3 sessions/week) Donepezil or saline i.p. injections 0.5 mg/kg Week 2 Week 3 Week 4 (d) TIAMG (e) ONC (c) 1 mg/kg Week 1 Visual stimulation test (3 sessions/week) Donepezil or saline i.p. injections 0.5 mg/kg Week 2 Week 3 Week 4 (d) TIAMG (e) g Visual stimulation test (3 sessions/week) ezil or saline i.p. injections 0.5 mg/kg Week 2 Week 3 We (d) V1 SC Ref. VEP recording (b) VIST training (a) k) Week 4 TIAMG (e) (d) (a) (e) (b) Figure 1: Experimental design. The study was performed over a period of 11 weeks: 5 weeks for the learning of the behavioural task training—VIST training, double-end arrow (a), 1 week for the baseline VEP recording (b) and the crush surgery (c), and 4 weeks for the repetitive behavioural and electrophysiological testing (d). Thallium autometallography (TlAMG) (e) was done one week after the last testing session. (a) The VIST test consisted of the detection of a bright stimulus (light gray square) among 6 openings (5 black and 1 gray square) on a touch screen. 1. Introduction Brieﬂy, the water-deprived rats were placed in a Skinner box having six equally sized openings in the front panel attached to an infrared touch screen and a green light and water dis- penser in the back panel. A trial consisted of illuminating 1. Introduction Partial ONC is an eﬀective model of diﬀuse axonal injury, which mimics visual impairment due to optic neuropathy or diﬀuse retinopathy. This model has been widely used to study neuronal damage, residual tissue left undamaged after the trauma or postinjury plasticity [22–24]. The spontaneous visual recovery after a mild ONC in rats has been characterized in behavioural stud- ies, in terms of rate and extent. Usually, partial recovery (up to 20%) develops within three weeks postlesion [3, 25–28]. The visual capacity was thus measured behaviourally before and after partial ONC using brightness discrimination task (VIST) and physiologically by recording visual evoked potentials (VEPs). In addition, thallium autometallography was used to evaluate the neuronal activity via the ex-vivo quantiﬁcation of visually induced cellular potassium uptake in the primary visual cortex (V1). The present result shows that the administration of DPZ in visually impaired rats induced a better recovery of brightness discrimination capac- ity in comparison to the non-DPZ treated animals. This result raises the possibility that DPZ may be used in processes of restoration of visual capacities. 2.2. Visual Stimulation Test. To quantify vision recovery, the visual stimulation test (VIST) technique [3] was used, where the visual performance of the rat was measured by the capacity of discriminating targets with diﬀerent levels of brightness and the percentage of correct choices. 13 levels of brightness were displayed: 100% (white, level 1), 94%, 88%, 81%, 75%, 69%, 62%, 56%, 50%, 44%, 37%, 31%, and 25% (dark gray, level 13). Brightness discrimina- tion was determined before ONC and after ONC during four consecutive weeks (3 sessions per week). Brieﬂy, the water-deprived rats were placed in a Skinner box having six equally sized openings in the front panel attached to an infrared touch screen and a green light and water dis- penser in the back panel. A trial consisted of illuminating 2.2. Visual Stimulation Test. To quantify vision recovery, the visual stimulation test (VIST) technique [3] was used, where the visual performance of the rat was measured by the capacity of discriminating targets with diﬀerent levels of brightness and the percentage of correct choices. 13 levels of brightness were displayed: 100% (white, level 1), 94%, 88%, 81%, 75%, 69%, 62%, 56%, 50%, 44%, 37%, 31%, and 25% (dark gray, level 13). Brightness discrimina- tion was determined before ONC and after ONC during four consecutive weeks (3 sessions per week). 2.3. VEP Recording 2.3. VEP Recording 2.3.1. Headstage Implantation. In order to monitor VEPs at diﬀerent time points, custom-made stainless steel Teﬂon- coated recording electrodes, 75 μm uncoated diameter and 140 μm coated with an impedance range of 100–500 kΩ (SS-3 T/HH; Science products GmbH), were chronically implanted in V1 (AP =−7 mm; ML= ±3 mm; DV =0 mm) and in the superﬁcial layer of the superior colliculus (Bregma coordinates: AP = −6.8; ML =±1 mm; DV = 3 mm) under 75 mg/kg ketamine and 0.5 mg/kg medetomidine anesthesia according to Paxinos and Watson (1998). The reference elec- trode was implanted into the nasal bone, and an extra screw was implanted into the parietal bone to ﬁx the headstage. Animals were allowed to recover during one week. 2.5. Drug Administration. DPZ (Sigma Aldrich, St-Louis, MO, USA) was prepared freshly in a sterile 0.9% NaCl solu- tion and administered i.p. at the end of the day to avoid any acute eﬀect. On the ﬁrst week post-ONC, a loading dose of DPZ (1mg/kg) was administered daily [12]. On the second week, a lower dose of DPZ (0.5 mg/kg) was admin- istered daily [18]. We have previously shown that both doses strongly enhance the visual cortex reactivity assessed by VEP [13]. As DPZ is eliminated from the body by renal excretion and as urination was reduced due to the water restriction regime, a maintain dose was given on weeks 3 and 4 (0.5 mg/kg of DPZ, once a week). The same volume of saline, but without DPZ, was injected in the control animals. 2.3.2. VEP Recording. The VEPs were recorded on awaken rats once before the sham or ONC surgery for baseline value and once every week after the sham or ONC surgery. Before the ﬁrst measurement and after a minimum of one-week recovery from stereotaxic surgery, rats were habituated to being handled and restrained in the electrophysiology set-up with opaque goggles ﬁxed on the headstage. The homemade metal goggles displayed a 1300 mcd homoge- neous illumination of the visual ﬁeld to each eye individu- ally by white 2.5 mm diameter light LED sources at 10 mm from the eyeball. To obtain VEP recordings, the pin sockets of the electrodes were connected with ﬂexible cables to allow the rat to freely move its heads [29]. A preampliﬁer was connected to the headstage. Recording was performed in a dark room. 2.3. VEP Recording Stimuli were delivered at 1 Hz for 2.5 min for one eye and then switched to the fel- low eye after a 1 min break. The trigger signal was created using an isolated pulse stimulator (A-M Systems, USA) and was sent to the ampliﬁer of the acquisition system as a marker. The signal was ampliﬁed ×20 and was band-pass ﬁltered between 0.1 Hz–2 kHz using an 8- channel Porti system (Twente Medical Systems International B.V., Netherlands) and digitized with a 2 kHz sampling rate. The VEPs were analyzed in Matlab (Mathworks, Nattick, MA, USA) and EEGlab toolbox [30]. VEP ampli- tudes were measured by peak-to-peak analysis subtracting the signal of the contralateral nonstimulated eye. The ﬁnal statistical analysis was performed only with the data of the rats that performed all the recordings during the 4-week post-ONC; some rats removed their headstage throughout 2.6. Thallium Uptake. A thallium-chelate solution was administered 1 week after the end of the behavioural study and the implantation of the catheter, in a dark room during visual stimulation with a visual ﬂickering in the left eye through the goggles to perform thallium autometallography (TlAMG). TlAMG is based on the bioaccumulation of thal- lium ions that substitute potassium ions and accumulate in cells and neurites during neuronal activation through ATPase channels. Thallium is then ﬁxed by the perfusion of a sodium sulﬁde solution and is developed with silver for visualization under a microscope, as previously described [32, 33]. Brieﬂy, catheters were implanted in the jugular vein. After 2-3 days postoperation, the catheter was connected to a polyethylene tube and 1 ml of a freshly prepared 0.05% thal- lium diethyldithiocarbamate solution in 0.9% NaCl was slowly injected over a period of 4 min during visual stimula- tion. After rinsing, 2 ml of sodium sulﬁde solution (0.32% Na2S in 100 mM phosphate buﬀer pH7.4) and a sulﬁde glu- taraldehyde solution (0.16% Na2S and 3% glutaraldehyde in 100 mM phosphate buﬀer pH 7.4) were bolus injected. The brains were then removed and immersed overnight in acro- lein solution for ﬁxation and then cryoprotected for 48h in 30% sucrose in 0.1 M phosphate buﬀer, pH 7.4 at 4°C. The brains were frozen and cut with a Leica cryostat into 25 μm thick sections. Sections were air dried and treated with 0.1 N HCl to remove zinc sulﬁde. Neural Plasticity Neural Plasticity 3 the testing weeks and were therefore excluded from the ﬁnal analysis (sham, n = 3; ONC/DPZ, n = 3; and ONC/ saline, n = 6). the testing weeks and were therefore excluded from the ﬁnal analysis (sham, n = 3; ONC/DPZ, n = 3; and ONC/ saline, n = 6). one opening for 7 sec that the rat had to poke with its nose. After each correct choice, the rat was rewarded with a drop of water. For each trial, the target stimulus was randomly presented at a diﬀerent location so that the rat had to track the position changes with repeated nose pokes until 4 consecutive correct choices were made, and then the next lower brightness level was presented. The task always started with the level 1 target then the level of brightness decreased. The performance was calculated by the following formula: 2.4. Optic Nerve Crush. A bilateral partial optic nerve crush was performed under ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia [3, 22, 31]. The optic nerve was exposed from the lateral side of the eye and then crushed with calibrated forceps at a distance of 2-3 mm from the eye for 30 sec. Retinal blood supply and dura were left intact. An antibiotic eye ointment (Aureomycin; Lederle Arzneimittel GmbH, Wolfratshausen, Germany) was topically applied on both eyes after the surgery to prevent inﬂammation. In the sham group, the same surgery steps were made but the optic nerve was not crushed. % correct choices = number of correct choices total × 100 1 2.3. VEP Recording % correct choices = number of correct choices total × 100 1 2.3. VEP Recording 2. Methods 2.1. Animal Preparation. A total of 20 adult Lister hooded rats (10 weeks of age when delivered to the animal facilities) were maintained in a 12h light/dark cycle with ad libitum access to food and water during the adaptation period. Animals were handled for 10 minutes every day for 4 days before the beginning of the experiments. The rats were water Neural Plasticity 2.3. VEP Recording Then, sections were stained in a standard arabic gum developer used for autometallogra- phy [33, 34] for 150 min in the dark for the diﬀerent groups 4 Neural Plasticity Neural Plasticity (brightness discrimination, F 1,1 = 134 068, p = 0 000; suc- cess rate, F 1,1 = 26 868, p = 0 001). However, there was no interactionbetweentimeanddrug(brightnessdiscrimination, F 1,11 = 0 891, p = 0 551; success rate, F 1,11 = 1 138, p = 0 341). (sham, n = 4; ONC/DPZ, n = 3; and ONC/Saline, n = 6); some rats were excluded because they removed their head- stage, and some died after the catheter implantation. (brightness discrimination, F 1,1 = 134 068, p = 0 000; suc- cess rate, F 1,1 = 26 868, p = 0 001). However, there was no interactionbetweentimeanddrug(brightnessdiscrimination, F 1,11 = 0 891, p = 0 551; success rate, F 1,11 = 1 138, p = 0 341). Sections of interest containing V1 and subcortical struc- tures were selected according to Paxinos and Watson (1998) and photographed with a Fuji FinePix S2 Pro digital camera mounted on a Leica DMR microscope. Photographs were displayed using the Adobe Photoshop software for Macintosh. The NIH image software (ImageJ for MacOS X) was used for the analysis of thallium uptake patterns. The coloured photomicrographs were converted to gray scale images using unweighted conversions in Adobe Photoshop. Gray levels were determined for each animal at the level of V1 and were compiled for analysis. High gray values corre- spond to low staining intensity, that is, low neuronal activity. 3.2. Electrophysiology Results. In order to evaluate whether the cholinergic treatment had an eﬀect on the visual cortex and superior colliculus response after the crush, the visual evoked potentials were compared between the 3 groups: sham, ONC/DPZ, and ONC/saline (Figure 3). A substantial decrease of the amplitude of the VEPs was observed after the crush in both crush groups (ONC/DPZ V1 and SC ≈80 mV, ONC/saline V1 ≈100 mV, and SC ≈90 mV) in contrast to the VEPs in the sham group that remained high. VEP values were signiﬁcantly decreased in the ONC compared to the sham groups (Kruskal-Wallis, testing week 5, V1, H (2,9) = 6.385, p = 0 041 and SC, J (2,9) = 6.231, p = 0 044). These results show that the electrophysiological response was not restored throughout the post-ONC period. 2.7. 2.3. VEP Recording Statistical Analysis. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). In order to reveal the eﬀect of the crush within each group, the bright- ness discrimination thresholds and percentage of correct choices before and after the ONC surgery were tested using pairwise t-test. Secondly, for the behavioural data, the bright- ness discrimination thresholds and percentage of correct choices were compared between the ONC groups for time and drug eﬀects using mixed model ANOVA, with p < 0 05 being considered signiﬁcant. For the VEP and optic density analysis, and given that the rat number was small, the between-group diﬀerence was evaluated using nonparamet- ric Kruskal-Wallis test. This test was performed for each time point of the VEP measurement after ONC and for the TlAMG measurement performed at the end of the behav- ioural experiment. The Bonferroni correction was applied to compensate for multiple testing conditions, and post hoc pairwise comparisons were carried out with the signiﬁcance level set at p < 0 05. 3.3. Thallium Autometallography (TlAMG). Neuronal activ- ity was assessed 5 weeks postcrush in V1 using TIAMG (Figure 4). The optical density was measured on coronal sec- tions, high gray levels corresponding to low staining inten- sity, that is, low neuronal activity. The statistical analysis between groups showed no signiﬁcant diﬀerence of optical density between the 3 groups at this time point (Kruskal- Wallis, H 2,11 = 2 007, p = 0 375). 4. Discussion We tested the eﬀect of DPZ administration on the recovery of brightness discrimination in rats after a bilateral partial ONC. All ONC groups showed a signiﬁcant impairment of brightness discrimination thresholds (reduction of 60% from initial value) after the crush, followed by a gradual restoration of brightness discrimination (up to 40% of the initial value in the ONC-DPZ group). The rats treated with DPZ had an overall better performance than the rats treated with saline. Both groups had spontaneous recovery of brightness dis- crimination and success rate during the 4 weeks of post- ONC testing. DPZ treatment did not improve V1 cortical activity measured by VEP after the ONC. Together, these results suggest that DPZ may help visual recovery by enhanc- ing visual processing eﬃciency. 3. Results 3.1. Visual Stimulation Test. All the animals learned the brightness discrimination task easily and reached a discrimi- nation performance of at least 25% of brightness before the ONC (Figure 2). The success rate (percentage of correct choices) was over 90% for all the animals (Figure 2(a)). These levels of discrimination performance and success rate were maintained in the sham group after the sham-ONC surgery and during the four weeks of VIST testing (Figures 2(b) and 2(d)). In contrast, both brightness discrimination thresh- old (ONC/DPZ: t 5 = −12 649, p = 0 000; ONC/saline: t 6 = −12 871, p = 0 000) and success rate (ONC/DPZ: t 5 = 8 447, p = 0 000; ONC/saline: t 6 = 8 578, p = 0 000) were signiﬁcantly impaired by ONC in both ONC/DPZ and ONC/saline groups (62–67% reduction) (Figures 2(a) and 2(c)). 4.1. Optic Nerve Crush Induces an Impairment Followed by a Gradual Recovery of the Visual Capacities. In this study, we focused on the extent of brightness discrimination recovery following an ONC. All ONC groups had a signiﬁcant drop of performance after the crush which was followed by a grad- ual but partial signiﬁcant function recovery throughout the post-ONC period. Moreover, sham and both ONC groups showed similar TI+ uptake as evaluated 5 weeks post-ONC with optical density, indicative of a substantial neuronal activity in V1 in both groups. These results are in agreement with previous studies showing the same dynamics for both the behavioural VIST measurements [3] and thallium uptake AmixedmodelANOVAwasfurtherusedtodeterminethe eﬀect of the time and drug through the 4 weeks of testing post- ONC in the ONC groups (Figures 2(b) and 2(d)). There was a main eﬀect of time (brightness discrimination, F 1,11 = 5 990, p = 0 000; success rate, F 1,11 = 3 505, p = 0 000) and of drug 5 5 Neural Plasticity 6 weeks after ONC [35]. 3. Results However, when measured at diﬀerent i i h f TI+ k i ﬁ d d i h d pathways from the retina to the cortex and superior c ill d d d h h VEP i i i i ONC/DPZ ONC/saline Sham 0 20 40 60 80 100 Percentage of correct choices (%) Pre-ONC Post-ONC # ⁎ (a) ONC/DPZ ONC/saline Sham 1 2 3 4 5 6 7 8 9 10 11 12 Testing post-ONC 0 20 40 60 80 100 (b) ONC/DPZ ONC/saline Sham 0 20 40 60 80 100 Brightness discrimination threshold ⁎ # Pre-ONC Post-ONC (c) Testing post-ONC 0 20 40 60 80 100 1 6 5 4 3 2 7 12 11 10 9 8 ONC/DPZ ONC/saline Sham (d) Figure 2: Brightness discrimination capacity and visual performance of the rats before and after the partial optic nerve crush or sham The visual performance evaluated by the VIST was characterized for the 3 groups: sham (blue), ONC/DPZ (red), and ONC/saline Two parameters were calculated: the success rate (percentage of correct choices (a), (b)) and the brightness discrimination thres (d). VIST was performed before (pre-ONC) and after the ONC for 4 weeks. (a), (c) In comparison to the pre-ONC value, the ON and ONC/DPZ group percentage of correct choices (a) and brightness discrimination (c) showed a signiﬁcant reduction ONC (∗p < 0 01, ONC/saline compared to pre-ONC; #p < 0 01, ONC/saline compared to pre-ONC). (b), (d) Success rate and b discrimination were partially restored after the crush in both ONC/saline and ONC/DPZ groups, but the last group was performi than ONC/saline group. Points in (b), (d) represent the testing session number (3 tests per week). 1 2 3 4 5 6 7 8 9 10 11 12 Testing post-ONC 0 20 40 60 80 100 Sham 0 20 40 60 80 100 Percentage of correct choices (%) Pre-ONC Post-ONC # ⁎ 1 2 3 4 5 6 7 8 9 10 11 12 Testing post-ONC 0 20 40 60 80 100 Testing post-ONC (a) 20 40 60 80 100 Brightness discrimination threshold ⁎ # Testing post-ONC 0 20 40 60 80 100 1 6 5 4 3 2 7 12 11 10 9 8 Testing post-ONC (c) (d) Figure 2: Brightness discrimination capacity and visual performance of the rats before and after the partial optic nerve crush or sham surgery. 3. Results Amplitude of the VEP recorded before and once a week for four weeks after the ONC in the visual cortex (a), (c) and in the superior colliculus (b), (d) for the 3 groups: sham (blue), ONC/DPZ (red), and ONC/saline (green). (a), (b) VEP tracing examples recorded at week 5 in the visual cortex (a) and the superior colliculus (b). VEP amplitudes show a substantial drop in both ONC groups after the crush and a signiﬁcant reduction of the VEP amplitude compared to sham groups both in the visual cortex (c) and in the superior colliculus (d). This indicates that cortical and subcortical visually evoked activity was not restored after the crush for any treated groups. the remapping and the functional reorganization throughout the brain may be involved in vision recovery. Surviving ganglion cells, in combination with stronger cortico-cortical connections, can be considered to provide the mechanism of spontaneous recovery that occurs approximately within the 3 weeks post-ONC [41]. on the one hand, there is a partial recovery of visual performance of the animals and neuronal activity in V1 but, on the other hand, no visually induced responses, suggesting that the behavioural recovery is rather due to plastic changes within cortical circuitry. These results are in agreement with a structural- functional mismatch between behaviour and anatomical changes in the damaged optic nerve [41, 42]. It is possible that plasticity of neuronal activity takes place in V1. Several studies in the past have used elaborated optic nerve or retinal lesions to document cortical reorganization [43–47]. Follow- ing the loss of visual input, recovery of visual capacities are accompanied by plasticity of cortical circuits and cross- modal innervation in or near the lesioned area [48]. A strengthening of cortico-cortical connections is reported in animal models of retinal lesions to compensate for the loss of retinal input [49–51]. Furthermore, in humans with optic nerve damage, a recovery of their vision is observed following treatment with brain current stimulation, associated with a partial restoration of the network organization [52, 53]. These studies show that following visual system damage, both long-range lateral connections and large-scale func- tional connectivity networks are altered. This indicates that 4.2. Donepezil Improves Visual Function following Optic Nerve Crush. Our ﬁnding that the ONC/DPZ group shows better brightness discrimination performance compared to the ONC/saline group gives the ﬁrst evidence that the cholin- ergic system may be critical for the recovery process. 3. Results The visual performance evaluated by the VIST was characterized for the 3 groups: sham (blue), ONC/DPZ (red), and ONC/saline (green). Two parameters were calculated: the success rate (percentage of correct choices (a), (b)) and the brightness discrimination threshold (c), (d). VIST was performed before (pre-ONC) and after the ONC for 4 weeks. (a), (c) In comparison to the pre-ONC value, the ONC/saline and ONC/DPZ group percentage of correct choices (a) and brightness discrimination (c) showed a signiﬁcant reduction after the ONC (∗p < 0 01, ONC/saline compared to pre-ONC; #p < 0 01, ONC/saline compared to pre-ONC). (b), (d) Success rate and brightness discrimination were partially restored after the crush in both ONC/saline and ONC/DPZ groups, but the last group was performing better than ONC/saline group. Points in (b), (d) represent the testing session number (3 tests per week). 6 weeks after ONC [35]. However, when measured at diﬀerent time points thereafter, TI+ uptake is ﬁrst reduced in the dorso- lateral geniculate nucleus and cortex after ONC but a normal cortical activity is restored 6 weeks after the crush [35]. Whether this cortical activity is related to visual function is not known. However, the amplitude of VEP evoked by visual stimulation is not restored at any time point after the ONC in V1 nor in the superior colliculus. This suggests that the visual pathways from the retina to the cortex and superior colliculus are still damaged and that the VEP is insensitive to any dynamic change that impacts the retina. Some recent studies show axonal regeneration following optic nerve crush in mice [36, 37]. However, the functional recovery in our animals occurred as early as on week three postinjury, which is well ahead of any possible axonal regrowth which could add to functional improvement [3, 29, 38–40]. 3. Results Thus, 6 Neural Plasticity Visual cortex 300 0 (ms) 0 ‒300 ‒100 100 300 300 0 (ms) ‒40 ‒20 0 20 40 ‒40 ‒20 0 20 40 300 0 (ms) (a) Superior colliculus 0 300 0 (ms) ‒300 ‒100 100 300 ‒40 0 300 ‒20 0 20 40 (ms) ‒40 ‒20 0 20 40 300 0 (ms) (b) ONC/DPZ ONC/saline Sham 0 200 400 600 Pre-ONC Testing weeks Amplitude in mV 2 3 4 5 (c) 0 200 400 600 Pre-ONC Testing weeks 2 3 4 5 ONC/DPZ ONC/saline Sham (d) Figure 3: Visual evoked potentials recording in the primary visual cortex and the superior colliculus before and after the partial optic nerve crush. Amplitude of the VEP recorded before and once a week for four weeks after the ONC in the visual cortex (a), (c) and in the superior colliculus (b), (d) for the 3 groups: sham (blue), ONC/DPZ (red), and ONC/saline (green). (a), (b) VEP tracing examples recorded at week 5 in the visual cortex (a) and the superior colliculus (b). VEP amplitudes show a substantial drop in both ONC groups after the crush and a signiﬁcant reduction of the VEP amplitude compared to sham groups both in the visual cortex (c) and in the superior colliculus (d). This indicates that cortical and subcortical visually evoked activity was not restored after the crush for any treated groups. Visual cortex 300 0 (ms) 0 ‒300 ‒100 100 300 300 0 (ms) ‒40 ‒20 0 20 40 ‒40 ‒20 0 20 40 300 0 (ms) Superior colliculus 0 300 0 (ms) ‒300 ‒100 100 300 ‒40 0 300 ‒20 0 20 40 (ms) ‒40 ‒20 0 20 40 300 0 (ms) Superior colliculus 0 300 0 (ms) ‒300 ‒100 100 300 ‒40 0 300 ‒20 0 20 40 (ms) ‒40 ‒20 0 20 40 300 0 (ms) Visual cortex 300 0 (ms) 0 ‒300 ‒100 100 300 300 0 (ms) ‒40 ‒20 0 20 40 ‒40 ‒20 0 20 40 300 0 (ms) 0 0 ‒300 ‒100 100 300 Superior colliculus Visual cortex (a) 0 200 400 600 Pre-ONC Testing weeks Amplitude in mV 2 3 4 5 (a) (b) 0 200 400 600 Pre-ONC Testing weeks 2 3 4 5 (b) Amplitude in mV (d) (c) Figure 3: Visual evoked potentials recording in the primary visual cortex and the superior colliculus before and after the partial optic nerve crush. 3. Results g y y y p It has already been shown that the cholinergic system is involved in reinforcing the thalamocortical connections. For example, presynaptic nicotinic receptors on thalamocor- tical ﬁbres boost thalamic input to the cortex [54] and more generally ACh strengthens the feedforward pathway therefore enhancing sensory performance [55–58]. Since the visual information arriving from the thalamocortical pathway to V1 is diminished by the ONC, the ACh should have an enhancing eﬀect on the remaining thalamocortical connections, therefore facilitating the cortical responses of the residual cells. However, the VEPs did not improve in 7 Neural Plasticity Neural Plasticity 0 50 100 150 200 Optical density Neuronal activity ONC/DPZ ONC/saline Sham ‒ + Figure 4: Cortical neuronal activity measured by thallium autometallography. Optical density (0 (black) to 255 (white) gray levels) was measured within V1 on coronal sections of the brain of rats having been perfused with a thallium solution during monocular ﬂicker visual stimulation (see text). The high gray values correspond to low neuronal activity gradient (scale on the right). The optical density within the visual cortex was not signiﬁcantly altered 5 weeks after the ONC, as shown for the ONC/DPZ (red) and ONC/saline (green) groups, compared to sham group (blue). 0 50 100 150 200 Optical density Neuronal activity ONC/DPZ ONC/saline Sham ‒ + studies [11, 13, 16, 69, 70]. Precisely, by combining visual exposure with electrical stimulation of the cholinergic system, rats showed an improvement of visual acuity in a water maze and the potentiation of the visual cortical respon- siveness. Moreover, exposing rats to 2 weeks of visual stimuli coupled with DPZ administration enhances the visual cortical response [13]. Additionally, administration of cholinergic enhancers signiﬁcantly improves behavioural performance in a visual task and contrast detectability in the healthy rats [12, 18]. In our study, DPZ induced better performance in the behavioural task, suggesting that this eﬀect of cholinergic enhancement on cortical plasticity is also observed in our model of visual impairment. Neuronal activity p The improved recovery of visual performance in ONC/ DPZ group might also be due to the role of the cholinergic system in attention. ACh is shown to aﬀect the strength of the connections in the visual cortex and enhance the relevant stimuli by facilitating glutamatergic feedback [71]. In fact, an enhancement of cholinergic concentration in the cortex pro- motes attention [72–74]. 3. Results Furthermore, studies show that attentional cueing tasks improve vision restoration on patients with visual ﬁeld loss therefore facilitating the visual perception recovery [75, 76]. Additionally, cholinergic enhancement induces potentiation in the cortical responsive- ness regardless of the type of stimuli [77, 78]. Previous stud- ies in healthy animals show that cholinergic enhancement potentiates visual cortical response and visual performance [10, 13, 18, 21, 69, 70]. In human studies, enhancing the cho- linergic system with AChEIs improves the performance in visual and behavioural tasks that require attention [79–83], suggesting an improvement of eﬃciency of the visual processing. In our study, DPZ allowed better detection of relevant stimuli after the crush, which could result from increased attention capacity. Therefore, the implication of the cholinergic system in attentional processes might have aﬀected visual recovery post-ONC. ONC/DPZ ONC/saline ONC/saline Figure 4: Cortical neuronal activity measured by thallium autometallography. Optical density (0 (black) to 255 (white) gray levels) was measured within V1 on coronal sections of the brain of rats having been perfused with a thallium solution during monocular ﬂicker visual stimulation (see text). The high gray values correspond to low neuronal activity gradient (scale on the right). The optical density within the visual cortex was not signiﬁcantly altered 5 weeks after the ONC, as shown for the ONC/DPZ (red) and ONC/saline (green) groups, compared to sham group (blue). the visual cortex nor in the superior colliculus for the 4 weeks post-ONC in either group (DPZ or saline), suggesting that either VEPs or physiological activity in the remaining retina- visual cortex pathways is insensitive to the changes—although after ONC, thesurviving ganglion cells adapt tothe needof the visual functions and have a better activity [26, 59, 60]. In addition, donepezil could by itself have a protective eﬀect of ganglion cells in the retina [61, 62] but the VEP recordings from the present study show that the retino-cortical function was not restored after 21 days after the crush. These results are in line with other studies using the ONC model in animals where the VEPs are deteriorated immediately after the crush and do not show any recovery for weeks [63, 64]. Additionally, inthecaseofopticdiseaseinhuman,whichismimickedbythe rat ONC model and which leads to partial vision loss, electro- physiology and imaging studies prove that diminished visual brain function related to the optic neuritis eyes is correlated with the extent of the optic nerve damage [65–67]. Conflicts of Interest The authors declare that there is no conﬂict of interest regarding the publication of this paper. 3. Results Since the VEPs did not improve after ONC in the ONC/saline nor in ONC/DPZ groups, it suggests that the expected cholinergic enhancement of thalamocortical input is not eﬃcient enough to sustain the behavioural better performance of the rats. 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https://openalex.org/W2416024274	https://actamedica.lfhk.cuni.cz/media/pdf/18059694.2018.115.pdf	English	null	Age and Sex Variability of Initial Parts of the QRS Complex Displayed in Isointegral Maps of Young People	Acta Medica	2,004	cc-by	3,300	BIOPHYSICAL DAYS BIOPHYSICAL DAYS Introduction (F3: 48 women; M3: 41 men). None of measured subjects suffered by any cardiovascular disease or had any abnor- mality found neither on 12–lead standard electrocardio- grams or M-mode echocardiograms. Physiologic values of electrocardiographic parameters are important to be known before stating the diagnosis. Individual characteristics of isointegral maps (IIMs) are in- fluenced by different factors such as age or sex. They may contribute to relatively high variability among measured parameters of patients or control subjects. Although it is important to know how these factors may affect used IIM characteristics, isointegral mapping of controls in different age and sex groups is very rare (8,9,10,11). Only a very little is known about IIM parameters in children (1,3,4,5,6,8,9). In our study we tried to fill in this gap with amplitude ana- lysis of IIMs of initial parts of the QRS complex in children and young adults. We used the limited 24–lead system after Barr implanted in the mapping system ProCardio (2,12) for the recording of the electric heart field and data processing. During each examination a record of one second duration was registered (a single heartbeat) in supine position. Linear baseline was taken through T-P segments. The onset and offset of the QRS complex was established manually from the root mean square signal (7). We compared extreme values of IIMs (maximum, mini- mum, and peak-to-peak value = maximum – minimum) (7). For construction of IIMs we used following integration in- tervals: the first 20 ms (IIM QRS20), the first 30 ms (IIM QRS30), and the first 40 ms of QRS complex (IIM QRS40), and the interval from 20 ms to 40 ms of QRS complex (IIM QRS20–40). AGE AND SEX VARIABILITY OF INITIAL PARTS OF THE QRS COMPLEX DISPLAYED IN ISOINTEGRAL MAPS OF YOUNG PEOPLE Katarína Kozlíková, Juraj Martinka University, Faculty of Medicine, Bratislava, Slovak Republic: Institute of Medical Physics and Biophysics Comenius University, Faculty of Medicine, Bratislava, Slovak Republic: Institute of Medical Phy Summary: Although body surface electrocardiographic mapping is used also in clinical practice, there are only a few papers concerning the isointegral maps (IIMs) in children. We constructed IIMs of 169 healthy young people during the initial parts of the QRS complex: the first 20 ms (QRS20), 30 ms (QRS30), 40 ms (QRS40), and from 20 ms to 40 ms (QRS20–40). Subjects were divided into 6 groups: 9–10 y (F1, M1), 13–14 y (F2, M2), 18–19 y (F3; M3). We analysed the extreme va- lues of each time integral. We found maxima and peak-to-peak values decreasing with age, while minima tended to increase (became less negative). Most age differences were found in IIM QRS20–40 and between peak-to-peak values. Least differen- ces were between minima. Maxima and peak-to-peak values were higher in males than in females. No significant differences were found in any extreme value between groups F1 and M1 except for IIM QRS20 maxima. Significant sex differences in- creased with age. The only significant difference found in minima was between F2 and M2. Most sex differences were found in IIM QRS20. We assume that our findings can be explained (at least in part) by the influence of heart – chest geo- metry. Key words: Body surface mapping; Isointegral map; QRS complex; Children; Statistical analysis Patients and Methods We recorded and analysed IIMs of 169 young healthy people (90 females and 79 males). They were chosen from over 200 records of young people from which subjects re- vealing any electrocardiographic abnormality or any tech- nical problem of the record were excluded. Remaining subjects were distributed into six groups after their age and sex: 9–10 years (group F1: 17 girls; group M1: 15 boys), 13–14 years (F2: 25 girls; M2: 23 boys), and 18–19 years Statistical analysis was performed in following way using the program Statgraphics 3 plus (13). All data were tested for normal distributions using skewness and kurtosis. If they were found normal, variance check was done using Bartlett’s test (age differences). If variances were found to be the same, analysis of variance was used with Fisher’s least significant differences. If any of previous conditions 317 ACTA MEDICA (Hradec Králové) 2004;47(2):317–321 Fig. 1: Mean IIMs of group F3. Each rectangle displays chest surface with the anterior chest on the right and back on the left. Maximal and minimal values are marked by plus and minus signs, respectively, and displayed above each map. Step concerns the difference between two successive isointegral lines. Template (group) maps are marked as QoQ3 (IIM QRS30), QoQ4 (IIM QRS40), QoX4 (IIM QRS20), and X4Q4 (IIM QRS20–40). Fig. 1: Mean IIMs of group F3. Each rectangle displays chest surface with the anterior chest on the right and back on the left. Maximal and minimal values are marked by plus and minus signs, respectively, and displayed above each map. Step concerns the difference between two successive isointegral lines. Template (group) maps are marked as QoQ3 (IIM QRS30), QoQ4 (IIM QRS40), QoX4 (IIM QRS20), and X4Q4 (IIM QRS20–40). Fig. 2: Mean IIMs of group M3. Next description as in Figure 1. Fig. 2: Mean IIMs of group M3. Next description as in Figure 1. Tab. Values were not distributed normally. Statistical significance: p < 0.05; &p < 0.01; p < 0.005; &&p < 0.001; && Results Mean IIMs of each group revealed smooth dipolar di- stributions with one maximum and one minimum. The only exception was in the IIM QRS40 where an indication of se- cond minimum was found at the right upper back in women (group F3, Figure 1) and in the right clavicular area in men (group M3, Figure 2). Group extreme values of isointegral maps are given in Tables 1–3 with statistically significant differences found between corresponding sex groups. Values are given in form Tab. 2: Values of minimum in isointegral maps in all studied groups and differences between s es of minimum in isointegral maps in all studied groups and differences between sexes of the same age. Tab. 2: Values of minimum in isointegral maps in all studied groups and differences between sexes of the same age. Group F1 F2 F3 M1 M2 M3 IIM (n=17) (n=25) (n=48) (n=15) (n=23) (n=41) QRS20 -3.6 ± 4.6 # -1.6 ± 0.6 -1.6 ± 0.4 -2.8 ± 1.1 -2.2 ± 0.6 * -1.8 ± 0.6 # (-2.4) (-1.5) (-1.6) (-2.5) (-2.2) (-1.8) < -21.0; -1.0> < -3.0; -0.8> < -2.5; -0.9> < -5.3; -1.4> < -3.5; -0.8> < -3.7; -0.8> QRS30 -2.7 ± 0.6 -2.6 ± 1.1 # -2.7 ± 0.8 3.4 ± 1.2 -2.9 ± 1.0 -3.0 ± 1.2 # (-2.6) (-2.3) (-2.7) (-3.1) (-2.8) (-2.7) < -4.0; -1.8> < -5.4; -0.7> < -4.4; -0.9> < -5.7; -1.3> < -4.8; -0.6> < -8.0; -1.3> QRS40 -6.6 ± 3.0 # -6.4 ± 2.4 -5.3 ± 2.5 # -6.3 ± 2.3 -6.9 ± 3.0 # -5.4 ± 2.2 # (-6.3) (-5.6) (-4.6) (-6.0) (-6.1) (-4.9) < -14.7; -2.3> < -12.9; -2.4> < -13.1; -1.6> < -12.2; -2.9> < -17.4; -3.3> < -12.5; -2.5> QRS20–40 -8.0 ± 3.7 # -7.6 ± 2.5 -5.9 ± 2.8 # -7.6 ± 3.0 # -7.7 ± 3.4 -6.0 ± 3.3 # (-7.1) (-6.6) (-5.0) (-6.9) (-6.4) (-5.2) < -18.3; -3.0> < -12.9; -4.9> < -15.1; -2.3> < -16.6; -4.0> < -18.0; -3.3> < -18.6; -2.1> # Values were not distributed normally. Statistical significance: p < 0.005. # Values were not distributed normally. Statistical significance: *p < 0.005. Tab. 3: Values of peak-to-peak in isointegral maps in all studied groups and differences between sexes of the same age. Tab. Patients and Methods Statistically significant differences were assumed for p < 0.05 or less. of mean ± standard deviation, median in parentheses, range is given below them in form of closed interval. Statistically significant differences concerning the age are given in Tables 4–6. If the comparison is not given, it was not signi- ficant. We found mean maxima and mean peak-to-peak values decreasing with age in all evaluated groups of both sexes (Tables 1 and 3). This trend was valid also for their medians. Minima behaved in a different way (Table 2). In most cases there were the lowest (deep) minima in the youngest child- ren except for IIM QRS40 and IIM QRS20–40 in group M2. The highest (shallow) minima tended to be in the oldest groups except for IIM QRS30 in males where it was found in group M2. Most age differences were found in IIM QRS20–40, which displayed the activation comprising the middle of the QRS complex, and between peak-to-peak va- lues. Least differences were between minima. As more than one third of data sets were not distributed normally, we have taken medians to perform these comparisons. Patients and Methods 1: Values of maxima in isointegral maps in all studied groups and differences between sexes of the s Group F1 F2 F3 M1 M2 M3 IIM (n=17) (n=25) (n=48) (n=15) (n=23) (n=41) QRS20 5.1 ± 1.8 4.1 ± 1.4 3.8 ± 1.3 # 6.7 ± 2.5 5.0 ± 1.5 * 4.6 ± 1.4 (5.4) (3.7) (3.8) (6.5) (4.9) * (4.4) & < 2.2; 8.6> < 1.7; 6.9> < 1.3; 9.3> < 3.0; 12.0> < 2.1; 7.6> < 2.6; 8.5> QRS30 12.9 ± 4.0 11.2 ± 4.0 9.9 ± 3.0 # 16.7 ± 6.7 14.0 ± 4.1 * 12.8 ± 4.1 (12.0) (10.4) (9.8) (15.7) (13.6) * (12.9) && < 5.7; 21.7> < 5.7; 19.3> < 3.7; 20.2> < 6.8; 29.7> < 6.7; 19.6> < 6.6; 21.8> QRS40 23.4 ± 8.0 18.8 ± 6.9 # 14.6 ± 5.2 # 23.3 ± 7.0 23.1 ± 6.7 20.7 ± 6.8 (21.6) (16.8) (14.1) (23.0) (22.5) * (21.2) &&& < 11.7; 42.2> < 7.0; 37.6> < 6.3; 32.7> < 12.6; 36.0> < 14.5; 38.3> < 9.2; 43.1> QRS20–40 24.4 ± 7.1 17.9 ± 6.2 # 13.5 ± 4.8 # 23.0 ± 5.7 22.6 ± 6.5 # 18.9 ± 6.5 # (22.9) (15.9) (12.1) (21.7) (21.1) ** (19.5) &&& < 12.6; 39.8> < 7.4; 35.4> < 6.0; 30.2> < 14.8; 30.8> < 14.6; 38.5> < 7.1; 42.5> # Values were not distributed normally. Statistical significance: p < 0.05; &p < 0.01; p < 0.005; &&p < 0.001; &&&p < 0.0001. Tab. 1: Values of maxima in isointegral maps in all studied groups and differences between sexes of the same age. # Values were not distributed normally. Statistical significance: p < 0.05; &p < 0.01; *p < 0.005; &&p < 0.001; &&&p < 0.0001. 318 was not fulfilled, Kruskal-Wallis test and Mann-Whitney test for medians were used. Sex differences were compared between corresponding age groups in pairs. For normal data Student’s t-test was used considering equal or non- equal variances after Fisher’s test. Else Mann-Whitney test for medians was used. Statistically significant differences were assumed for p < 0.05 or less. was not fulfilled, Kruskal-Wallis test and Mann-Whitney test for medians were used. Sex differences were compared between corresponding age groups in pairs. For normal data Student’s t-test was used considering equal or non- equal variances after Fisher’s test. Else Mann-Whitney test for medians was used. Values were not distributed normally. Statistical significance: p < 0.05; p < 0.005; &&p < 0.001; p < 0.0 Results IIM F1–F2 F2–F3 F1–F3 M1–M2 M2–M3 M1–M3 QRS20 p < 0.01 NS p < 0.001 p < 0.01 NS p < 0.0005 QRS30 NS NS p < 0.01 NS NS NS QRS40 NS p < 0.005 p < 0.0005 NS NS NS QRS20–40 p < 0.01 p < 0.0005 p < 0.0001 NS p < 0.05 p < 0.05 NS: not significant Tab. 6: Significant age differences between medians of groups for peak-to-peak values. IIM F1–F2 F2–F3 F1–F3 M2–M3 M1–M3 QRS20 p < 0.005 NS p < 0.0001 NS p < 0.001 QRS40 NS p < 0.05 NS p < 0.05 NS QRS20–40 NS p < 0.005 p < 0.05 p < 0.05 p < 0.05 NS: not significant Tab. 5: Significant age differences between medians of groups for minima. IIM F1–F2 F2–F3 F1–F3 M1–M2 M1–M3 QRS20 NS NS p < 0.01 p < 0.05 p < 0.005 QRS30 NS NS p < 0.01 NS NS QRS40 p < 0.05 p < 0.01 p < 0.0001 NS NS QRS20–40 p < 0.005 p < 0.005 p < 0.0001 NS p < 0.05 Tab. 4: Significant age differences between medians of groups for maxima. Tab. 4: Significant age differences between medians of groups for maxima. IIM F1–F2 F2–F3 F1–F3 M2–M3 M1–M3 QRS20 p < 0.005 NS p < 0.0001 NS p < 0.001 QRS40 NS p < 0.05 NS p < 0.05 NS QRS20–40 NS p < 0.005 p < 0.05 p < 0.05 p < 0.05 NS i ifi Tab. 5: Significant age differences between medians of groups for minima. Tab. 5: Significant age differences between medians of groups for minima. NS: not significant IIM F1–F2 F2–F3 F1–F3 M1–M2 M2–M3 M1–M3 QRS20 p < 0.01 NS p < 0.001 p < 0.01 NS p < 0.0005 QRS30 NS NS p < 0.01 NS NS NS QRS40 NS p < 0.005 p < 0.0005 NS NS NS QRS20–40 p < 0.01 p < 0.0005 p < 0.0001 NS p < 0.05 p < 0.05 NS: not significant Tab. 6: Significant age differences between medians of groups for peak-to-peak values. Tab. 6: Significant age differences between medians of groups for peak-to-peak values. Mean maxima and mean peak-to-peak values were high- er in males than in corresponding females except for groups F1 and M1 in IIM QRS40 and IIM QRS20–40 (Tables 1 and 3). This was also valid for medians. Results No significant differen- ces were found in any extreme value when comparing the youngest girls and boys except for maxima in IIM QRS20. The number of significant sex differences increased with in- creasing age. The only significant difference found in sex minima comparison was between groups F2 and M2 (Table 2). Most sex differences were found at the very beginning of the QRS complex (IIM QRS20). years) were found in IIMs during the whole QRS complex and corresponded with our findings (8,9). These maps were recorded in sitting position using 80 chest electrodes. When looking at these findings, there still remains the question why we found decreasing values with increasing age in young people. One possible answer is the influence of heart geometry, as well as the geometry heart – chest. Or are the changes during puberty responsible for the diffe- rences? Results 3: Values of peak-to-peak in isointegral maps in all studied groups and differences between sexes Group F1 F2 F3 M1 M2 M3 IIM (n=17) (n=25) (n=48) (n=15) (n=23) (n=41) QRS20 8.7 ± 4.9 # 5.7 ± 1.5 5.4 ± 1.4 # 9.5 ± 2.6 7.2 ± 1.8 * 6.4 ± 1.7 (7.3) (5.7) (5.5) (9.5) (7.3) & (6.2) *** < 5.0; 26.0 > < 3.5; 8.8 > < 2.2; 10.7 > < 5.3; 15.3 > < 3.6; 9.8 > < 3.6; 10.4 > QRS30 15.6 ± 4.2 13.7 ± 4.7 12.6 ± 3.5 # 20.1 ± 7.4 16.9 ± 4.6 * 15.8 ± 4.8 (14.4) (12.7) (12.2) (18.5) (16.3) * (15.8) ** < 8.2; 25.5 > < 7.2; 23.1 > < 4.6; 24.5 > < 8.1; 35.4 > < 8.9; 22.6 > < 8.5; 26.2 > QRS40 29.9 ± 9.9 25.3 ± 8.8 # 19.9 ± 7.1 # 29.6 ± 8.3 30.0 ± 7.8 26.1 ± 8.4 (26.5) (23.2) (18.4) (29.6) (28.0) * (27.6) *** < 14.0; 53.6 > < 9.4; 47.8 > < 7.9; 43.8 > < 16.7; 43.7 > < 19.5; 47.1 > < 12.0; 53.2 > QRS20–40 32.4 ± 9.4 25.4 ± 7.9 # 19.4 ± 7.1 # 30.6 ± 7.2 30.3 ± 8.3 # 24.9 ± 9.1 # (30.0) (22.1) (17.7) (30.5) (28.3) * (23.6) && < 15.6; 52.2 > < 14.0; 46.2 > < 8.5; 43.0 > < 18.8; 39.9 > < 19.9; 51.9 > < 9.2; 56.6 > # Values were not distributed normally. Statistical significance: p < 0.05; p < 0.005; &&p < 0.001; *p < 0.0005. # Values were not distributed normally. Statistical significance: p < 0.05; p < 0.005; &&p < 0.001; *p < 0.0005. 319 IIM F1–F2 F2–F3 F1–F3 M1–M2 M1–M3 QRS20 NS NS p < 0.01 p < 0.05 p < 0.005 QRS30 NS NS p < 0.01 NS NS QRS40 p < 0.05 p < 0.01 p < 0.0001 NS NS QRS20–40 p < 0.005 p < 0.005 p < 0.0001 NS p < 0.05 NS: not significant Tab. 4: Significant age differences between medians of groups for maxima. Discussion and Conclusions 1. Asano Y, Izumida N, Kiyohara K et al. Diagnosis of right ventricular overload by body surface QRST isointegral maps in children with postoperative right bundle branch block. J Electrocardiol 1995;28(3):209–21. We did not found published results of IIMs during the initial parts of QRS complex in children. Similar sex diffe- rences were found in two 2 published papers. In the first one, Q-zone maps (from the beginning of the QRS complex to its middle, comparable with our IIM QRS40) were ana- lysed in adults (20–46 years old) (10). Forty male subjects had significantly higher maxima than 15 females. Dif- ferences between minima were not significant. Published maps were recorded with 117 chest leads. In the second paper, QR maps (comparable with our IIM QRS40) were analysed in 18 men and 25 women (18–19 years old) (11). Higher extreme values were found in men. 2. Barr RC, Spach MS, Herman-Giddens GS. Selection of the number and positions of measuring locations for electrocardiography. IEEE Trans Biomed Eng 1971; 18(2):125–38. 3. Izumida N, Asano Y, Kiyohara K et al. QRST isointegral map reflects the sudden reduction of right ventricular pressure after balloon pulmonary valvuloplasty. J Electrocardiol 1995;28(3):223–9 4. Izumida N, Asano Y, Kiyohara K et al. Precordial leads QRST time integrals for evaluation of right ventricular overload in children with congenital heart diseases. J Electrocardiol 1997;30(3):257–64. 5. Izumida N, Asano Y, Wakimoto H et al. Analysis of T wave changes by activation recovery interval in patients with atrial septal defect. Int J Cardiol 2000; 74(2–3):115–24. 6. Izumida N, Kiyohara K, Asano Y et al. The body surface QRST isointegral maps in infants with right ventricular overload. Jpn Circulat J 1993;57(2): 123–30. 7. Kozlíková K. Body surface integral maps, their characteristics and methods of quantitative analysis. (in Slovak) Bratisl Med J 1990;91(11):815–23. Age differences were analysed in 2 papers. In both, sig- nificantly higher values of maxima in younger children (9–10 years) and deeper minima in older children (13–14 8. Kozlíková K, Turzová M, Tyšler M et al. Body surface integral mapping of ven- tricular activation in boys during puberty. (in Slovak) Bratisl Med J 1988; 89(9):694–705. 320 9. 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RNDr. Katarína Kozlíková, Ph.D., Comenius University, Faculty of Medicine, Institute of Medical Physics and Biophysics, Sasinkova 2, SK 813 72 Bratislava, Slovak Republic. e-mail: katarina.kozlikova@fmed.uniba.sk Doc. RNDr. Katarína Kozlíková, Ph.D., Comenius University, Faculty of Medicine, Institute of Medical Physics and Biophysics, Sasinkova 2, SK 813 72 Bratislava, Slovak Republic. e-mail: katarina.kozlikova@fmed.uniba.sk Slovak Republic. 321 321
https://openalex.org/W2273397289	http://internext.espm.br/internext/article/download/310/242	Portuguese	null	Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas	Internext	2,015	cc-by	8,865	Revista Eletrônica de Negócios Internacionais São Paulo, v.10, n. 3, p. 1-14, set./dez. 2015 \| e-ISSN: 1980-4865 \| http://internext.espm.br Revista Eletrônica de Negócios Internacionais São Paulo, v.10, n. 3, p. 1-14, set./dez. 2015 \| e-ISSN: 1980-4865 \| http://internext.espm.br RESUMO DETALHES DO ARTIGO Histórico do artigo: Recebido em 14 de janeiro de 2015 Aceito em 27 de julho de 2015 Disponível online em 19 de dezembro de 2015 Sistema de Revisão “Double Blind Review” Editor científico: Felipe Mendes Borini Palavras-chaves: Distância institucional Determinantes de exportação Recursos da empresa Histórico do artigo: Recebido em 14 de janeiro de 2015 Aceito em 27 de julho de 2015 Disponível online em 19 de dezembro de 2015 Sistema de Revisão “Double Blind Review” Editor científico: Felipe Mendes Borini Palavras-chaves: Distância institucional Determinantes de exportação Recursos da empresa Histórico do artigo: Recebido em 14 de janeiro de 2015 Aceito em 27 de julho de 2015 Disponível online em 19 de dezembro de 2015 Sistema de Revisão “Double Blind Review” Editor científico: Felipe Mendes Borini Palavras-chaves: Distância institucional Determinantes de exportação Recursos da empresa O objetivo deste artigo é avaliar o efeito da distância sobre o desempenho exportador das empresas de Santa Catarina e em que medida tal efeito é moderado pelas características dos recursos organizacionais. Foi utilizada regressão linear múltipla e análise de variância a partir de levantamento da percepção dos gestores de exportação em uma amostra final de 49 empresas produtoras exportadoras. Os constructos exibiram validade interna e possibilitam a análise dos dados. Os resultados apresentaram apenas evidências sobre o efeito da distância psíquica, apontando para uma relação positiva com o desempenho de exportação. Além disso, a estimação do modelo mostrou que os recursos da organização moderam a relação entre distância e desempenho exportador. Finalmente, o estudo mostra também que a equipe de exportação e a estrutura organizacional moderam o efeito da distância sobre o desempenho e expectativa futura de desempenho das empresas Palavras-chaves: Distância institucional Determinantes de exportação Recursos da empresa © 2015 Internext \| ESPM. Todos os direitos reservados! © 2015 Internext \| ESPM. Todos os direitos reservados! 1 Contado do autor: Email: givanildo@givanildo.adm.br São Paulo, v.10, n. 3, p. 1-14, set./dez. 2015 \| e-ISSN: 1980-4865 \| http://internext.espm.br Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas Givanildo Silva1A, Mohamed AmalA, Gérson TontiniA e Gustavo da Rosa BorgesB A Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil B Universidade Federal do Pampa – UFP, Dom Pedrito, RS, Brasil DETALHES DO ARTIGO 1. Introdução sociedade; e regras formais: constituições, leis, regras, sanções e direitos de propriedade (NORTH, 1991). A distância institucional é influenciada pelas percepções dos gestores, por seus entendimentos e opiniões sobre a política e as condições institucionais dos países estrangeiros (GHEMAWAT, 2001). Distância cultural é relacionada às semelhanças nos valores, normas e religião que facilitam a interação e a comunicação (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). As multinacionais precisam acessar e utilizar o conhecimento relacionado aos costumes locais e outras condições externas afetadas pela cultura local (BARKEMA; BELL; PENNINGS, 1996). Uma maior distância cultural aumenta os desafios e o tempo necessário para a empresa se ajustar de forma eficaz no novo ambiente, ou seja, pode-se incorrer em maiores custos de ajuste dinâmico (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). Em estudo realizado na China, o desempenho superior das exportações está alinhado com a visão baseada em recursos e a distância institucional (HE; BROUTHERS; FILATOTCHEV, 2012). No Brasil, verificou-se efeito negativo moderador do efeito da distância psíquica na rentabilidade das exportações, que pode ser explicada considerando que alguns custos tendem a ser menores nos países desenvolvidos, enquanto outros tendem a serem mais elevados (CARNEIRO; ROCHA; SILVA, 2011). Além de considerar as diferenças culturais, a distância institucional trata de restrições concebidas pelas pessoas por meio de estruturas políticas, interações econômicas e sociais, e pode ser classificada em forças informais: valores, crenças, sanções, tabus, costumes, tradições, códigos de conduta, comportamentos comuns e práticas da Na visão baseada em recursos (VBR), as capacidades podem ser vistas como conjuntos de ativos tangíveis e intangíveis, incluindo as competências de gestão da empresa, processos, © 2015 Internext \| ESPM. Todos os direitos reservados! DOI: dx.doi.org/10.18568/1980-4865.1031-14 G. Silva, M. Amal, G. Tontini e G. R. Borges 2 rotinas organizacionais, informações e conhecimentos. Nos negócios internacionais, a VBR ajuda a especificar a natureza dos recursos necessários para superar o passivo da internacionalização (liability of foreignness- LOF) e indica um método para investigar os recursos referentes à definição de produtos e diversificação internacional (BARNEY; WRIGHT; KETCHEN, 2001). rotinas organizacionais, informações e conhecimentos. Nos negócios internacionais, a VBR ajuda a especificar a natureza dos recursos necessários para superar o passivo da internacionalização (liability of foreignness- LOF) e indica um método para investigar os recursos referentes à definição de produtos e diversificação internacional (BARNEY; WRIGHT; KETCHEN, 2001). 2.1 Desempenho exportador Os conceitos apresentados por Platchek, Floriani e Borini (2012, p. 73) explicam o desempenho exportador, pois citam Aulakh, Kotabe e Teegen (2000), que avaliaram três dimensões, sendo, “estratégia competitiva, nível de padronização versus adaptação da oferta e grau de diversificação geográfica das exportações”. Os autores também mencionaram Leonidou et al. (2002), que decompuseram em três níveis de análise, sendo, “influência das variáveis de estratégia de exportação sobre o desempenho em geral, efeito das variáveis de estratégia de exportação sobre medidas específicas de desempenho e variações dos efeitos das variáveis de estratégia de exportação sobre o desempenho”; bem como apontaram Madsen (1989), que selecionou fatores que explanassem o desempenho exportador, sendo, “a estratégia de marketing de exportação e as características da empresa”. Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 1. Introdução das exportações, crescimento das receitas da empresa, intensidade de exportação, experiência internacional da empresa, grau de diversificação geográfica, estratégia de marketing de exportação, atratividade do mercado externo, barreiras à exportação, distância geográfica, distância psíquica, expectativas e características dos gerentes, entre outros (PLATCHEK; FLORIANI; BORINI, 2012). e organizacionais, Pesquisa com 195 empresas industriais exportadoras da China concluiu que o desempenho superior das exportações está alinhado com fatores baseados em recursos e a distância institucional. A análise empírica sugere que, depois de considerar a influência moderadora da distância institucional, alinhando recursos internos com o canal de exportação, há maior desempenho das exportações. Os resultados sugerem que a distância institucional entre os mercados domésticos e de exportação modera esta relação, aumentando valor ao se usar canais hierárquicos quando as empresas possuem fortes capacidades de orientação para o mercado ou a necessidade de parceiros quando as empresas não têm tais capacidades (HE; BROUTHERS; FILATOTCHEV, 2012). O presente trabalho tem como objetivo de analisar os determinantes de desempenho exportador a partir dos efeitos das distâncias psíquica, cultural e institucional, e pela perspectiva da visão baseada em recursos. Para atender este objetivo, a pergunta de pesquisa deste estudo é: Quais os efeitos das distâncias sobre o desempenho exportador de empresas? E, em que medida, tais efeitos são moderados pelos recursos da empresa? Este artigo compõe-se de cinco seções. Após esta introdução apresenta-se a revisão de literatura e as hipóteses de pesquisa. A terceira seção descreve o método, técnica de pesquisa, constructos, variáveis, modelo moderador e amostra. Na quarta seção são expostos e discutidos os resultados das análises de regressão linear múltipla e rotação Varimax. As considerações finais, os impactos perante demais estudos e limitações desta investigação são mostrados ao final. Estudo realizado com amostra final de 389 grandes exportadores brasileiros de produtos manufaturados verificou efeito negativo moderado da distância psíquica na rentabilidade das exportações, que pode ser explicado considerando que alguns custos tendem a ser menores nos países desenvolvidos (transporte e questões legais), enquanto outros tendem a serem mais elevados (publicidade e promoção). Também era esperada uma relação negativa dos efeitos da distância, contudo não foram encontrados efeitos significativos. Uma possível explicação é que os gestores possam prestar mais atenção às operações em mercados psiquicamente distantes, com o objetivo de se preparar para dificuldades maiores do que o encontrado (CARNEIRO; ROCHA; SILVA, 2011). 2.3 Distância cultural Uma das principais dificuldades relacionadas a expansão internacional é a distância entre os novos contextos locais e os ambientes em que a organização já atua, sendo que a dimensão crítica é a distância cultural (GHEMAWAT, 2001). A distância cultural é relacionada às semelhanças nos valores, normas e religião que facilitam a interação e a comunicação entre pessoas de países distintos, por meio de significados compartilhados, entendimentos tácitos, expectativas de contextos e de comportamentos. Desta forma, na medida em que as culturas são diferentes, tais entendimentos ficam debilitados e a comunicação torna-se mais difícil (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). Quanto maior a distância cultural entre os países exportadores e importadores, mais difícil será identificar e interpretar corretamente as informações disponíveis sobre o mercado externo (SOUSA; BRADLEY, 2006). As multinacionais precisam acessar e utilizar os conhecimentos relacionados aos costumes locais e outras condições externas afetadas pela cultura local (BARKEMA; BELL; PENNINGS, 1996). O processo de internacionalização de empresas de Uppsala propõe que as empresas multinacionais iniciam suas atividades exportadoras pelos países mais próximos antes de buscarem mercados em regiões com maior distância psíquica (JOHANSON; VAHLNE, 1977). Este modelo foi revisado considerando que não importam as adversidades de ser uma empresa estrangeira em um país distante, mas sim o apoio recebido de um parceiro local. Pode ser difícil de iniciar a expansão internacional caso a multinacional não tenha aderência as redes de negócios em contextos locais desconhecidos. Desta forma, as relações existentes em um novo ambiente são mais importantes do que as distâncias entre os países da matriz e da filial (JOHANSON; VAHLNE, 2009). Portanto, quanto mais familiar for o país estrangeiro, menor será a distância psíquica. Contudo, ao se subestimar o grau de adaptação necessária ao entrar em um mercado que se percebe como familiar e semelhante ao mercado doméstico, pode-se incorrer no paradoxo distância psíquica decorrente de assimetrias nas percepções psíquicas de distância. Por exemplo, a expansão de empresas dos EUA sobre o Canadá, bem como, no resto do mundo, relacionado a grandes países e seus pequenos vizinhos (HÅKANSON; AMBOS, 2010). Pesquisas indicam que as percepções, comportamentos e crenças das pessoas são orientados pela sua cultura em nível nacional, ou seja, os indivíduos de países diferentes pensam e agem de forma diversa, devido a sua cultura (KIRKMAN; LOWE; GIBSON, 2006; LEUNG et al., 2005). Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 3 dos custos de movimentação de bens físicos (transporte, tarifas e outros) (BECKERMAN, 1956). O fenômeno da empresa “nascida global” que rapidamente entra em mercados muito distantes sugere, por exemplo, que a variância nas percepções de distâncias psíquicas de gestores individuais talvez aumentasse, dramaticamente, ao longo do tempo (HÅKANSON; AMBOS, 2010). Outra abordagem teórica e operacionalmente mais simples é medir a distância psíquica direta e, posteriormente, avaliar em que medida a distância cultural, as diferenças linguísticas e outros fatores, impactam nestas percepções (SOUSA; BRADLEY, 2006). A distância psíquica trata da percepção implícita de como o indivíduo avalia as diferenças entre os países estrangeiros por meio das formas de relações familiares, hábitos e costumes (DICHTL; KOEGLMAYR; MUELLER, 1990). Extrapolando para a teoria de negócios internacionais, a distância psíquica trata da percepção média referente a determinado país, que contribui para interpretar o comportamento médio das empresas daquele país, sendo que as normas de interação e comunicação empresarial podem ser altamente influenciadas pelo nível de desenvolvimento econômico dos países (DOW; KARUNARATNA, 2006). 2.2 Distância psíquica e o modelo de Uppsala revisitado A distância psíquica foi observada na tendência de países concentrarem suas relações comerciais em países mais próximos, em que os contatos estrangeiros já foram pessoalmente estabelecidos e desenvolvidos, muito além do que a distância econômica pode explicar. Desta forma, a distância psíquica foi conceituada pelo seu aspecto subjetivo em relação a distância econômica objetiva, que trata O desempenho exportador pode ser mensurado pelo volume de receitas de exportação, lucratividade Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 2.4 Distância institucional A premissa básica da teoria institucional é que as tendências das empresas em relação a conformidade com normas e tradições predominantes em cada contexto social leva a homogeneidade entre as empresas, em suas estruturas e atividades, e que as bem sucedidas são aquelas que ganham apoio e legitimidade, em conformidade com as pressões sociais (MEYER; ROWAN, 1977; OLIVER, 1997). Todos os atores institucionais podem impor diferentes pressões coercivas, miméticas e normativas sobre os gestores. As pressões coercitivas têm forças impostas pelos governos. As pressões miméticas ocorrem por meio da imitação organizacional ou por modelagem de normas e práticas no campo institucional da organização. E, as pressões normativas têm suas origens na profissionalização da indústria ou de membros do setor que tentam definir as condições e métodos para seu trabalho, buscando legitimar sua autonomia profissional (DIMAGGIO; POWELL, 1983; OLIVER, 1997). Instituições são restrições concebidas pelas pessoas, por meio de estruturas políticas e interações econômicas e sociais, podendo ser classificadas em forças informais (valores, crenças, sanções, tabus, costumes, tradições, códigos de conduta, comportamentos comuns e práticas da sociedade) e regras formais (constituições, leis, regras, sanções, direitos de propriedade), com o objetivo de criar ordem e reduzir a incerteza econômica (NORTH, 1991). A partir dos estudos sobre distância e desempenho exportador, apresentamos a primeira hipótese desta investigação que, logo em seguida, é dividida nas distâncias psíquica, cultural e institucional: Outra perspectiva de análise das instituições pode ser a área de política (corrupção, transparência), a legislação (liberalização econômica, regime regulatório) e a sociedade (normas éticas, atitudes em relação ao empreendedorismo). As instituições podem ser analisadas como variáveis independentes, em uma visão baseada em estratégia institucional, que se centra na interação dinâmica entre instituições e organizações, e considera-se as escolhas estratégicas como resultado dessa interação (PENG; WANG; JIANG, 2008). As instituições nacionais fornecem uma estrutura de incentivos para a economia que, por meio da evolução, moldam a direção da mudança para o crescimento econômico, estagnação ou declínio (NORTH, 1991).  Hipótese 1: Quanto maior a distância entre o país de origem da empresa exportadora e o país de destino, menor é o desempenho exportador da organização.  Hipótese 1a: Quanto maior a distância psíquica entre o país de origem da empresa exportadora e o país de destino, menor é o desempenho exportador da organização. 2.3 Distância cultural Portanto, negócios internacionais em culturas distantes precisam considerar a gestão de conflitos, tomada de decisões, interação dos membros das equipes, fornecedores e clientes, que apresentam diferentes crenças, percepções e processos cognitivos (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). Acredita-se que as distâncias psíquicas, como convencionalmente medidas pelas distâncias culturais, não são simétricas, ou seja, as distâncias percebidas de A para B e de B para A não são necessariamente iguais (SHENKAR, 2001). Dados de pesquisa descritiva confirmam a existência de considerável assimetria e a análise estatística sugere algumas das fontes desse problema (HÅKANSON; AMBOS, 2010). A diversidade cultural existente, caracterizada pelo conjunto de contextos locais em que a organização opera atualmente, tem efeito negativo sobre a taxa de expansão internacional, pois aumenta Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 G. Silva, M. Amal, G. Tontini e G. R. Borges G. Silva, M. Amal, G. Tontini e G. R. Borges 4 a complexidade da governança interna e ambiental, no tocante a gerenciar as relações internas da rede de empresas da multinacional, bem como as relações com as partes interessadas externas e, pode, portanto, desestimular o aumento ainda maior da distância cultural (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). políticas e judiciais pouco desenvolvidas, onde os costumes podem ser regidos por regras informais e convenções que podem parecer estranhas, ineficientes ou mesmo corruptas e imorais (GHEMAWAT, 2001). As atividades das multinacionais podem sofrer pressões institucionais em nível interorganizacional, que surgem de fontes externas, tais como governos, mercados e sociedade (HOFFMAN, 2001), ou em nível organizacional, a partir da cultura, sistemas de crenças compartilhadas e processos políticos (DIMAGGIO; POWELL, 1983), e também dos acionistas (HENRIQUES; SADORSKY, 1996). 2.5 Visão Baseada em Recursos A visão baseada em recursos contribui para especificar a natureza dos recursos necessários para superar a responsabilidade do estrangeirismo (BARNEY; WRIGHT; KETCHEN, 2001). A multinacional deve recombinar sua base de recursos existentes aqueles necessários no ambiente exterior, o que é um processo demorado dado a responsabilidade do parceiro local (JOHANSON; VAHLNE, 2009). O desafio é, portanto, desenvolver experiência gerencial que possa suportar tal recombinação de recursos e a coordenação em vários contextos. A empresa multinacional também deve aumentar sua base de recursos existentes e construir novas capacidades organizacionais, tanto para gerenciar a complexa estrutura interna, bem como a sua rede externa (MEYER; MUDAMBI; NARULA, 2011). Os estudos sobre gestão estratégica sugerem que as empresas obtêm vantagens competitivas sustentáveis por meio da implementação de estratégias que exploram suas forças e evitam suas fraquezas internas, enquanto neutralizam as ameaças externas. Os quatro indicadores empíricos dos recursos potenciais das organizações para gerar vantagem competitiva sustentável proposta são: ser valioso, raro entre os concorrentes, difícil de imitar e sem substitutos estrategicamente equivalentes. O papel dos gestores na interpretação e análise das capacidades dos recursos controlados pela organização é fundamental para a obtenção de vantagem competitiva sustentada (BARNEY, 1991). A visão baseada em recursos não considera o contexto social em que as decisões são tomadas, referente a seleção e incorporação de recursos com objetivo de heterogeneidade da organização. Desta forma, faz-se necessário um modelo de vantagem competitiva sustentável que considere a convergência entre a teoria institucional e a visão baseada em recursos. Em nível individual, o modelo proposto por Oliver (1997) analisa a racionalidade normativa, referente a custos cognitivos e conformidade com as tradições. Em nível organizacional, consideram-se os fatores institucionais, tais como, cultura e política adotadas internamente. E, em nível social, tratam-se das pressões isomórficas, no tocante ao governo, profissões, alianças entre as empresas, entre outros (OLIVER, 1997). Conclui-se que os gestores precisam examinar cuidadosamente antes de empreender muitos projetos de expansão internacional em novos contextos locais, e avaliar a probabilidade efetiva de implementação, considerando-se a distância cultural adicionada. Bem como, é importante estar ciente de que, com o aumento da diversidade cultural, o ritmo e o alcance da expansão internacional da multinacional podem ser reduzidos (HUTZSCHENREUTER; VOLL; VERBEKE, 2011). A visão baseada em recursos pode ser analisada pelas características dos recursos da organização, que podem moderar o efeito da distância sobre o desempenho exportador. 2.4 Distância institucional  Hipótese 1b: Quanto maior a distância cultural entre o país de origem da empresa exportadora e o país de destino, menor é o desempenho exportador da organização. A distância institucional é influenciada pelas percepções dos gestores, por seus entendimentos e opiniões sobre a política, e as condições institucionais dos países estrangeiros. Essas diferenças relacionam- se ao ambiente regulatório de um país, que pode ser transparente e com estruturas de governança eficientes. Desta forma, mais fáceis de compreender e se relacionar. Ou, por outro lado, com instituições  Hipótese 1c: Quanto maior a distância institucional entre o país de origem da empresa exportadora e o país de destino, menor é o desempenho exportador da organização. Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 5 Figura 1. Descrição gráfica do modelo Fonte: Autores Figura 1. Descrição gráfica do modelo Fonte: Autores Figura 1. Descrição gráfica do modelo 3.1 Questionário O questionário com 46 perguntas sobre os determinantes de desempenho exportador utilizou escala Likert de 7 pontos, e baseou-se no formulário de Carneiro, Rocha e Silva (2011). A partir da revisão de literatura, as adaptações do questionário tratam dos atributos relacionados às hipóteses de pesquisa, sendo, distâncias, recursos organizacionais e desempenho exportador. O questionário consolidou- se após aplicação de pré-teste com gerente de exportação de empresa de grande porte, com objetivo de avaliar a linguagem e a compreensão das questões do instrumento de pesquisa.  Hipótese 2b: A estrutura organizacional tem um efeito moderador positivo na relação entre a distância e o desempenho exportador.  Hipótese 2c: O departamento de exportação tem um efeito moderador positivo na relação entre a distância e o desempenho exportador. A Figura 1 descreve o modelo referente as hipóteses das relações entre os efeitos das distâncias psíquica, cultural e institucional sobre os determinantes de desempenho exportador, bem como, as influências moderadoras dos recursos da organização: sendo, características da equipe de exportação, estrutura da empresa e o departamento de exportação. O questionário também contou com 12 perguntas sobre produto, país de exportação, viagem ao país, tipo de exportação, perspectivas futuras da atividade, receita total, percentual de exportação e outras, bem como, 8 questões referente a informações gerais da empresa. Os resultados são apresentados no perfil da amostra por meio de estatística descritiva. 2.5 Visão Baseada em Recursos Neste estudo, os recursos da organização foram analisados em termos de características da: (1) da equipe de exportação, (2) estrutura organizacional e (3) departamento de Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 G. Silva, M. Amal, G. Tontini e G. R. Borges 6 exportação. Desta forma, apresentamos a Hipótese 2 deste estudo: variáveis moderadoras de recursos da organização sobre a relação das variáveis independentes de distâncias e variáveis dependentes de desempenho de exportação. Não foram realizadas as rotações Varimax, pois tratam-se de variáveis moderadoras.  Hipótese 2: Os recursos da organização tem um efeito moderador positivo na relação entre a distância e o desempenho exportador.  Hipótese 2a: A equipe de exportação tem um efeito moderador positivo na relação entre a distância e o desempenho exportador. 3.2 Constructos e variáveis 3.2.1 Variáveis dependentes O desempenho exportador foi mensurado a partir da percepção de satisfação dos gestores em relação aos resultados da exportação do principal produto informado pelas empresas, no tocante ao valor total das exportações, margem de lucro, crescimento no volume e, resultados gerais (lucro, volume, posição competitiva, relacionamento com clientes, etc.) de 2011 a 2013. 3. Metodologia Este estudo trata de pesquisa descritiva que apresenta determinada situação por meio de medições de um fenômeno, atividade ou evento (HAIR Jr. et al., 2005). As hipóteses foram testadas pela análise de regressão linear múltipla, que “é um modelo mais realista porque, no mundo em que vivemos, a previsão quase sempre depende de vários fatores, não de um só” (HAIR Jr. et al., 2005, p. 326). A análise de variância foi verificada pela ANOVA que “é usada para examinar diferenças estatísticas entre as medidas de dois ou mais grupos” (HAIR Jr. et al., 2005, p. 321). Foi realizada rotação Varimax para confirmação das dimensões das variáveis independentes de distâncias, sendo, psíquica, institucional e cultural. Tabela 1.1 á Tabela 1.1 Variáveis Dependentes - Desempenho Exportador Desempenho de Exportação Exportação para Outros Países Expectativa Resultados Futuros Valor total Valor total Resultados gerais Margem de lucro Crescimento Quantidade Crescimento Lucratividade Crescimento Resultados gerais Quantidade X Concorrente Lucratividade Alfa de Cronbach: 0,81 0,69 0,88 Fonte: Dados da pesquisa. Tabela 1.1 Variáveis Dependentes - Desempenho Exportador Variáveis Dependentes - Desempenho Exportador Conforme Vieira e Faia (2014), para moderação dos recursos organizacionais sobre a relação entre distância e desempenho de exportação, aplicaram-se os seguintes passos: (1) todas as variáveis foram padronizadas, ou seja, normalizadas com a centralização das variáveis contínuas; (2) criaram-se termos multiplicativos entre as diversas variáveis moderadoras e as variáveis e; (3) testaram-se as regressões destas novas variáveis em conjunto com as moderadoras e independentes. Considerando o mesmo período, analisaram-se as percepções referentes aos resultados de exportação Foi aplicado método de inserção das variáveis por etapas (stepwise) para cálculo dos efeitos das Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 7 Tabela 1.2 Variáveis Independentes - Efeitos da Distância Constructo Variável Autores Alfa de Cronbach Distância Psíquica Confiança nas pessoas Dow and Karunaratna (2006) 0.66 Grau de profissionalização Grau de exigência dos produtos Distância Cultural Costumes e tradições Hutzschenreuter et al. (2011) 0.79 Ética e códigos de conduta Normas e práticas de negócios Comunicação e linguagem Distância Institucional Estrutura política North (1991) 0.84 Estrutura econômica Estrutura tecnológica Ambiente regulatório Fonte: Dados da pesquisa. confiança nas pessoas daquele país, grau de profissionalização das pessoas naquele país, estrutura política do país de exportação, estrutura econômica, estrutura tecnológica, ambiente regulatório com a constituição e leis daquele país, e grau de exigência dos produtos daquele país. comparativamente as exportações deste produto para outros países em relação ao valor total, crescimento do volume e lucratividade. A quantidade do produto exportado foi analisada em relação aos resultados dos concorrentes. E, para os próximos 3 anos, verificou-se as expectativas de resultados futuros de exportação, considerando uma taxa de câmbio em torno de R$ 2,10/dólar, referente a resultados gerais, quantidade, crescimento da quantidade e lucratividade. A Tabela 1.1 resume as variáveis dependentes referente ao desempenho exportador, avaliadas nesta pesquisa. A Tabela 1.2 apresenta os constructos das distâncias, variáveis, autores e consistência interna por meio do Alfa de Cronbach. E, a Tabela 1.3 confirma os componentes por meio da rotação Varimax das distâncias. Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 3.2.2 Variáveis independentes Acredita-se que a equipe de exportação é um dos recursos que as empresas podem utilizar para diminuir as distâncias psíquica, cultural e institucional. As equipes foram avaliadas referentes à suas práticas em relação à experiência internacional, habilidade em idiomas, tempo gasto em viagens As distâncias foram mensuradas a partir dos resultados referentes a costumes locais e tradições do país de exportação, ética e códigos de conduta, normas e práticas de negócios daquele país, comunicação e linguagem do país de exportação, Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Tabela 1.3 Rotação Varimax das Distâncias Variáveis Componentes Institucional Cultural Psíquica Estrutura política 0.89 Estrutura econômica 0.87 Ambiente regulatório 0.81 Estrutura tecnológica 0.59 Ética códigos de conduta 0.86 Costumes e tradições 0.85 Normas práticas negócios 0.61 Grau de exigência 0.9 Confiança nas pessoas 0.76 % Variação 31.46 22.36 20.45 Alfa Cronbach 0.84 0.79 0.66 Fonte: Dados da pesquisa. Tabela 1.3 G. Silva, M. Amal, G. Tontini e G. R. Borges 8 Tabela 1.4 Variáveis Moderadoras - Recursos da Organização Equipe de Exportação Estrutura da Organização Departamento de Exportação Experiência internacional da equipe Tipo de produto Grau de autonomia Habilidade em idiomas Tamanho da empresa Regras de comportamento Tempo gasto atualmente em viagens internacionais Idade da empresa Níveis hierárquicos Conhecimento do mercado do país de exportação Experiência em exportação Especialização dos cargos Domínio das formas de negociação Inovação atual em produtos Local do departamento Domínio das finanças internacionais Competências e capacidades Domínio dos aspectos econômicos Planejamento estratégico de exportação Suporte ao cliente estrangeiro Domínio da legislação internacional Cronbach Alpha: 0.86 0.79 0.89 Fonte: Dados da pesquisa. Tabela 1.4 Fonte: Dados da pesquisa. Ministério do Desenvolvimento, Indústria e Comércio Exterior, 2014). No mês de junho de 2014, foram enviados 870 questionários pelos correios, e realizados 4 envios para listagem com 636 e-mails e efetuadas 329 ligações telefônicas para solicitar o preenchimento dos formulários desta pesquisa. Obtivemos 49 respostas, sendo que a amostra é composta por 3,24% empresas produtoras que exportaram seus produtos nos últimos anos. internacionais e conhecimento do mercado do país de exportação. As equipes também foram analisadas referentes ao domínio das formas de negociação, finanças internacionais, aspectos econômicos e legislação internacional do país de exportação. A estrutura da empresa é outro recurso importante na avaliação das distâncias, sendo mensurada quanto ao tipo de produto, tamanho e idade da empresa, tempo de experiência em exportação, inovação de produtos, competências e capacidades da empresa e o planejamento estratégico de exportação. Outro recurso considerado é o departamento de exportação, relativo ao grau de autonomia, regras de comportamento da equipe, níveis hierárquicos, especialização dos cargos, local do departamento de exportação e o suporte prestado ao cliente estrangeiro. A Tabela 1.4 resume as variáveis moderadoras desta pesquisa. Em média, as empresas respondentes exportam há 16 anos (de 2 a 60 anos de atividade exportadora) para uma média de 11 países diferentes (até 72 países). No total, as 49 empresas possuem 15.142 funcionários no Brasil e 4.457 empregados no exterior. Os principais produtos exportados pelas empresas respondentes são máquinas e equipamentos mecânicos, peças fundidas em aço, compensados, móveis, portas de madeiras, esquadrias, molduras, têxteis, revestimentos cerâmicos, papel seda, calçados, sucos, duchas eletrônicas e outros. Fonte: Dados da pesquisa Tabela 2 Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Tipo de exportação Freq. % % válida % acum. Indireta (por meio de trading ou de empresa comercial exportadora ou de agente de compra sediados no Brasil) 6 12.2 12.2 12.2 Direta via agentes (por meio de agentes ou representantes da própria empresa sediados no exterior) 16 32.7 32.7 44.9 Direta sem agentes (para distribuidores ou para empresas de importação sediados no exterior; ou para subsidiárias de distribuição ou instalações da sua própria empresa no exterior; ou diretamente para clientes no exterior) 27 55.1 55.1 100 Total 49 100 100 Fonte: Dados da pesquisa 3.3 Amostra Para 27 empresas o tipo de exportação, apresentado na Tabela 2, é exportação direta sem agentes. Outras 16 empresas exportam de forma No estado de Santa Catarina constam 1.512 empresas exportadoras ativas em 2013 (MDIC- Tabela 2 Tipo de exportação Tabela 3 Fonte: Dados da pesquisa. (Tabela 3). Na etapa 2, verifica-se o efeito moderador dos recursos organizacionais (Tabela 4.1). Na etapa 3, procede-se ao teste do efeito moderador da equipe de exportação (Tabela 4.2). Nas etapas 4 e 5, respectivamente testam-se os efeitos da estrutura organizacional (Tabela 4.3) e do departamento de exportação (Tabela 4.4). direta via agentes (por meio de agentes ou representantes da própria empresa sediados no exterior). Demais 6 exportam de forma indireta (por meio de trading ou de empresa comercial exportadora, ou de agente de compra sediados no Brasil). Nenhuma utiliza consórcio de exportação (por meio de ação cooperada entre um grupo de empresas). As hipóteses H1, H1a, H1b e H1c tratam dos efeitos da distância sobre o desempenho exportador, em que, quanto maiores as distâncias psíquica, cultural e institucional entre o país de origem da empresa exportadora e o país de importação o qual é o destino dos produtos, menor é o desempenho exportador da organização. E, no instrumento de pesquisa respondido pelas 49 empresas produtoras 4. Estimação do modelo e discussão dos resultados A estimação do modelo de análise dos efeitos das distâncias sobre o desempenho exportador dá-se em quatro principais etapas. Na primeira etapa, estimam-se apenas os efeitos das três distâncias Total Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 9 Tabela 3 Efeitos das distâncias sobre os desempenhos Modelo 1a Desempenho Exportação Modelo 1b Exportação Outros Países Modelo 1c Desempenho Futuro B Sig. VIF B Sig. VIF B Sig. VIF Dist. Psíquica 0,224 0,177 1,304 0,297 0,068 1,310 0,058 0,733 1,310 Dist. Cultural 0,108 0,549 1,587 0,096 0,585 1,593 0,051 0,789 1,593 Dist. Institucional 0,022 0,891 1,268 0,038 0,807 1,274 -0,040 0,814 1,274 F 1,470 2,336 0,114 R2 0,091 0,135 0,008 ajustado 0,029 0,077 -0,059 Fonte: Dados da pesquisa. Tabela 3 Efeitos das distâncias sobre os desempenhos Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo Fonte: Dados da pesquisa. Tabela 4.1 Tabela 4.1 Moderação dos recursos organizacionais sobre a relação entre distância e desempenho Modelo 1a Desempenho Exportação Modelo 1b Exportação Outros Países Modelo 1c Desempenho Futuro B Sig. B Sig. B Sig. Distância Psíquica ns ns ns ns ns ns Distância Cultural ns ns ns ns ns ns Distância Institucional ns ns ns ns ns ns Equipe de Exportação ns ns ns ns 0.293 0.041* Estrutura Organização 0.429 0.002* ns ns ns ns Departamento Export. ns ns ns ns ns ns Psíquica X Equipe ns ns 0.410 0.003* ns ns Cultural X Equipe ns ns ns ns ns ns Institucional X Equipe ns ns ns ns ns ns Psíquica X Estrutura ns ns ns ns ns ns Cultural X Estrutura ns ns ns ns ns ns Institucional X Estrutura ns ns ns ns ns ns Psíquica X Depto ns ns ns ns ns ns Cultural X Depto ns ns ns ns ns ns Institucional X Depto ns ns ns ns ns ns F 10.600 9.514 4.423 R2 0.187 0.168 0.086 ajustado 0.170 0.151 0.067 Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo Fonte: Dados da pesquisa. Moderação dos recursos organizacionais sobre a relação entre distância e desempenho Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 G. Silva, M. Amal, G. Tontini e G. R. Borges 10 Tabela 4.2 Moderação da equipe de exportação sobre a relação entre distância e desempenho Modelo 1a Desempenho Exportação Modelo 1b Exportação Outros Países Modelo 1c Desempenho Futuro B Sig. B Sig. B Sig. Distância Psíquica ns ns ns ns ns ns Distância Cultural ns ns ns ns ns ns Distância Institucional ns ns ns ns ns ns Equipe de Exportação ns ns ns ns 0.293 0.041* Psíquica X Equipe 0.322 0.024* 0.410 0.003* ns ns Cultural X Equipe ns ns ns ns ns ns Institucional X Equipe ns ns ns ns ns ns F 5.435 9.514 4.423 R2 0.106 0.168 0.086 ajustado 0.086 0.151 0.067 Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo. Fonte: Dados da pesquisa. Tabela 4.1 bela 4.2 deração da equipe de exportação sobre a relação entre distância e desempenho Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo. Fonte: Dados da pesquisa. Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo. Fonte: Dados da pesquisa. Analisando o conjunto de variáveis independentes, o F também possui pouca significância (Sig. 1,470, 2,336 e 0,114), tampouco, a variação nas variáveis independentes (R2 0,091, 0,135 e 0,008), explicando, no máximo, apenas 13,5% do desempenho exportador comparativamente com outros países. Portanto, os resultados não confirmam as hipóteses H1b e H1c, para a amostra desta pesquisa. Analisando o conjunto de variáveis independentes, o F também possui pouca significância (Sig. 1,470, 2,336 e 0,114), tampouco, a variação nas variáveis independentes (R2 0,091, 0,135 e 0,008), explicando, no máximo, apenas 13,5% do desempenho exportador comparativamente com outros países. Portanto, os resultados não confirmam as hipóteses H1b e H1c, para a amostra desta pesquisa. exportadoras, as questões referente as distâncias foram mensuradas com escalas referente ao desempenho de exportação, sendo de 1 (forte barreira) até 7 (forte incentivo). Conforme Tabela 3, somente a distância psíquica (H1a) se relaciona com o desempenho de exportação do mesmo produto para outros países (Sig. 0,068). E, constata-se que nenhuma das demais médias das variáveis dos constructos das distâncias psíquica, cultural e institucional apresentaram significância estatística com as médias das variáveis de determinantes de desempenho, sendo, desempenho entre 2011 e 2013, desempenho exportador comparativamente com outros países, e expectativa de desempenho exportador para os próximos 3 anos. A Tabela 4.1 apresenta a análise da influência dos recursos organizacionais sobre a relação entre distância e desempenho. Foi utilizada a regressão linear múltipla com método de inserção das variáveis em etapas (stepwise) e, na amostra pesquisada, verificou-se relação entre a estrutura da organização e o desempenho de exportação (Sig. 0,002). A variável multiplicadora “distância psíquica X equipe de exportação” também apresenta relação com a Tabela 4.4 Tabela 4.4 Moderação do departamento de exportação sobre a relação entre distância e desempenho Modelo 1a Desempenho Exportação Modelo 1b Exportação Outros Países Modelo 1c Desempenho Futuro B Sig. B Sig. B Sig. Distância Psíquica ns ns 0.352 0.013* ns ns Distância Cultural ns ns ns ns ns ns Distância Institucional ns ns ns ns ns ns Departamento Export. ns ns ns ns ns ns Psíquica X Depto 0.309 0.031* ns ns ns ns Cultural X Depto ns ns ns ns ns ns Institucional X Depto ns ns ns ns ns ns F 4.968 6.628 ns R2 0.097 0.124 ns ajustado 0.078 0.105 ns Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo Fonte: Dados da pesquisa. Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo Fonte: Dados da pesquisa. positiva com a expectativa de desempenho futuro (Sig. 0,041), admitindo em parte a hipótese H2c. Referente a hipótese H2b, apenas a variável multiplicadora demonstrou relação significativa. Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 11 exportação para países que não são o destino principal das exportações (Sig. 0,003). Por fim, a equipe de exportação apresenta relação com a expectativa de desempenho futuro nos próximos três anos (Sig. 0,041). desempenho exportador atual, exportação para outros países e expectativa de resultados futuros também apresentaram bons indicadores de Alfa de Cronbach. Finalmente, os constructos das variáveis moderadoras de equipe de exportação, estrutura da organização e departamento de exportação, igualmente possuem validade interna, o que possibilita a análise das hipóteses deste estudo. Para análise da influência da equipe de exportação sobre a relação entre a distância e o desempenho de exportação, a Tabela 4.2 apresenta este efeito moderador. A variável multiplicadora “distância psíquica X equipe de exportação” exibe relaçãoempenho de exportação (Sig. 0,024) e a exportação para outros países (Sig. 0,003). E, a equipe de exportação também apresenta relação com a expectativa de desempenho futuro (Sig. 0,041). Somente a distância psíquica possui relação positiva com o desempenho de exportação para outros países (Sig. 0,068). Portanto, para a amostra pesquisada, apresentaram-se apenas evidências sobre a hipótese H1a. As demais hipóteses H1, H1b e H1c não foram demonstradas, pois, não há significância entre as distâncias cultural e institucional em relação aos desempenhos de exportação atual, para outros países e desempenho futuro. A moderação da estrutura da organização destaca relação com o desempenho de exportação (Sig. 0,002), conforme Tabela 4.3, assim como, a distância psíquica aponta relação com a exportações para outros países (Sig. 0,013). As hipóteses H2, H2a, H2b, e H2c tratam do efeito moderador dos recursos da organização sobre a relação entre a distância e o desempenho exportador. Na análise de todas as variáveis moderadoras em conjunto, a estrutura da organização assinala relação positiva com o desempenho de exportação (Sig. 0,002), confirmando parcialmente a hipótese H2a. Assim como, a equipe de exportação destaca relação Por fim, na Tabela 4.4, a análise da influência do departamento de exportação sobre a relação entre a distância e o desempenho, apresenta relação entre a variável multiplicadora “distância psíquica X departamento de exportação” e o desempenho exportador (Sig. 0,031) e relação entre a distância psíquica e a exportação para outros países (Sig. 0,013). Tabela 4.3 Tabela 4.3 Moderação da estrutura da organização sobre a relação entre distância e desempenho Modelo 1a Desempenho Exportação Modelo 1b Exportação Outros Países Modelo 1c Desempenho Futuro B Sig. B Sig. B Sig. Distância Psíquica ns ns 0.352 0.013* ns ns Distância Cultural ns ns ns ns ns ns Distância Institucional ns ns ns ns ns ns Estrutura Organização 0.429 0.002* ns ns ns ns Psíquica X Estrutura ns ns ns ns ns ns Cultural X Estrutura ns ns ns ns ns ns Institucional X Estrutura ns ns ns ns ns ns F 10.600 6.628 ns R2 0.187 0.124 ns ajustado 0.170 0.105 ns Método de inserção das variáveis: Em etapas (Critérios: Probabilidade de F a ser inserido <= ,090, Probabilidade de F a ser removido >= ,100). ** para p<0,01; * para p<0,05; ns: não significativo Fonte: Dados da pesquisa. Moderação da estrutura da organização sobre a relação entre distância e desempenho Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 Efeitos das distâncias e recursos da empresa no desempenho exportador de empresas 5. Considerações Finais Na amostra pesquisada, pode-se afirmar que os constructos dos efeitos das distâncias psíquica, cultural e institucional possuem consistência interna. Da mesma forma, as variáveis dependentes de Na análise somente do efeito moderador da equipe Na análise somente do efeito moderador da equipe Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 G. Silva, M. Amal, G. Tontini e G. R. Borges 12 de exportação sobre a relação entre distância e desempenho, demonstram-se evidências sobre a hipótese H2c, em que a equipe de exportação possui relação positiva com o desempenho futuro (Sig. 0,041). A variável multiplicadora “distância psíquica X equipe” apontou significância com o desempenho de exportação e, também, com a exportação para outros países, confirmando em parte o efeito moderador previsto nas hipóteses H2a e H2b. Por fim, não é possível comparar o estudo de Hutzschenreuter, Voll e Verbeke (2011), pois este trata dos efeitos da distância cultural existente e adicionada, em relação a diversificação e a expansão internacional no período subsequente. As conclusões elencadas limitam-se a amostra pesquisada, e conforme apresentado, precisam-se considerar as quantidades das variações nas variáveis dependentes associadas com todas as variáveis independentes consideradas em conjunto (medida de adequação). A principal limitação deste estudo foi o baixo interesse das empresas exportadoras em participar da pesquisa e responder o questionário enviado pelos correios e listagem de e-mails. No exame da influência moderadora da estrutura da organização sobre a relação entre distância e desempenho, considera-se parcialmente aceita a hipótese H2a, pois, a estrutura da organização aponta relação positiva com o desempenho exportador (Sig. 0,002). Nesta moderação, a distância psíquica apresenta relação positiva com a exportação para outros países (Sig. 0,013). 6. Referências  AULAKH, P.; KOTABE, M.; TEEGEN, H. Export Strategies and Performance of Firms from Emerging Economies: Evidence from Brazil, Chile, and Mexico. Academy of Management Journal, v.43, n.3, p.342-361, 2000. E, por fim, a moderação do departamento de exportação sobre a relação entre distância e desempenho não apresenta resultados que coadunam com as hipóteses do estudo. Apenas a variável multiplicadora “distância psíquica X departamento” possui relação com o desempenho exportador e, a distância psíquica apresenta significância com a exportação para outros países.  BARKEMA, Harry; BELL, John; PENNINGS, J. M. E. Foreign entry, cultural barriers and learning. Strategic Management Journal, v. 17, n. 2, p. 151-166, 1996. O presente estudo não corrobora as conclusões de He, Brouthers e Filatotchev (2012), em que o desempenho superior das exportações está alinhado com os fatores baseados em recursos e a distância institucional. A análise empírica sugere que, depois de considerar a influência moderadora da distância institucional, alinhando os recursos internos com o canal de exportação, há maior desempenho das exportações (HE; BROUTHERS; FILATOTCHEV, 2012).  BARNEY, Jay; WRIGHT, Mike; KETCHEN, David J. The resource-based view of the firm: Ten years after 1991. Journal of Management, v. 27, n. 6, p. 625-641, 2001. DOI 10.1177/014920630102700601  BARNEY, Jay. Firm resources and sustained competitive advantage. Journal of Management, v. 17, n. 1, p. 99-120, 1991.  BECKERMAN, W. Distance and the pattern of intra- European trade. The Review of Economics and Statistics, v. 38, n. 1, p. 31-40, 1956. DOI 10.2307/1925556 Ainda, esta pesquisa não confirma os achados de Carneiro, Rocha e Silva (2011) com 389 grandes exportadores brasileiros de produtos manufaturados, que verificou efeito negativo moderado da distância psíquica na rentabilidade das exportações. Segundo estes autores, as receitas de exportação mostraram uma forte associação positiva com o estado da atividade exportadora e uma forte associação negativa com barreiras no país de acolhimento. A rentabilidade das exportações mostrou uma forte associação positiva com a sistematização de planejamento de exportação e uma associação negativa moderada com a distância psíquica e com barreiras no país de acolhimento (CARNEIRO; ROCHA; SILVA, 2011).  CARNEIRO, Jorge; ROCHA, Angela da; SILVA, Jorge Ferreira da. Determinants of export performance: a study of large Brazilian manufacturing firms. BAR - Brazilian Administration Review, v. 8, n. 2, p. 107- 132, 2011. DOI 10.1590/s1807- 76922011000200002  CARNEIRO, Jorge; ROCHA, Angela da; SILVA, Jorge Ferreira da. 6. Referências Determinants of export performance: a study of large Brazilian manufacturing firms. BAR - Brazilian Administration Review, v. 8, n. 2, p. 107- 132, 2011. 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DOI 10.1057/jibs.2009.24  PLATCHEK, Renata Bertoldi; FLORIANI, Dinorá Eliete; BORINI, Felipe Mendes. A Influência do Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015 14 G. Silva, M. Amal, G. Tontini e G. R. Borges  SOUSA, Carlos MP; BRADLEY, Frank. Cultural distance and psychic distance: two peas in a pod? Journal of International Marketing, v. 14, n. 1, p. 49- 70, 2006.  SOUSA, Carlos MP; BRADLEY, Frank. Cultural distance and psychic distance: two peas in a pod? Journal of International Marketing, v. 14, n. 1, p. 49- 70, 2006. Grau de Internacionalização no Desempenho das Empresas Têxteis. Revista Gestão Organizacional, v. 5, n. 1, p. 70-81, 2012.  SHENKAR, Oded. Cultural distance revisited: Towards a more rigorous conceptualization and measurement of cultural differences. Journal of International Business Studies, v. 32, n. 3, p. 519- 535, 2001. DOI 10.1057/palgrave.jibs.8490982  VIEIRA, Valter Afonso; FAIA, Valter da Silva. Efeitos Moderadores Duplos e Triplos na Análise de Regressão. XXXVIII Encontro da Anpad. Rio de Janeiro, 2014.  VIEIRA, Valter Afonso; FAIA, Valter da Silva. Efeitos Moderadores Duplos e Triplos na Análise de Regressão. XXXVIII Encontro da Anpad. Rio de Janeiro, 2014. Sobre os autores  Givanildo Silva é Doutorando em Administração pela Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: givanildo@givanildo.adm.br  Givanildo Silva é Doutorando em Administração pela Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: givanildo@givanildo.adm.br  Mohamed Amal é Doutor em Engenharia da Produção. Professor no Programa de Pós-graduação em Administração e no departamento de Economia na Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: mohamedamal.amal@gmail.com  Mohamed Amal é Doutor em Engenharia da Produção. Professor no Programa de Pós-graduação em Administração e no departamento de Economia na Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: mohamedamal.amal@gmail.com  Gérson Tontini é Doutor em Engenharia Mecânica. Professor titular da Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: gersontontini@gmail.com  Gérson Tontini é Doutor em Engenharia Mecânica. Professor titular da Fundação Universidade Regional de Blumenau – FURB, Blumenau, SC, Brasil. E-mail: gersontontini@gmail.com  Gustavo da Rosa Borges é Doutor em Administração. Professor titular da Universidade Federal do Pampa – UFP, Dom Pedrito, RS, Brasil. E-mail: gustavodarosaborges@gmail.com  Gustavo da Rosa Borges é Doutor em Administração. Professor titular da Universidade Federal do Pampa – UFP, Dom Pedrito, RS, Brasil. E-mail: gustavodarosaborges@gmail.com ABSTRACT Article history: Received 14 January de 2015 Accepted 27 July 2015 Available online in 19 December 2015 Double Blind Review System Scientific Editor Felipe Mendes Borini Keywords: Institutional distance Export determinants Company resources Article history: Received 14 January de 2015 Accepted 27 July 2015 Available online in 19 December 2015 Double Blind Review System Scientific Editor Felipe Mendes Borini Keywords: Institutional distance Export determinants Company resources Article history: Received 14 January de 2015 Accepted 27 July 2015 Available online in 19 December 2015 The purpose of this article is to evaluate the effect of distance on the export performance of companies in Santa Catarina, and to what extent this effect is moderated by organizational resource characteristics. Multiple linear regression and variance analysis were used for a perception survey of export managers with a final sample of 49 exporting producers. The constructs showed internal validity and allowed the data to be analyzed. The results only revealed evidence regarding the effect of psychic distance, showing a positive relationship with export performance. Also, the model estimation showed that the organization's resources moderate the relation between distance and export performance. Finally the study also shows that the export team and the organizational structure moderate the effect of distance on the performance and future performance expectations of the companies. © 2015 Internext \| ESPM. All rights reserved! © 2015 Internext \| ESPM. All rights reserved! Internext \| São Paulo, v.10, n. 3, p. 1-14, set./dez., 2015
https://openalex.org/W2991274106	https://europepmc.org/articles/pmc7005080?pdf=render	English	null	Clinical outcome of standardized 177Lu-PSMA-617 therapy in metastatic prostate cancer patients receiving 7400 MBq every 4 weeks	European journal of nuclear medicine and molecular imaging	2,019	cc-by	6,171	Abstract 15 weeks, P < 0.0001) and OS (median survival not reached vs. 52 weeks, P < 0.001) than patients with no PSA reduction. Moreover, patients with reduction in PSA levels ≥50% (median survival not reached vs. 52 weeks, P < 0.0001) and ≥80% (median survival not reached vs. 87 weeks, P = 0.008) lived significantly longer. While hemoglobin did not change during treatment, levels of platelets (236 ± 71 g/L vs. 193 ± 67 g/L) and leucocytes (6.5, range 2.9–13.7 g/L vs. 4.8, range 1.5–12.3 g/L) decreased significantly, both P < 0.001. Two grade 3 leukocytopenia and one grade 3 anemia were observed. Conclusion Intense PSMA-RLT regime with four weekly intervals between the cycles is well-tolerated and offers favorable response rates, PFS, and survival rates for patients with mCRPC. Conclusion Intense PSMA-RLT regime with four weekly intervals between the cycles is well-tolerated and offers favorable response rates, PFS, and survival rates for patients with mCRPC. Keywords PSMA therapy . PSMA PET . Prostate cancer . mCRPC . PSA response European Journal of Nuclear Medicine and Molecular Imaging (2020) 47:713–720 https://doi.org/10.1007/s00259-019-04584-1 European Journal of Nuclear Medicine and Molecular Imaging (2020) 47:713–720 https://doi.org/10.1007/s00259-019-04584-1 European Journal of Nuclear Medicine and Molecular Imaging (2020) 47:713–720 https://doi.org/10.1007/s00259-019-04584-1 ORIGINAL ARTICLE Clinical outcome of standardized 177Lu-PSMA-617 therapy in metastatic prostate cancer patients receiving 7400 MBq every 4 weeks Sazan Rasul1 & Marcus Hacker1 & Elisabeth Kretschmer-Chott1 & Asha Leisser1 & Bernhard Grubmüller2 & Gero Kramer2 & Shahrokh Shariat2,3,4,5,6 & Wolfgang Wadsak1,7 & Markus Mitterhauser1,8 & Markus Hartenbach1 & Alexander R. Haug1,9 Received: 26 June 2019 /Accepted: 16 October 2019 /Published online: 28 November 2019 # The Author(s) 2019 Abstract b a Purpose [177Lu]Lu-PSMA-617 radio-ligand therapy (PSMA-RLT) is emerging in patients with an advanced metastatic castration-resistant prostate cancer (mCRPC). Here, we aimed to estimate the results of PSMA-RLT in terms of response, progression-free survival (PFS), and overall survival (OS) in patients receiving a highly standardized treatment regimen due to mCRPC. The toxicity of PSMA-RLT has also been evaluated. Patients and methods Fifty-four patients (mean age 72 ± 7 years, median PSA at time of initial therapy 66 [range 1.0–4890 μg/L]), receiving three PSMA-RLT cycles (mean 7315 ± 573 MBq) at four weekly intervals, were included in this retrospective analysis. Hematological and biochemical parameters were regularly determined in every patient. Kaplan-Meier estimates were used to assess PFS and OS and a Cox proportional hazard model was used to analyze significant associations. Treatment response was based on PSA measurements 4 weeks after the 3rd treatment. Results The majority of patients were previously treated with abiraterone/enzalutamide (69%) and docetaxel/cabazitaxel (67%). In total, 79% of the patients showed a decrease in PSA (median PSA decrease from 66 to 19.8, range 0.7–4563 μg/L, P < 0.001) 1 month after the 3rd therapy cycle. Among them, 58% and 35% demonstrated a PSA-decline of > 50% and > 80%, respectively. Median OS was 119 weeks; median PFS was 25 weeks. Patients presenting with a PSA decline had significantly longer PFS (27 vs. 15 weeks, P < 0.0001) and OS (median survival not reached vs. 52 weeks, P < 0.001) than patients with no PSA reduction. Moreover, patients with reduction in PSA levels ≥50% (median survival not reached vs. 52 weeks, P < 0.0001) and ≥80% (median survival not reached vs. 87 weeks, P = 0.008) lived significantly longer. While hemoglobin did not change during t t t l l f l t l t (236 ± 71 /L 193 ± 67 /L) d l t (6 5 2 9 13 7 /L 4 8 1 5 12 3 /L) j y p p y ( ) ( ) In total, 79% of the patients showed a decrease in PSA (median PSA decrease from 66 to 19.8, range 0.7–4563 μg/L, P < 0.001) 1 month after the 3rd therapy cycle. Among them, 58% and 35% demonstrated a PSA-decline of > 50% and > 80%, respectively. Median OS was 119 weeks; median PFS was 25 weeks. Patients presenting with a PSA decline had significantly longer PFS (27 vs. Lu-PSMA RLT regime The therapy was composed of 3 cycles of an intravenous ad- ministration of [177Lu]Lu-PSMA-617 that has been acquired from ABX GmbH (Radeberg, Germany) and prepared accord- ing to methods that have previously been described [4]. In all patients, the interval between each cycle was 4 weeks. During each therapy, patients obtained 1-l normal saline infusion at 300 ml/h, 30 min before administration of an average of 7315 ± 573 MBq PSMA-RLT. Moreover, to protect salivary glands from the effect of the therapy, every patient received prophy- lactically cool-packs, which have regularly been changed, on their salivary glands 30 min before and up to 6 h after injection of the radiopharmaceutical. Introduction Therefore, [68Ga]Ga-PSMA ligand positron emission tomography (PET) is nowadays avail- able and expansively used as a sensitive non-invasive diagnostic method to image prostate tumor and its related metastasis. In addition, [177Lu]Lu-PSMA-617 radio-ligand therapy (PSMA- RLT) offers an emerging therapy in patients with an advanced metastatic castration-resistant prostate cancer (mCRPC) [3]. PSMA is strongly increased up to thousand-fold in prostatic cancer and particularly in aggressive as well as metastatic and hormone-resistant prostate cancer. Therefore, [68Ga]Ga-PSMA ligand positron emission tomography (PET) is nowadays avail- able and expansively used as a sensitive non-invasive diagnostic method to image prostate tumor and its related metastasis. In addition, [177Lu]Lu-PSMA-617 radio-ligand therapy (PSMA- RLT) offers an emerging therapy in patients with an advanced metastatic castration-resistant prostate cancer (mCRPC) [3]. Since the rise of PSMA-RLT, many studies could demon- strate its effectiveness and safeness in mCRPC [4–8]. In a metanalysis of 17 studies with collectively 744 mCRPC pa- tients, PSMA-RLT was commonly well-tolerated by the pa- tients and was associated with only mild degrees of hemato- logical and nephrological toxicities [9]. However, with the exception of the Hofmann et al. study, where all patients re- ceived 7500 MBq PSMA-RLT per cycle at 6-week intervals [10], most of the other reported studies in this metanalysis did not have a standardized treatment protocol for PSMA-RLT in mCRPC and the treated patients have received the therapy either as a single dose or in multiple cycles at 8–12-week intervals. Moreover, the treatment plans in these studies were heterogeneous even in terms of therapy activity, as the activity of [177Lu]Lu-PSMA ranged from about 1.1 to 9.3 GBq per cycle [9]. So far, it seems there is no established therapeutic protocol concerning PSMA-RLT in mCRPC patients and this might complicate the assessment of the potential of this ther- apy. Hence, the objectives of this retrospective study were to evaluate rate of response and therapeutic impact of the PSMA- RLT on the progression-free survival (PFS) and overall sur- vival (OS) in mCRPC patients that were all treated with a highly standardized regime and in regular short cycle inter- vals. Furthermore, we aimed to determine degrees of hemato- logical toxicities associated with the therapy in these patients. Patients and methods The software IBM SPSS Statistics version 24.0 was used for all data entry and analysis. Prior to any analysis, all data were sub- jected to Kolmogorov-Smirnov test to determine their distribu- tion. Normally distributed data were expressed as mean ± stan- dard deviation, whereas not normally distributed data were pre- sented as median and range and were log-10 transformed for analysis. Categorical variables were shown in percentages and number of reported cases. For comparisons of data between pa- tients responded to those not responded to the therapy, an independent-samples t test was used. Kaplan-Meier estimates and a Cox proportional hazard model were used to assess PFS and OS in all patients. PFS was indicated as the time from the first therapy to the detection of the PSA progression and OS was defined from the date of first cycle to the date of the death or to the date of the last hospital visit. In all analysis, a value of P < 0.05 was considered statistically significant. Introduction It is estimated that prostatic cancer is the second most common type of cancer and the third leading cause of death in males [1]. The 5-year survival rate among these patients depends on the stage of the tumor and varies from 100% at early stage of the disease to only about 30% in case of presence of advanced me- tastasis [2]. Over the last few years, the prostate-specific mem- brane antigens (PSMA), which are enzymes and receptors on the surface of prostatic cells and also known as glutamate carboxy- peptidase II, have widely attracted attentions as a target for im- aging and treating patients with prostate cancer. Expression of Markus Hartenbach and Alexander R. Haug are senior authors who contributed equally to this work. This article is part of the Topical Collection on Oncology – Genitourinary * Markus Hartenbach * Alexander R. Haug alexander.haug@meduniwien.ac.at Extended author information available on the last page of the article Markus Hartenbach and Alexander R. Haug are senior authors who contributed equally to this work. Markus Hartenbach and Alexander R. Haug are senior authors who contributed equally to this work. This article is part of the Topical Collection on Oncology – Genitourinary * Alexander R. Haug alexander.haug@meduniwien.ac.at Eur J Nucl Med Mol Imaging (2020) 47:713–720 714 performed for all patient to illustrate presence of PSMA- overexpression in all lesions [11]. Patients with PSMA- negative metastases have been excluded from this study (n = 2). During each hospital admission, patients subjected to a proper physical examination by an experienced medical doctor and, accordingly, Karnofsky-Index und Eastern Cooperative Oncology Group (ECOG) Status, were determined for every patient. Routinely measured laboratory parameters including complete blood counts, biochemistry, and PSA levels were regularly measured for every patient in each hospital visit and 1 month after the last 3rd therapy. Toxicities were assessed following the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0. Furthermore, another [68Ga]Ga-PSMA whole-body PET scan was done for all pa- tients 4–6 weeks after the last PSMA-RLT to evaluate and imaging therapy efficacy [11]. The Ethics Committee of Medical University of Vienna has approved the study (EK: 1143/2019) and a written informed consent was provided from patients prior to each therapy. PSMA is strongly increased up to thousand-fold in prostatic cancer and particularly in aggressive as well as metastatic and hormone-resistant prostate cancer. Results Generally, all patients tolerated the therapy very well and no acute adverse effects were reported during the hospital stay. Moreover, although serum levels of hemoglobin did not differ significantly before and after receiving 3 cycles of PSMA- RLT (11.7 ± 1.9 g/dL vs. 11.4 ± 1.9 g/dL, P = 0.11), levels of thrombocytes and leucocytes were significantly lower after the 3rd therapy than before therapy (236 g/L ± 71 vs. 193 g/L ± 67 and 6.5 g/L [range 2.9–13.7] vs. 4.8 g/L [range 1.5– 12.3], P < 0.001), respectively. Nevertheless, no thrombocyte levels lower than 75 g/L and no leucocyte levels lower than 1.5 g/L were observed among the treated patients. In general, no grade 3 thrombocytopenia was observed, but 2 patients developed grade 3 leukocytopenia (leucocyte levels were be- tween 1.9 and 1.5 g/L) and only one patient revealed grade 3 anemia (hemoglobin 6.9gm/dL) after the 3rd PSMA RLT. No grade 4 hematological toxicity occurred. No statistically sig- nificant differences were observed in levels of creatinine among the patients before and 1 month after receiving 3 cycles of PSMA-RLT. Laboratory parameters of all studied patients before and after PSMA-RLT are presented in Table 2. Collectively, 54 patients were included in this study. Clinical characteristics of these 54 patients (aged 72 ± 7 years, weight 82 ± 13 kg) are shown in Table 1. Thirty-three percent and 67% of the patients presented with a Karnofsky score lower than 80% and higher or equal than 80%, respectively. ECOG- Index was 0 in 2%, 1 in 90%, and 2 in 8% of the patients. In total, 69% and 67% of the patients had a previous history of receiving enzalutamide and/or abiraterone and chemotherapy with docetaxel and/or cabazitaxel, respectively. Furthermore, distribution of the PSMA-avid metastases was as follows: bone only in 27.7% (n = 15) of the patients, local recurrence with lymph nodes in 14.8% (n = 8), bone and lymph nodes in 40.7% (n = 22), bone with hepatic and lymph node metastases in 9.5% (n = 5), and bone with pulmonary and lymph node metastases in 7.4% (n = 4) of the patients. Patients This retrospective study included all patients, who have been referred to the Department of Nuclear Medicine, Medical University of Vienna, Vienna General Hospital, between September 2015 and September 2018 for receiving PSMA- RLT due to mCRPC. The median duration of follow-up for patients included in this study was 24 months and ranged be- tween 6 and 40 months. All patients were discussed in an interdisciplinary tumor board with the recommendation of PSMA-RLT. The treatments were conducted according to §8 of the Austrian pharmaceutical law (AMG). We have included all patients who were intended to receive three treatments with about 7400 MBq [177Lu]Lu-PSMA-617 every 4 weeks. Prior to therapy, a [68Ga]Ga-PSMA whole-body PET scan was Eur J Nucl Med Mol Imaging (2020) 47:713–720 715 Survival rate estimation Among all treated patients, 79% (43 out of 54) of them showed PSA-response in terms of any PSA decline, whereas 58% and 35% presented with a PSA-decline of ≥50% and ≥ 80%, respectively. The median serum levels of PSA 1 month after receiving the last (3rd) cycle of Lu-PSMA RLT were significantly lower than before start of treatment (19.8 μg/L [range 0.7–4563] vs. 66 μg/L [range 1.0–4890], P < 0.001). The median OS among the entire treated cohort was 119 weeks (95%CI, 91.7–130.0) and the median PFS was 25 weeks (95%CI, 23.1–26.8) (Fig. 1). Moreover, results of Kaplan-Meier analysis indicated that patients with any PSA decline after 3 cycles of PSMA-RLT had a significantly longer PFS than patients with no PSA reduction (27 vs. 15 weeks, P < 0.0001). A decline of PSA of ≥50% and ≥80% was asso- ciated with increased PFS (≥50%, 27 weeks vs. 17 weeks, P = 0.01; ≥80%, 38 weeks vs. 22 weeks, P = 0.034) (all Fig. 2). Furthermore, patients with any PSA decrease showed a sig- nificantly longer OS than patients with increasing PSA (52 weeks vs. median survival not reached, P < 0.0001) (Fig. 3). Likewise, patients with reduction in PSA levels ≥50% (me- dian survival not reached vs. 52 weeks (95%CI, 25.0–66.8), P < 0.0001) and a PSA reduction of ≥80% (median survival not reached vs. 87 weeks (95%CI, 68.4–115.8), P = 0.008) after the last cycle of the therapy lived significantly longer than patients with no PSA decrease (Figs. 3 and 4). Representative examples of therapy response with [68Ga]Ga- PSMA PET images are illustrated in Fig. 5. Table 1 Clinical characteristics of all studied mCRPC patients prior to PSMA-RLT therapy Parameters Values Patients (n) 54 Age (mean ± SD) 72 ± 7 years Weight (mean ± SD) 82 ± 13 kg [177Lu]Lu-PSMA-617 (mean ± SD) 7315 ± 573 MBq Karnofsky score (n) < 80% ≥80% (18) 33 % (36) 67 % ECOG-Index (n) 0 1 2 (1) 2 % (49) 90 % (4) 8 % Patients with previous enzalutamid/abirateron 69 % Patients with previous docetaxel/cabazitaxel 67 % Metastatic lesions (n) Bone Local recurrence + lymph node Bone + lymph node Bone + lymph node + liver Bone + lymph node + lung (15) 27.7% (8) 14.8% (22) 40.7% (5) 9.2% (4) 7.4% Table 1 Clinical characteristics of all studied mCRPC patients prior to PSMA-RLT therapy Discussion The introduction of PSMA-RLT has further expanded the treatment options for patients with mCRPC [7]. In this study, we analyzed for the first-time data of mCRPC patients, the majority pretreated with chemo- and/or antihormonal thera- pies, and received 3 cycles of highly standardized PSMA- RLT at 4 weekly intervals. We mainly concentrated on the Eur J Nucl Med Mol Imaging (2020) 47:713–720 716 Table 2 Laboratory parameters of mCRPC studied patients before and after 3 cycles of PSMA-RLT at 4 weekly intervals Table 2 Laboratory parameters of mCRPC studied patients before and after 3 cycles of PSMA-RLT at 4 weekly intervals Parameters Before therapy After therapy P value PSA μg/L 66 (1.0–4890) 19.8 (0.7–4563) < 0.001 Hemoglobin g/dL (mean ± SD) 11.7 ± 1.9 11.4 ± 1.9 NS Thrombocytes g/L (mean ± SD) 236 ± 71 193 ± 67 < 0.001 Leucocytes g/L 6.5 (2.9–13.7) 4.8 (1.5–12.3) < 0.001 Creatinine mg/dL 0.9 (0.6–1.6) 0.86 (0.6–1.8) NS Alkaline phosphatase U/L (mean ± SD) 66 (27–469) 68 (27–580) NS LDH (mean ± SD) U/L 189 (136–620) 197 (125–323) NS Data with no Gaussian distribution, presented in median (range) and log10-transformd for analysis. NS not significant. P < 0.05 = statically significant * Data with no Gaussian distribution, presented in median (range) and log10-transformd for analysis. NS not significant. P < 0.05 = statically significant PSA-decline of ≥50% and ≥80%, respectively. Probably because of differences in degrees of tumor stage among the treated patients and differences in applied therapies prior the PSMA-RLT, this considerable finding is slightly superior to results of previous studies that have demonstrated response rates to PSMA-RLT of about 60–70% in the treated mCRPC patients [4, 6, 14–16]. Nevertheless, this result is in line with results of a previous phase II trial study that included 30 mCRPC patients with progressed disease treated with PSMA-RLT at 6 weekly interval after failure of the standard treatments and indicated a PSA reduction of ≥50% in 57% among the studied participants [10]. Furthermore, in the study of Emmett et al. that included 18 mCRPC patients receiving a mean dose of 7.0 GBq PSMA-RLT with 6 weeks interval between the cycles, a therapy response in terms of PSA reduction in 71% of the treated patients was reported [17]. In 36% of them, there was a reduction in PSA level of ≥ 50%. In Baum et al. Table 2 Laboratory parameters of mCRPC studied patients before and after 3 cycles of PSMA-RLT at 4 weekly intervals Discussion [18], rate of response could be seen in 80% (45 out of 56) of the treated patients; here, the numbers of the applied cycles were very heterogeneous and varied from 1 to 5 cycles and the interval between the cycles was not defined. safety as well as efficacy, rate of response, and the clinical outcome of this therapy protocol among the treated patients. Although, there is an ongoing approach to standardize PSMA- RLT protocols [12], the recent meta-analysis of Yadav et al. [9] has briefly summarized data of several PSMA-RTL studies and showed inhomogeneity of in these studies concerning doses and intervals of the provided therapies. Up to the pres- ent, there is no information about safety and effectivity of the PSMA-RLT in mCRPC patients received equally high-dose of the therapy with merely 4 weeks interval between the cycles and, to the best of our knowledge, we are, at the moment, the only clinical center applying this therapeutic protocol. The rationales for developing such a protocol were, on the one hand, the promising results of safety and toxicity with an approximately 6.0 GBq every 8 weeks protocol from another working group [13] and on the other hand, the hypothesis that a follow-up therapy after circa 4 half-lives of 177Lutetium will keep up the effective dose in the tumor more efficiently than an 8-week gap, following a concept similar to a fractionated radiation therapy. The results of our study revealed response rates to the PSMA-RLT in terms of any PSA decline in 79% of the treated cases and 58% and 35% of them presented with a safety as well as efficacy, rate of response, and the clinical outcome of this therapy protocol among the treated patients. Although, there is an ongoing approach to standardize PSMA- RLT protocols [12], the recent meta-analysis of Yadav et al. [9] has briefly summarized data of several PSMA-RTL studies and showed inhomogeneity of in these studies concerning doses and intervals of the provided therapies. Up to the pres- ent, there is no information about safety and effectivity of the PSMA-RLT in mCRPC patients received equally high-dose of the therapy with merely 4 weeks interval between the cycles and, to the best of our knowledge, we are, at the moment, the only clinical center applying this therapeutic protocol. Discussion The rationales for developing such a protocol were, on the one hand, the promising results of safety and toxicity with an approximately 6.0 GBq every 8 weeks protocol from another working group [13] and on the other hand, the hypothesis that a follow-up therapy after circa 4 half-lives of 177Lutetium will keep up the effective dose in the tumor more efficiently than an 8-week gap, following a concept similar to a fractionated radiation therapy. The results of our study revealed response rates to the PSMA-RLT in terms of any PSA decline in 79% of the treated cases and 58% and 35% of them presented with a Fig. 1 Kaplan-Meier plot shows median overall survival and median progression-free survival among the entire studied cohort. Patients received 3 cycles of highly standardized PSMA-RLT at 4 weekly intervals: The median overall survival was 119 weeks and the median progression-free survival was 25 weeks Fig. 1 Kaplan-Meier plot shows median overall survival and median progression-free survival among the entire studied cohort. Patients received 3 cycles of highly standardized PSMA-RLT at 4 weekly Fig. 1 Kaplan-Meier plot shows median overall survival and median progression-free survival among the entire studied cohort. Patients received 3 cycles of highly standardized PSMA-RLT at 4 weekly intervals: The median overall survival was 119 weeks and the median progression-free survival was 25 weeks intervals: The median overall survival was 119 weeks and the median progression-free survival was 25 weeks Moreover, we observed no significant differences in serum hemoglobin levels before and after receiving 3 cycles of Lu- PSMA-RLT and although thrombocytes and leucocytes levels were significantly lower after the therapy, only two cases de- veloped grade 3 leukocytopenia and no grade 3 thrombocyto- penia was observed among the patients. Additionally, no relevant nephrological effects were noticed among the patients before and 1 month after receiving 3 cycles of PSMA-RLT. Similarly, various previously published studies indicated ei- ther low, minimal, or reversible hemato- and nephrotoxicity effects of PSMA-RLT in the treated patients [5, 18, 19]. Ahmadzadehfar et al., in their first experience with PSMA- Fig. 2 Kaplan-Meier plot shows progression free survival of the patients stratified by PSA response. Patients with any PSA reduction revealed significantly longer PFS than patients with no PSA decline (27 vs. 15 weeks, P < 0.0001). Patients with a PSA reduction of ≥80% had significantly longer PFS than patients with no PSA reduction (38 vs. Discussion 22 weeks, P = 0.03) Eur J Nucl Med Mol Imaging (2020) 47:713–720 717 Eur J Nucl Med Mol Imaging (2020) 47:713–720 717 Fig. 2 Kaplan-Meier plot shows progression free survival of the patients stratified by PSA response. Patients with any PSA reduction revealed significantly longer PFS than patients with no PSA decline (27 vs. 15 weeks, P < 0.0001). Patients with a PSA reduction of ≥80% had significantly longer PFS than patients with no PSA reduction (38 vs. 22 weeks, P = 0.03) g g Fig. 2 Kaplan-Meier plot shows progression free survival of the patients stratified by PSA response. Patients with any PSA reduction revealed significantly longer PFS than patients with no PSA decline (27 vs. 15 weeks, P < 0.0001). Patients with a PSA reduction of ≥80% had significantly longer PFS than patients with no PSA reduction (38 vs. 22 weeks, P = 0.03) Moreover, we observed no significant differences in serum hemoglobin levels before and after receiving 3 cycles of Lu- PSMA-RLT and although thrombocytes and leucocytes levels were significantly lower after the therapy, only two cases de- veloped grade 3 leukocytopenia and no grade 3 thrombocyto- penia was observed among the patients. Additionally, no Moreover, we observed no significant differences in serum hemoglobin levels before and after receiving 3 cycles of Lu- PSMA-RLT and although thrombocytes and leucocytes levels were significantly lower after the therapy, only two cases de- veloped grade 3 leukocytopenia and no grade 3 thrombocyto- penia was observed among the patients. Additionally, no Moreover, we observed no significant differences in serum hemoglobin levels before and after receiving 3 cycles of Lu- PSMA-RLT and although thrombocytes and leucocytes levels were significantly lower after the therapy, only two cases de- veloped grade 3 leukocytopenia and no grade 3 thrombocyto- penia was observed among the patients. Additionally, no relevant nephrological effects were noticed among the patients before and 1 month after receiving 3 cycles of PSMA-RLT. Similarly, various previously published studies indicated ei- ther low, minimal, or reversible hemato- and nephrotoxicity effects of PSMA-RLT in the treated patients [5, 18, 19]. Ahmadzadehfar et al., in their first experience with PSMA- Eur J Nucl Med Mol Imaging (2020) 47:713–720 718 Fig. 3 Kaplan-Meier survival plot of the patients stratified by PSA response. Discussion In comparison to patients with no PSA decline, patients with any PSA reduction up to 50% and more lived significantly longer (median survival not reached vs. 52 weeks, P < 0.0001) RLT, reported early side-effects in terms of some degrees of hematotoxicity in only few treated cases without evidence of relevant nephro- or hepatotoxicity 8 weeks after receiving a single dose of the therapy [4]. In another study, they could confirm safety of PSMA-RLT in 24 mCRPC patients obtaining repeated cycles of the therapy [14]. previously treated with chemotherapy or points towards a su- perior efficacy of high doses of 3 cycles PSMA-RTL in short 4-week interval. Furthermore, it has previously been demon- strated that administrations of repeated therapy cycles will increase the rate of response to the PSMA-RLTand this might consequently increase the OS, as about one third of treated mCRPC patients reveal late therapy response [20]. However, we (Fig. 3) and Rahbar et al. could observe a nearly identical longer survival among patients exhibiting any PSA decline than patients with PSA progression [16]. Similarly, depending on presence and the degree of PSA decline after the therapy, our and other studies could show that PFS and OS are signif- icantly longer in responding patients [5, 7, 10, 21, 22]. The observations of very low toxicity and comparatively high re- sponse rates among treated patients with this presented thera- peutic scheme might enable treatments with even higher In addition, we found that the median OS for the entire treated patients was 119 weeks and the median PFS was 25 weeks. Rahbar et al. showed recently a median OS of 56 weeks in the entire 104 mCRPC patients who received an inhomogeneous number of PSMA-RLT ranged between 1 and 8 cycles at 8 weeks interval [16]. This notable difference in patient’s median OS between these 2 studies might either reflect the disparity of the patient’s population and their tumor burden as our study included almost 30% chemotherapy naïve patients, while in Rahbar et al., the entire studied cohort was Fig. 4 Kaplan-Meier survival plot of patients with a PSA drop ≥ 80%. Patients with a PSA reduction of ≥80% after therapy have significantly longer survival than patients with no PSA decrease (median survival not reached vs. 87 weeks, P = 0.008) ≥ l ) Fig. 4 Kaplan-Meier survival plot of patients with a PSA drop ≥ 80%. Discussion Patients with a PSA reduction of ≥80% after therapy have significantly longer survival than patients with no PSA decrease (median survival not reached vs. 87 weeks, P = 0.008) Eur J Nucl Med Mol Imaging (2020) 47:713–720 719 b μg/L); the arrows indicate residual metastatic areas. b Patient with therapy response and PSA decline of ≥80% (initial PSA 70.68 μg/L, after therapy PSA 6.4 μg/L) a Fig. 5 [68Ga]Ga-PSMA PET images of mCRPC patients before and after 3 cycles of PSMA-RLT: a patient with therapy response and PSA drop of about 70% (initial PSA 80.95 μg/L, after therapy PSA 22.95 b b a a a Fig. 5 [68Ga]Ga-PSMA PET images of mCRPC patients before and after 3 cycles of PSMA-RLT: a patient with therapy response and PSA drop of about 70% (initial PSA 80.95 μg/L, after therapy PSA 22.95 μg/L); the arrows indicate residual metastatic areas. b Patient with therapy response and PSA decline of ≥80% (initial PSA 70.68 μg/L, after therapy PSA 6.4 μg/L) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. PSMA-RLT doses, as this has also been shown by Rathke et al. [23] and could encourage systematic dose-finding stud- ies for this treatment. Although this study included a homogeneous group of mCRPC patients treated with a homogeneous scheme of PSMA-RLT, the retrospective design is the main limitation of this study. Moreover, degrees of therapy toxicity observed among patients included in this study might have been affect- ed by the differences in patient’s population in terms of pre- treatment and disparities in tumor stage. Therefore, further larger prospective studies with a more homogeneous patient population using this promising therapy regime are essential to confirm our results and to move forward towards global standardization of this therapy in patients with mCRPC. Conclusion Results of this study showed that mCRPC patients treated with a highly standardized PSMA-RLT regime at four weekly intervals routinely applied in our clinic have a considerable response rate and a favorable PFS and survival. In addition, results demonstrated good tolerability of PSMA-RLT. 5. Tagawa ST, Milowsky MI, Morris M, Vallabhajosula S, Christos P, Akhtar NH, et al. Phase II study of Lutetium-177-labeled anti-pros- tate-specific membrane antigen monoclonal antibody J591 for met- astatic castration-resistant prostate cancer. Clin Cancer Res. 2013;19:5182–91. 6. McBean R, O'Kane B, Parsons R, Wong D. Lu177-PSMA therapy for men with advanced prostate cancer: Initial 18 months experience at a single Australian tertiary institution. J Med Imaging Radiat Oncol. 2019. Funding information Open access funding provided by Medical University of Vienna. 7. Rahbar K, Bode A, Weckesser M, Avramovic N, Claesener M, Stegger L, et al. 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Results of a Prospective Phase 2 Pilot Trial of (177)Lu- 1 Department of Biomedical Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria Affiliations Sazan Rasul1 & Marcus Hacker1 & Elisabeth Kretschmer-Chott1 & Asha Leisser1 & Bernhard Grubmüller2 & Gero Kramer2 & Shahrokh Shariat2,3,4,5,6 &Wolfgang Wadsak1,7 &Markus Mitterhauser1,8 &Markus Hartenbach1 &Alexander R. Haug1,9 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. Sazan Rasul1 & Marcus Hacker1 & Elisabeth Kretschmer-Chott1 & Asha Leisser1 & Bernhard Grubmüller2 & Gero Kramer2 & Shahrokh Shariat2,3,4,5,6 &Wolfgang Wadsak1,7 &Markus Mitterhauser1,8 &Markus Hartenbach1 &Alexander R. Haug1,9 1 Department of Biomedical Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria 2 Department of Urology, Comprehensive Cancer Center, Vienna General Hospital, Medical University of Vienna, Vienna, Austria 3 Department of Urology, Weill Cornell Medical College, New York, NY, USA 4 Department of Urology, Second Faculty of Medicine, Charles University, Prague, Czech Republic 5 Institute for Urology and Reproductive Health, I.M. Sechenov First Moscow State Medical University, Moscow, Russia 6 Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA 7 CBmed GmbH, Center for Biomarker Research in Medicine, Graz, Austria 8 Ludwig Boltzmann Institute Applied Diagnostics, Vienna, Austria 9 Christian Doppler Laboratory for Applied Metabolomics (CDL AM), Medical University of Vienna, Vienna, Austria 1 Department of Biomedical Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria 5 Institute for Urology and Reproductive Health, I.M. Sechenov First Moscow State Medical University, Moscow, Russia 6 Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2 Department of Urology, Comprehensive Cancer Center, Vienna General Hospital, Medical University of Vienna, Vienna, Austria 7 CBmed GmbH, Center for Biomarker Research in Medicine, Graz, Austria 3 Department of Urology, Weill Cornell Medical College, New York, NY, USA 8 Ludwig Boltzmann Institute Applied Diagnostics, Vienna, Austria 4 Department of Urology, Second Faculty of Medicine, Charles University, Prague, Czech Republic 9 Christian Doppler Laboratory for Applied Metabolomics (CDL AM), Medical University of Vienna, Vienna, Austria
https://openalex.org/W2073431172	https://hal.inrae.fr/hal-02605249/document	English	null	Deriving a per-field land use and land cover map in an agricultural mosaic catchment	Earth system science data	2,014	cc-by	9,970	To cite this version: B. Seo, C. Bogner, P. Poppenborg, E. Martin, M. Hoffmeister, et al.. Deriving a per-field land use and land cover map in an agricultural mosaic catchment. Earth System Science Data, 2014, 6 (2), pp.339-352. 10.5194/essd-6-339-2014. hal-02605249 Deriving a per-field land use and land cover map in an agricultural mosaic catchment B. Seo, C. Bogner, P. Poppenborg, E. Martin, M. Hoffmeister, M. Jun, T. Koellner, Björn Reineking, C.L. Hope, J. Tenhunen Deriving a per-ﬁeld land use and land cover map in an agricultural mosaic catchment Deriving a per-ﬁeld land use and land cover map in an agricultural mosaic catchment B. Seo1,2, C. Bogner2, P. Poppenborg3, E. Martin4, M. Hoffmeister5,6, M. Jun7, T. Koellner3, B. Reineking1,8, C. L. Shope9, and J. Tenhunen10 1Biogeographical Modelling, BayCEER, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany 2Ecological Modelling, BayCEER, University of Bayreuth, Dr.-Hans-Frisch-Straße 1–3, 95448 Bayreuth, Germany 3Professorship of Ecological Services, BayCEER, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany 4Department of Animal Ecology and Tropical Biology, Biocentre, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 5Department of Organismic Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria 6Institute of Sensory Ecology, Department of Biology, Heinrich Heine University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany 7Research Institute for Gangwon, Junangro 5-GA, Chuncheon, Republic of Korea 8UR EMGR Écosystèmes Montagnards, Irstea, 2 rue de la Papeterie-BP 76, 38402, St-Martin-d’Hères, France 9US Geological Survey, 2329 W. Orton Circle, Salt Lake City, UT 84119-2047, USA 10Plant Ecology Department, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany Correspondence to: B. Seo (bumsuk.seo@uni-bayreuth.de) Received: 5 March 2014 – Published in Earth Syst. Sci. Data Discuss.: 30 April 2014 Revised: 4 September 2014 – Accepted: 9 September 2014 – Published: 30 September 2014 B. Seo1,2, C. Bogner2, P. Poppenborg3, E. Martin4, M. Hoffmeister5,6, M. Jun7, T. Koellner3 2Ecological Modelling, BayCEER, University of Bayreuth, Dr.-Hans-Frisch-Straße 1–3, 95448 Bayreuth, Germany 3Professorship of Ecological Services, BayCEER, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany 3Professorship of Ecological Services, BayCEER, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany 4Department of Animal Ecology and Tropical Biology, Biocentre, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 5Department of Organismic Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria 6Institute of Sensory Ecology, Department of Biology, Heinrich Heine University Düsseldorf, 7Research Institute for Gangwon, Junangro 5-GA, Chuncheon, Republic of Korea 8UR EMGR Écosystèmes Montagnards, Irstea, 2 rue de la Papeterie-BP 76, 38402, St-Martin-d’Hères, France 9US Geological Survey, 2329 W. Orton Circle, Salt Lake City, UT 84119-2047, USA 10Plant Ecology Department, University of Bayreuth, Universitätsstraße 30, 95447 Bayreuth, Germany Correspondence to: B. Seo (bumsuk.seo@uni-bayreuth.de) Correspondence to: B. Seo (bumsuk.seo@uni-bayreuth.de) Received: 5 March 2014 – Published in Earth Syst. Sci. Data Discuss.: 30 April 2014 Revised: 4 September 2014 – Accepted: 9 September 2014 – Published: 30 September 2014 Abstract. Detailed data on land use and land cover constitute important information for Earth system models, environmental monitoring and ecosystem services research. HAL Id: hal-02605249 https://hal.inrae.fr/hal-02605249v1 Submitted on 16 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ doi:10.5194/essd-6-339-2014 © Author(s) 2014. CC Attribution 3.0 License. 2.1 Study area The study area, Haean catchment, is located at the bor- der between North and South Korea (128◦1′33.101′′ E, 38◦28′6.231′′ N). It is a small agricultural catchment (64.4 km2) with rice paddies, annual and perennial dry ﬁelds and orchard farms. Approximately 1200 inhabitants live in Haean, mostly commercial farmers running their own small farms in the catchment. Agricultural ﬁelds in this area are typically smaller than 40 ha, and agricultural practice is in- tensive in terms of fertilisation and tillage. Remote sensing has been increasingly used to derive bet- ter LULC data for the past few decades (Bartholomé and Belward, 2005; Loveland et al., 2000; Loveland and Bel- ward, 2010; Friedl et al., 2010). Nevertheless, because avail- able global land cover products are still limited thematically, continuous efforts to improve the LULC products are being made (Blanco et al., 2010; Colditz et al., 2011; Fernandes et al., 2004). The altitudes in the Haean catchment range from approx- imately 500 to 1200 m. Due to its characteristic bowl shape, land use changes, consisting predominantly of rice paddies at the valley bottom and dry-ﬁeld farming on moderate slopes. The higher altitudes are covered by deciduous and mixed forests. Particularly for agricultural landscapes, detailed land cover information is often lacking (Potgieter et al., 2007; Pittman et al., 2010; Fritz et al., 2013). In fact, the most widely used land cover databases such as GlobCover or Mod- erate Resolution Imaging Spectroradiometer (MODIS) land cover only have a few crop-related classes (Loveland et al., 2000; Bontemps et al., 2011; US Geological Survey, 2012). Especially for heterogeneous arable zones, such as irrigated ﬁelds (e.g. Conrad et al., 2010), land cover products based on remote sensing are underdeveloped (Colditz et al., 2011). The average annual air temperature is approximately 8 ◦C, and the mean annual precipitation ranges from 1200 to 1300 mm, with more than 60 % of rainfall occurring during the summer monsoon between June and August (Korean Me- teorological Administration, http://web.kma.go.kr/eng). Be- tween 1999 and 2010 the maximum daily rainfall during summer reached up to 223 mm. Furthermore, spatial resolution of LULC data is often re- stricted. This limitation is particularly pronounced in hetero- geneous landscapes, such as mixed-farming areas, due to the complex mosaic of crop/non-crop land use and land cover types (Schulp and Alkemade, 2011). Unlike a homogeneous landscape (e.g. 2.1 Study area plantation farm), this type of agricultural mo- saic needs a comprehensive number of LULC classes in a rel- atively small area. Therefore, spatial resolution up to several hundred metres might be too coarse for this type of land- scape. Longitudinal land cover data also constitute an im- portant element when agricultural land use changes rapidly. MODIS Land Cover Type (MCD12Q1) is the only product that provides annual information. It has been widely used for analysing land cover changes (Loveland et al., 2000). This area has been studied intensively as it shows a typ- ical conﬂict between agriculture and environmental protec- tion (Martin et al., 2013; Poppenborg and Koellner, 2013; Thanh Nguyen et al., 2012). The downstream water qual- ity was heavily degraded by the agricultural activity occur- ring in the catchment (Shope et al., 2013; Meusburger et al., 2013). The local government pursued different policy mea- sures concerning this conﬂict, such as subsidising perennial crops, which caused rapid LULC changes in land use and land cover. B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 340 1 Introduction In our study we address some of the above-mentioned problems and provide thematically and spatially rich land use and land cover data. We censused a small agroecosys- tem with complex agricultural land use. We recorded ﬁeld- by-ﬁeld land use and land cover type; hence, the unit entity of our data set is a single land parcel and we call it “per-ﬁeld”. We followed Conrad et al. (2010), who deﬁned “per-ﬁeld” data as a data set based on actual agricultural ﬁelds. Agricultural land use affects ecosystem services, such as the provision of drinking water or the control of soil ero- sion. Inappropriate agricultural practice can lead to serious soil loss and pollution of surface water and groundwater by agrochemicals. Detailed data on land use and land cover (LULC) in an agricultural landscape constitute basic infor- mation for environmental monitoring and pollution control (Conrad et al., 2010; Potgieter et al., 2007; Pittman et al., 2010). In this paper we introduce the data and their collection and post-processing protocol. Additionally we compared our data with MCD12Q1. The data are now available at the public repository Pangaea (doi:10.1594/PANGAEA.823677). In general, precise information on land cover is required for running Earth system models (Ottlé et al., 2013) be- cause land use change directly affects numerous climate pa- rameters such as albedo, CO2 cycling and hydrologic cycles (Matthews, 1983; Mahecha et al., 2010). Additionally, LULC information is crucial for ecosystem services research, deci- sion making and studies on global change in general (Hansen et al., 2013; Schulp and Alkemade, 2011; Martin et al., 2013; Poppenborg and Koellner, 2013). Deriving a per-ﬁeld land use and land cover map in an agricultural mosaic catchment Global land cover products are evolving rapidly; however, there is still a lack of information particularly for heterogeneous agricultural landscapes. We censused land use and land cover ﬁeld by ﬁeld in the agricultural mosaic catchment Haean in South Korea. We recorded the land cover types with additional information on agricultural practice. In this paper we introduce the data, their collection and the post-processing protocol. Furthermore, because it is important to quantitatively evaluate available land use and land cover products, we compared our data with the MODIS Land Cover Type product (MCD12Q1). During the studied period, a large portion of dry ﬁelds was converted to perennial crops. Compared to our data, the forested area was underrepresented and the agricultural area overrepresented in MCD12Q1. In addition, linear landscape elements such as waterbodies were missing in the MODIS product due to its coarse spatial resolution. The data presented here can be useful for earth science and ecosystem services research. The data are available at the public repository Pangaea (doi:10.1594/PANGAEA.823677). Published by Copernicus Publications. 2 Material and methods 2.2 Preparation of data collection Prior to the ﬁeld campaign, we collected pre-existing infor- mation to create an initial “base map”. It served as a ﬁeld template and was particularly useful for gaining access to As a consequence, for an agricultural mosaic landscape with frequently changing land use, the only way to obtain detailed land cover information is surveying the study area. Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 341 isolated patches. We used a SPOTMaps image (Astrium Ser- vices, http://www.astrium-geo.com), a mosaic of multiple SPOT 5 images, with a ground resolution of 2.5 m. Fur- thermore, we worked with aerial photographs and a land cover map from the Korean Ministry of Environment (KME) (http://egis.me.go.kr) to complement the SPOTMaps image. From the KME land cover map, we extracted vector-based linear elements such as road and stream networks. An addi- tional land use map by the Research Institute For Gangwon (http://gdri.re.kr) from 2007 provided information on previ- ously surveyed agricultural land use. The data sources are summarised in Table 1. isolated patches. We used a SPOTMaps image (Astrium Ser- vices, http://www.astrium-geo.com), a mosaic of multiple SPOT 5 images, with a ground resolution of 2.5 m. Fur- thermore, we worked with aerial photographs and a land cover map from the Korean Ministry of Environment (KME) (http://egis.me.go.kr) to complement the SPOTMaps image. From the KME land cover map, we extracted vector-based linear elements such as road and stream networks. An addi- tional land use map by the Research Institute For Gangwon (http://gdri.re.kr) from 2007 provided information on previ- ously surveyed agricultural land use. The data sources are summarised in Table 1. To record a landscape element, we marked vertices and edges for each spatial entity as GPS waypoints (WPs) and tracks. The WP IDs were written on the printed base map and corresponding information in the ﬁeld data book. GPS tracks were continuously stored in the device as we moved around and gave us complementary data for drawing polygon edges and polylines. We used several GPS devices (Garmin CSX60, Garmin Colorado 300 and Garmin eTrex 30) simultaneously to re- trieve location information. The use of multiple devices as a back-up secured the data against sudden power loss. For devices capable of loading custom maps, we loaded the base map in order to simultaneously review newly recorded WPs. 2.4.1 Digitising the ﬁeld records We digitised the ﬁeld records into polygons and polylines with LULC type labels. The base map served as background information to complement the ﬁeld records. In addition to LULC classes, we stored other descriptive information in an attribute table. In the corresponding columns, quality as- surance (QA) was recorded as: “?” (questionable), “*” (un- known) and “/” (not valid). For instance, a question mark was assigned if we could not identify the crop reliably. Gap-ﬁlled data were also marked by a question mark. A forward slash indicates that the data were collected but was unreliable (e.g. incorrect identiﬁcation). For further information, the reader is referred to the readme ﬁle of the data set at the Pangaea repository. 2.3 Data collection The main goal of the data collection campaign was to survey LULC information in the entire catchment. We carried out annual campaigns in 2009–2011 to census the entire land- scape. The term “census” is adopted here in contrast to the term “sampling” because we recorded LULC information from the whole study area and not from a subset of land parcels. Accordingly, we mapped the complete set of land parcels and documented land cover type together with addi- tional information on data quality and spatial and temporal mixture of land use types (e.g. double dry-ﬁeld cropping per year or mixed dry ﬁelds). In contrast to 2009 and 2010, we were only able to map the northern half of the catchment in 2011 due to time and budget limitations. Therefore, we did not consider these data when calculating descriptive statistics or analysing land use change and only compared the years 2009 and 2010. 2.2 Preparation of data collection The images selected for the base map were only mod- erately well georeferenced. The SPOTMaps image, for ex- ample, had an approximated location error of 10–15 m ac- cording to the speciﬁcation, and the other spatial data also revealed a substantial location error. Therefore, we georef- erenced them again using 14 ground control points (GCPs) distributed over the entire catchment. They were established along linear elements, such as roads, and deﬁned by the Global Positioning System (GPS) coordinates averaged over several measurements. After georeferencing by the ﬁrst- order polynomial (afﬁne) transformation, the horizontal root mean squared error (RMSE) of the ﬁnal base map image equalled 9.62 m. www.earth-syst-sci-data.net/6/339/2014/ 2.4.3 Deﬁnition of LULC classes 2.4.3 Deﬁnition of LULC classes We deﬁned a LULC classiﬁcation scheme with 67 land use and land cover classes to adequately represent the agriculture mosaic in the catchment. If several LULC types coexisted in one polygon, we assigned it to the LULC type that made up the largest portion and recorded mixture information in the attribute table. The scheme incorporates a large number of regional crop types as well as several natural and seminatural land cover classes found in the area. In the following we call this detailed classiﬁcation scheme S1. These reclassiﬁed LULC data can be used together with global products such as MCD12Q1 or GlobCover that fol- low the IGBP and FAO-LCCS schemes, respectively. For the IGBP classes, we reclassiﬁed some of the perennial crops as non-crop types (forest or shrub) to be consistent with the IGBP system (e.g. “orchard ﬁeld” coded as “open shrub”) (Friedl et al., 2002). We also reclassiﬁed rice paddies as “croplands”, unlike in S2, which distinguishes “paddy ﬁeld” from other agricultural types. For vegetative classes, we also recorded information on life form, life cycle and crop type following the land cover classiﬁcation system (LCCS) developed by the FAO (Food and Agricultural Organization of the United Nations) (Di Gregorio, 2005). We categorised the life cycle of a class as “perennial”, “annual” or their mixture “annual/perennial” based on the life cycle of the plant species and the local cul- tivation practice. In other words, if a perennial crop was har- vested after one growing season we classiﬁed it as “annual”. We distinguished between the life forms “woody”, “herba- ceous” and “lichens/mosses”, or a combination of them. Crop type patches were further subdivided into 12 different crop types (Supplement Table S1 at Pangaea repository). We as- signed mixed crop type values for patches where various crop/non-crop vegetation coexisted. B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 342 Table 1. Data used for the base map and gap ﬁlling. SPOTMaps served as the main background information for data collection. Maps by the Korean Ministry of Environment (KME) and by the Research Institute For Gangwon (RIG) provided previously recorded land use information and were also used for gap ﬁlling. Table 1. Data used for the base map and gap ﬁlling. SPOTMaps served as the main background information for data collection. Maps by the Korean Ministry of Environment (KME) and by the Research Institute For Gangwon (RIG) provided previously recorded land use information and were also used for gap ﬁlling. Table 1. Data used for the base map and gap ﬁlling. SPOTMaps served as the main background information for data collection. Maps by the Korean Ministry of Environment (KME) and by the Research Institute For Gangwon (RIG) provided previously recorded land use information and were also used for gap ﬁlling. Name Format Temporal Description Source coverage SPOTMaps Raster (Geographic Tagged Image File Format, GeoTIFF) 2009 Mosaic of multiple SPOT 5 images, reso- lution 2.5 m, natural colours (three bands) Astrium Services, http://www. astrium-geo.com/spotmaps KME land cover map Vector (polygon) 2000 21 classes based on Landsat Thematic Mapper (TM) images combined with large-scale vector maps Ministry of Environment, Republic of Ko- rea (KME), http://me.go.kr RIG land use map Vector (polygon) 2007 70 classes based on ﬁeld observation Research Institute For Gangwon, Repub- lic of Korea (RIG), http://gdri.re.kr extrapolated information are of interest, the QA ﬂag can be used to ﬁlter out transferred land cover information. Because the LULC data were recorded yearly, the gaps differed from year to year. Therefore, we ﬁlled them separately for each year. regrouped the S1 classes into eight major types (Supple- ment Table S2 at Pangaea repository). Two of the eight classes are relevant for crop distinction. Finally, we classiﬁed our data according to the International Geosphere–Biosphere Programme (IGBP) Discover land cover system which con- tains 17 classes, two of which are crop classes (Loveland et al., 2000; Loveland and Belward, 2010; Friedl et al., 2010). Thus, the schemes S1, S2, FAO-LCCS and IGBP differ in the total number of classes and the number of crop classes (Ta- ble 2). 2.4.2 Gap ﬁlling After digitising the ﬁeld records, some gaps remained be- tween polygons. They occurred mostly around patches that were irregularly shaped and therefore difﬁcult to map. We ﬁlled these gaps using the KME land cover map (Table 1) and our own data on linear elements. First, we added the main road and stream networks ex- tracted from the KME land cover map. Subsequently, we created two major linear elements, namely seminatural ﬁeld edges and a stream network from our GPS track data. For this purpose, we converted the GPS tracks of ﬁeld edges and non-paved agricultural pathways, which were initially poly- lines, into polygons by creating 6 m wide buffers encompass- ing the tracks. Similarly, based on the GPS tracks recorded along small streams, we created the stream network buffers of 5 m width and assigned them to the existing “inland water” polygons. We divided landscape elements into two categories, namely patches and linear elements. The former included agricultural and non-agricultural ﬁelds, forest, waterbodies and all other areal land cover types best represented by a polygon. In general, we visited patches once per year. How- ever, patches with a spatially or temporally mixed land use type were inspected multiple times. Linear elements com- prised roads, stream networks, ﬁeld edges or any other ele- ment that can be represented by a polyline. They were inves- tigated during the whole project period from 2009 to 2011 because of their large extent and relative temporal stability. Finally, we used the KME land cover map to ﬁll the re- maining gaps. Forest areas that were inaccessible due to mil- itary restrictions made up the major part of the transferred land cover information. We updated the QA information during the gap-ﬁlling procedure. If only original observations without any Earth Syst. Sci. Data, 6, 339–352, 2014 3.1 Local classiﬁcation scheme S1 Q = PJ g=1 qg 2 (2) and Q = PJ g=1 qg 2 The ﬁeld survey resulted in vector geographic information system (GIS) data with 67 LULC classes (S1). Overall, the study site can be characterised as an extremely heteroge- neous agricultural landscape with a large number of LULC types in its central part (Fig. 1; proportions in the Supplement Table S3 at Pangaea repository). We provide more details on the LULC types in the meta information of the data set (cf. Supplement to the data set at Pangaea repository). (2) and and A = PJ g=1 ag 2 , (3) (3) where qg and ag are quantity disagreement and allocation disagreement in the LULC class g. They are calculated as qg = J X i=1 pig ! − J X j=1 pgj ! (4) The data have 3377 polygons with an average size of 0.019 km2. Because in 2011 we only surveyed the northern half of the catchment, 12.3 % of the values were lacking for this year. (4) and and ag = 2min " J X i=1 pig ! −pgg , J X j=1 pgj ! −pgg # , (5) “Deciduous forest” at the steep hill slopes was stable dur- ing the studied period. It occupied more than half of the study area and was therefore the most dominant type (55.6 %, 2- year average). The moderate slopes from the forest edges to the ﬂat centre were dominated by dry-ﬁeld farms which oc- cupied 16.3 % (2-year average) of the total catchment. The major dry-ﬁeld crops among the total of the 42 we recorded were soybean, ginseng, potato, radish, European and Chinese cabbages and maize. Rice paddies (8.3 %, 2-year average) were prevalent in the central part and surrounded the small urban core (0.86 %, 2-year average). (5) where pig is the proportion of the area of class g in the ref- erence map, pgj is its proportion in the comparison map and pgg is the proportion classiﬁed correctly. The overall quantity disagreement Q indicates the differ- ence between a reference map and a comparison map due to the less than perfect match in the proportions of the cat- egories. The overall allocation disagreement A shows the difference between a reference map and a comparison map caused by the less than optimal match in the allocation of the categories. 3.1 Local classiﬁcation scheme S1 Finally, the total disagreement D is the sum of Q and A. 3.1.1 Major changes in land use During the study period, dry ﬁelds and rice farming de- creased and orchards and ginseng cultivation increased (Ta- ble 6 and Supplement Table S3 at Pangaea repository). In fact, “Ginseng” almost doubled from 2009 to 2010 (1.26 to 2.48 %). It is consistent with the rapid ginseng expansion re- ported by Jun and Kang (2010), who suggested replacing an- nual dry crops by perennial crops to stabilise soils and thus prevent erosion. An expected reduction of soil erosion due to B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Table 2. Characteristics of the different land use and land cover classiﬁcation schemes. Name Description Total Classes related to agriculture classes S1 LULC types observed 67 Individual crops recorded S2 Locally deﬁned grouping 10 “Dry ﬁeld”, “paddy ﬁeld” and “orchard ﬁeld” FAO-LCCS FAO-LCCS major land cover classes 8 “Cultivated terrestrial” and “cultivated aquatic” IGBP IGBP Discover system 17 “Croplands” and “cropland/ natural vegetation mosaics” Characteristics of the different land use and land cover classiﬁcation schemes. Table 2. Characteristics of the different land use and land cover classiﬁcation schemes. GEOS (Geometry Engine - Open Source) (GEOS Develop- ment Team, 2014) through the package rgeos (Bivand and Rundel, 2014). where po is the proportion of pixels in which the two data sets agreed and pc is the proportion of pixels for which agree- ment is expected by chance. Recently, κ has been criticised because of its limited use in remote sensing (Pontius Jr. and Millones, 2011). There- fore, we also provide Pontius’s quantity disagreement Q and allocation disagreement A. They are deﬁned as 2.4.4 Comparison with MODIS land cover We compared the proportions of different classes in our data set with those provided in MCD12Q1 Land Cover Type 1 (IGBP). Additionally, we compared maps derived from our data with those provided in MCD12Q1 for 2009 and 2010. Therefore, we rasterised our survey data at the same spatial resolution (MODIS 500 m sinusoidal grid). We determined the LULC class label of a grid cell covered by multiple poly- gons based on the exact area size. Therefore, we calculated the fraction of the occupied area in the projected (Euclidean) space and assigned the LULC class labels based on the high- est proportion. In addition to the S1 scheme containing 67 classes, we reclassiﬁed the LULC information according to three sim- pler schemes. First, we generated a locally optimised scheme with 10 classes (called S2 in the following) that reﬂects the edaphic and socio-economic conditions in the study area. It consists of the classes “barren”, “dry ﬁeld”, “forest”, “green- house”, “inland water”, “orchard ﬁeld”, “paddy ﬁeld”, “sem- inatural” and “urban”. Then, based on the FAO-LCCS we To measure the agreement between maps, we derived con- fusion matrices and calculated Cohen’s non-weighted κ (Co- hen, 1960): κ = po −pc 1 −pc , (1) (1) Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ www.earth-syst-sci-data.net/6/339/2014/ www.earth-syst-sci-data.net/6/339/2014/ 343 www.earth-syst-sci-data.net/6/339/2014/ B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 345 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Table 3. Changes in the FAO-LCCS category life form. Note that the survey data of 2011 are incomplete. Table 3. Changes in the FAO-LCCS category life form. Note that the survey data of 2011 are incomplete. Table 5. Proportions of crop types deﬁned according to the FAO- LCCS crop types. Note that the survey data of 2011 are incomplete. Life form Survey (%) 2009 2010 2011 Herbaceous 30.17 29.64 19.13 Herbaceous/woody 6.82 6.91 7.03 Non-vegetated 2.85 2.81 2.56 Woody 60.15 60.60 59.01 Missing data 0.02 0.03 12.27 Table 4. Changes of the FAO-LCCS category life cycle. Note that the survey data of 2011 are incomplete. Crop types Survey (%) 2009 2010 2011 Cereals and pseudocereals 9.25 8.34 4.77 Cereals and pseudocereals/fodder crops 0.26 0.77 0.93 Fodder crops 0.07 0.09 0.59 Fruit and nuts 1.07 1.48 0.91 Fruit and nuts/pulses and vegetables 0.00 0.01 0.04 Industrial crops 3.76 5.35 4.27 Mixed crops 4.50 2.74 2.26 Non-crop vegetation 69.08 71.96 67.97 Non-vegetated 2.73 2.81 2.54 Pulses and vegetables 2.57 1.70 0.95 Roots and tubers 6.69 4.71 2.50 Missing data 0.02 0.03 12.27 Table 4. Changes of the FAO-LCCS category life cycle. Note that the survey data of 2011 are incomplete. Life cycle Survey (%) 2009 2010 2011 Annual 19.87 17.45 10.58 Annual/perennial 15.85 16.93 13.14 Non-vegetated 2.73 2.81 2.54 Perennial 61.53 62.78 61.46 Missing data 0.02 0.03 12.27 bers”, “pulses and vegetables”, “fruits and nuts”, “fodder crops” and “industrial crops” (Supplement Table S1 at Pan- gaea repository). We used combinations of them if multiple crop types were identiﬁed on the same patch. Occasionally, the class “mixed crops” was assigned when the combination was not precisely recorded. Field-level land use change was more pronounced than the change of the proportions due to crop rotation, which is common for the annual crops in the region. The annual crops are rarely cultivated in successive years and the dry- ﬁeld crops commonly have a 3-year portfolio (e.g. potato– cabbage–soybean). This pattern is most distinctive in the northern part of the arable zone where the colours (LULC types) are displaced between 2009 and 2010 (Fig. 1). How- ever this displacement is not reﬂected in the proportions. For some crops, the most suitable type was difﬁcult to ﬁnd. B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Indeed, the LCCS manual classiﬁes “soybean” as an indus- trial crop, while in the region it is often used as a vegetable because the green part is popular in local cuisine. “Wild sesame” is another example of a crop with multiple purposes, namely “pulses and vegetables” and “industrial crops”. In our study we deﬁned “soybean” and “wild sesame” as “industrial crops”. The 3 years of crop type information are shown in Fig. 4 and summarised in Table 5. “Cereals and pseudocereals” and “roots and tubers” diminished as “rice paddy”, “white radish” and “potato” cultivation decreased. In contrast, “fruit and nuts” and “industrial crops” increased because the or- chards and a few other industrial crops such as “ginseng” expanded due to the governmental promotion of perennial crops. Additionally, “non-crop vegetation” rose from 2009 to 2010 (69.1 to 72.0 %) as a consequence of an increased num- ber of fallow ﬁelds in preparation for future ginseng farming. 3.1.2 Life form and life cycle For vegetated patches, “herbaceous” vegetation dominated the central agricultural area in contrast to the surrounding for- est which was entirely “woody” (Fig. 2). “lichens/mosses” type vegetation was not recorded. The life form did not change over the period studied (Table 3), possibly because land use changes mainly occurred within the “herbaceous” category (i.e. in the agricultural area). The distribution of life cycles changed from 2009 to 2010 (Table 4). “Annual”-type vegetation dropped from 19.87 to 17.45 % due to decreasing rice paddies and dry ﬁelds. In con- trast, natural “perennial” vegetation expanded over a larger area (61.53 % in 2009 to 62.78 % in 2010). These changes are clearly visible in the mid-western part of the area (Fig. 3) and are probably due to the governmental policy of replacing dry ﬁelds by perennial crops such as ginseng and orchards. 2.4.5 Software We processed the data in GNU R v3.0.2 (R Core Team, 2013) and provide the R code along with the data set in the repos- itory Pangaea (doi:10.1594/PANGAEA.823677). For the re- classiﬁcation, we used the package raster (Bivand and Run- del, 2014). For the rasterisation, we used the geometry engine Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ 344 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Figure 1. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S1 containing 67 classes. this land use change was discussed in Kettering et al. (2012); A h ld l (2013) R idi h l (2013) d Sh l farming could start. The ginseng company Korea Ginseng C i l i i h f h h B. Seo et al.: Deriving a per-ﬁeld land use and land cover map B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 344 Figure 1. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S1 containing 67 classes. nd cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S Figure 1. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S1 containing 67 classes. this land use change was discussed in Kettering et al. (2012); Arnhold et al. (2013); Ruidisch et al. (2013) and Shope et al. (2014). farming could start. The ginseng company Korea Ginseng Corporation only signs a contract with farmers when those regulations are fulﬁlled. Additionally, fallow ﬁelds increased in 2010 (4.8 %) com- pared to 2009 (1.9 %) and replaced a large number of dry ﬁelds. We attribute these changes partially to the subsidy for fallow ﬁelds and partially to corporal regulations requiring at least 3 years of fallow or organic farming before ginseng Compared to the patches, linear elements such as “semi- natural” (6.0 %), “transportation” (0.78 %) and “inland wa- ter” (0.32 %) made up a small proportion in 2009 and 2010. Nevertheless, they covered the whole catchment (Fig. 1). Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ www.earth-syst-sci-data.net/6/339/2014/ 3.2 Classiﬁcation schemes S2 and FAO-LCCS 3.2 Classiﬁcation schemes S2 and FAO-LCCS The coarser classiﬁcation scheme S2 summarises the main changes in land use in the study area (Fig. 5 and Table 6). Actually, “dry ﬁeld” dropped from 2009 (17.83 %) to 2010 (14.83 %) and the “seminatural” type increased from 11.35 % to 14.18 %. We attribute the latter change to the spread of fallow ﬁelds. The three dominant FAO-LCCS types, namely “natural and seminatural terrestrial vegetation”, “cultivated and man- aged terrestrial area” and “cultivated aquatic or regularly ﬂooded areas” covered 97.2 % (2-year average) of the total 3.1.3 Crop types We found 6 of the 12 FAO-LCCS crop types in the study area, namely “cereals and pseudocereals”, “roots and tu- www.earth-syst-sci-data.net/6/339/2014/ Earth Syst. Sci. Data, 6, 339–352, 2014 346 B. Seo et al.: Deriving a per-ﬁeld land use and land cover m Figure 2. Life form of the vegetation cover according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. B. S B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 346 gure 2. Life form of the vegetation cover according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Figure 2. Life form of the vegetation cover according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Figure 3. Life cycle of the vegetation cover according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Figure 3. Life cycle of the vegetation cover according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Table 6. Changes in land use and land cover based on the classiﬁ- cation scheme S2. Note that the survey data of 2011 are incomplete. When applying the FAO-LCCS scheme to our data, the classiﬁcation of “rice paddy” was challenging. In actual fact, in Haean, rice is sometimes irrigated with water from deep wells. However, although the “cultivated aquatic or regu- larly ﬂooded areas” class excludes irrigated cultivated areas (Di Gregorio, 2005), we assigned rice to this type as it is mostly rainfed. Class Survey (%) 2009 2010 2011 Barren 0.31 0.22 0.08 Dry ﬁeld 17.83 14.83 11.07 Forest 57.74 57.79 57.11 Greenhouse 0.77 0.84 0.58 Inland water 0.69 0.86 0.89 Inland wetland 0.00 0.00 0.00 Orchard ﬁeld 1.07 1.48 0.91 Paddy ﬁeld 8.50 8.04 4.65 Seminatural 11.35 14.18 10.86 Urban 1.72 1.72 1.57 Missing data 0.02 0.03 12.27 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 347 Figure 4. Crop types according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Figure 5. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S2. Figure 4. Crop types according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. 10 d ( ) 2011 Figure 4. Crop types according to the FAO-LCCS in (a) 2009, (b) 2010 and (c) 2011. Figure 5. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S2. Figure 5. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S2. he Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to the classiﬁcation scheme S2. Figure 5. Land use and land cover in the Haean catchment in (a) 2009, (b) 2010 and (c) 2011 according to th in the MODIS land cover while they represent only 29.2 % in our survey data (2-year averages). For “croplands” the MODIS product shows a moderate agreement with the survey data (29.0 % vs. 24.3 %, 2-year averages). The mosaic class “cropland/natural vegetation mosaics” type was not found in our survey data while in the MODIS data set it comprises 10.97 % in 2009 and 5.34 % in 2010. MODIS assigns this class to pixels containing a mixed of croplands, forests, shrubland and grasslands as long as no single component comprises more than 60 % of the area (Friedl et al., 2002). By deﬁnition, this mixture class is am- biguous (Friedl et al., 2002, 2010). In contrast, we explicitly recorded the individual classes for smaller patches instead of assigning the mosaic class for a larger area. In contrast, the forested area is underrepresented by MODIS as “deciduous broadleaf forests” and “mixed forests” add up to 49.7 % in the MODIS land cover while they cover 57.5% in our survey, averaged over 2 years. In- dividually, in our survey, the area of “deciduous broadleaf forests” is larger (55.4 % vs. 31.2 %) and the area of “mixed forests” is substantially smaller (2.08 % vs. 18.5 %) com- pared to MCD12Q1 (averaged over 2 years). 3.3.1 Comparison between MODIS land cover and the original survey data We found 10 IGBP classes in our study area, namely “waterbodies”, “evergreen needleleaf forests”, “deciduous broadleaf forests”, “mixed forests”, “closed shrublands”, “open shrublands”, “grasslands”, “croplands”, “urban and built-up lands” and “barren or sparsely vegetated”. In con- trast, MCD12Q1 contained only ﬁve classes: “deciduous broadleaf forests”, “mixed forests”, “grasslands”, “crop- lands” and “cropland/natural vegetation mosaics”. The ﬁrst row of Fig. 7 shows the original survey data and the third shows MCD12Q1. In addition, Table 8 summarises area pro- portions in both data sets. area. The “natural and seminatural terrestrial vegetation” pre- vailed (70.6 %, 2-year average) and increased from 2009 to 2010 (Table 7). In contrast, “cultivated and managed terres- trial area” and “cultivated aquatic or regularly ﬂooded areas” decreased, probably due to reduced dry-ﬁeld and rice farm- ing, respectively. Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ www.earth-syst-sci-data.net/6/339/2014/ Earth Syst. Sci. Data, 6, 339–352, 2014 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 348 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Figure 6. Reclassiﬁed land use and land cover in (a) 2009, (b) 2010 and (c) 2011 according to the FAO-LCCS eight major land cover classes. The annual proportions are shown in Supplement Table S2 at Pangaea repository. These classes are deﬁned by the stratiﬁed structure with three dichotomous levels: presence of vegetation, edaphic condition and artiﬁciality of cover. 348 Figure 6. Reclassiﬁed land use and land cover in (a) 2009, (b) 2010 and (c) 2011 according to the FAO-LCCS eight major land cover classes. The annual proportions are shown in Supplement Table S2 at Pangaea repository. These classes are deﬁned by the stratiﬁed structure with three dichotomous levels: presence of vegetation, edaphic condition and artiﬁciality of cover. Table 7. Annual proportions of the reclassiﬁed land use and land cover data according to the FAO-LCCS eight major land cover classes. Note that the survey data of 2011 are incomplete. Table 7. Annual proportions of the reclassiﬁed land use and land cover data according to the FAO-LCCS eight major land cover classes. Note that the survey data of 2011 are incomplete. Table 7. Annual proportions of the reclassiﬁed land use and land cover data according to the FAO-LCCS eight major land cover classes. Note that the survey data of 2011 are incomplete. f the reclassiﬁed land use and land cover data according to the FAO-LCCS eight major land cover classes. 1 are incomplete. LCCS eight major classes Survey (%) 2009 2010 2011 Artiﬁcial surfaces and associated area 1.72 1.72 1.57 Artiﬁcial waterbodies, snow and ice 0.07 0.23 0.24 Bare area 0.22 0.08 0.05 Cultivated and managed terrestrial area 19.68 17.15 12.56 Cultivated aquatic or regularly ﬂooded areas 8.50 8.04 4.65 Natural and seminatural aquatic or regularly ﬂooded vegetation 0.09 0.09 0.07 Natural and seminatural terrestrial vegetation 69.09 72.02 67.94 Natural waterbodies, snow and ice 0.62 0.63 0.65 Missing data 0.02 0.03 12.27 either as pure cropland or pure natural vegetation. However, analysing this effect is beyond the scope of this work. either as pure cropland or pure natural vegetation. However, analysing this effect is beyond the scope of this work. For the 2-year average, the total disagreement D is 0.42, the quantity disagreement Q is 0.36 and the allocation dis- agreement A is 0.053. Thus, quantity disagreement accounts for 87 % of the overall disagreement. 3.3.2 Comparison between MODIS land cover and the rasterised survey data 3.3.2 Comparison between MODIS land cover and the rasterised survey data After rasterisation, six IGBP classes were found in the survey data, namely “deciduous broadleaf forests”, “mixed forests”, “closed shrublands”, “grasslands”, “croplands” and “urban and built-up lands”. “Urban and built-up lands” were missing in the MODIS data while “cropland/natural vegetation mo- saics” does not exist in our data. Figure 7 shows the rasterised ground observations (in the middle row) and MCD12Q1 (in the bottom row). B. Seo et al.: Deriving a per-ﬁeld land use and land cover map The disagreement in the agricultural and the forest types may be due to the coarser resolution of the MODIS product (500 m). This becomes more problematic for land cover types smaller than the MODIS pixel in its typical dimension. In- deed, linear elements such as “waterbodies” and “urban and built-up lands” were not found in the product. The shrubland classes as well as the cropland classes are relevant to agriculture as some of the perennial crop types were classiﬁed as “closed shrublands” and “open shrub- lands”. We have more than 5 % of shrubland classes in the survey data which are not found in the MODIS product for the 2-year period. We note that, for the forest classes, our limited access to the surrounding forest may have caused inaccuracies in our data. Moreover, the agreement between the two data sets could be higher if we used the mosaic class “crop- land/natural vegetation mosaics” for our data. There may have been patches that are better described as mixtures of cropland and natural vegetation than by reclassifying them There is an overrepresentation of the agricultural area in MCD12Q1 compared to our ground observations. If we combine all the agriculturally relevant classes, namely “croplands”, “cropland/natural vegetation mosaics”, “closed shrublands” and “open shrublands”, these add up to 37.1 % www.earth-syst-sci-data.net/6/339/2014/ Earth Syst. Sci. Data, 6, 339–352, 2014 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map B. Seo et al.: Deriving a per-ﬁeld land use and land cover map This suggests that MCD12Q1 may fail to evaluate the quantity of different LULC classes in complex agricultural landscapes. 4 Data structure and data access The data set and its description are available at the Pan- gaea repository under the Creative Commons Attribution- NonCommercial 3.0 Unported license. The data contain LULC observations and ancillary information in a single ESRI polygon shape ﬁle (ESRI Inc., http://esri.com). The LULC type, QA, management and double-cropping and mixed-use information are provided on an annual basis. The deﬁnition of classes and the reclassiﬁcation table are given separately in a legend table. For each polygon, LULC in- formation for 3 years is given in separate columns (e.g. LULC2009, LULC2010 and LULC2011). Note that multi- ple entries in a LULC type column occur in cases when the polygon exhibited mixed land uses spatially or temporally. To compare the two maps, we derived confusion matri- ces, Cohen’s κ and Pontius’s Q and A for 2009 (Supplement Table S3 at Pangaea repository) and 2010 (Supplement Ta- ble S4 at Pangaea repository). We excluded the year 2011 due to a lack of ground observations. The mean κ for the 2 years equals 0.41, which indicates a fair but not substantial agreement. Earth Syst. Sci. Data, 6, 339–352, 2014 www.earth-syst-sci-data.net/6/339/2014/ B. Seo et al.: Deriving a per-ﬁeld land use and land cover map B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 349 B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 349 Figure 7. Land use and land cover reclassiﬁed according to the IGBP 17-class system: the original survey data in (a) 2009, (b) 2010 and (c) 2011; the rasterised survey data in (d) 2009, (e) 2010 and (f) 2011; MODIS Land Cover Type product (MCD12Q1) in (g) 2009, (h) 2010 and (i) 2011. Note that the IGBP system does not distinguish the paddy ﬁeld from a general cultivated zone. Note that “interrupted areas” is a special mask for Goode’s interrupted area (US Geological Survey, 2012). Figure 7. Land use and land cover reclassiﬁed according to the IGBP 17-class system: the original survey data in (a) 2009, (b) 2010 and (c) 2011; the rasterised survey data in (d) 2009, (e) 2010 and (f) 2011; MODIS Land Cover Type product (MCD12Q1) in (g) 2009, (h) 2010 and (i) 2011. Note that the IGBP system does not distinguish the paddy ﬁeld from a general cultivated zone. Note that “interrupted areas” is a special mask for Goode’s interrupted area (US Geological Survey, 2012). www.earth-syst-sci-data.net/6/339/2014/ B. Seo et al.: Deriving a per-ﬁeld land use and land cover map 350 Table 8. Changes of land use and land cover according to the IGBP 17-class system. The columns under “survey” refer to the survey data and those under “MODIS” to MODIS Land Cover Type (MCD12Q1) following the same classiﬁcation system. Note that the “waterbodies” and “urban” classes were not detected by MODIS, presumably as a result of coarse resolution (500 m). Note that the survey data of 2011 are incomplete. Table 8. Changes of land use and land cover according to the IGBP 17-class system. The columns under “survey” refer to the survey data and those under “MODIS” to MODIS Land Cover Type (MCD12Q1) following the same classiﬁcation system. Note that the “waterbodies” and “urban” classes were not detected by MODIS, presumably as a result of coarse resolution (500 m). Note that the survey data of 2011 are incomplete. IGBP 17 classes Survey (%) MODIS (%) 2009 2010 2011 2009 2010 2011 Waterbodies 0.69 0.86 0.89 0.00 0.00 0.00 Evergreen needleleaf forests 0.29 0.29 0.30 0.00 0.00 0.00 Evergreen broadleaf forests 0.00 0.00 0.00 0.00 0.00 0.00 Deciduous needleleaf forests 0.00 0.00 0.00 0.00 0.00 0.00 Deciduous broadleaf forests 55.39 55.41 54.73 34.73 27.73 27.41 Mixed forests 2.06 2.08 2.08 12.45 24.58 25.57 Closed shrublands 3.60 3.67 3.14 0.00 0.00 0.00 Open shrublands 1.06 1.48 0.93 0.00 0.00 0.00 Woody savannas 0.00 0.00 0.00 0.00 0.00 0.00 Savannas 0.00 0.00 0.00 0.00 0.00 0.00 Grasslands 7.89 10.80 7.82 10.67 15.67 17.01 Permanent wetlands 0.00 0.00 0.00 0.00 0.00 0.00 Croplands 26.09 22.54 15.58 31.19 26.68 26.35 Urban and built-up lands 2.49 2.57 2.15 0.00 0.00 0.00 Cropland/natural vegetation mosaics 0.00 0.00 0.00 10.97 5.34 3.67 Snow and ice 0.00 0.00 0.00 0.00 0.00 0.00 Barren or sparsely vegetated 0.44 0.22 0.09 0.00 0.00 0.00 Interrupted areas 0.00 0.00 0.00 0.00 0.00 0.00 Missing data 0.02 0.03 12.27 0.00 0.00 0.00 use of general cropland classes may be inappropriate in complex agricultural landscapes. For example, Berger et al. (2013a) pointed out the lack of paddy soil and subsoil stud- ies despite their potential impact on global carbon and nitro- gen cycles. Recent studies in the same area repeatedly sug- gested that complex agricultural landscapes needed greater attention (Shope et al., 2014; Arnhold et al., 2013; Berger et al., 2013a, b; Kim et al., 2014; Kettering et al., 2012; Ruidisch et al., 2013). B. Seo et al.: Deriving a per-ﬁeld land use and land cover map Thus, thematic improvement of global land cover databases is of great importance. Acknowledgements. We thank Hamada Elsayed Ali, Sebastian Arnhold, Jaesung Eum and Ralf Geyer for their help in the labo- ratory and during data collection. This research was supported by the International Research Training Group of Germany and South Korea (DFG/KOSEF, Complex TERRain and ECOlogical Hetero- geneity (TERRECO), GRK 1565/1). MODIS data were obtained from https://lpdaac.usgs.gov, main- tained by the NASA Land Processes Distributed Active Archive Center (LP DAAC) at the USGS/Earth Resources Observation and Science (EROS) Center. g p There have been ongoing efforts to extend MODIS land cover databases (Biggs et al., 2006; Potgieter et al., 2007; Wardlow et al., 2007; Wardlow and Egbert, 2008; Pittman et al., 2010; He and Bo, 2011; Gumma et al., 2011). For nat- ural vegetation, global high-resolution databases are becom- ing available (e.g. Hansen et al., 2013). Our vector-form data can be useful in developing/validating high-resolution data sets for complex agricultural landscapes because the data in- clude detailed crop type information with a consistent and complete description established by the FAO (Di Gregorio, 2005). Additionally, our data contains different classiﬁcation systems and can be transformed to any raster grid. Due to this detailed information, our data could be used for regional environmental modelling as well as for ecosystem services research and decision making analysis. Edited by: D. Carlson 5 Summary and conclusions resents forest types compared to our ground observations. Linear elements such as “waterbodies” were missing in the remote-sensing product due to its coarse spatial resolution. We measured the agreement between the rasterised ground truth and the MODIS land cover. The agreement was fair but not substantial for the primary land cover type. We provide an annual per-ﬁeld land use and land cover data set for the agricultural mosaic catchment Haean (South Ko- rea). During the study period many dry ﬁelds were con- verted to perennial crops such as ginseng and orchards, prob- ably due to governmental policy measures. 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https://openalex.org/W4312557472	https://periodicos.ufmg.br/index.php/geografias/article/download/19738/30266	Portuguese	null	Cidade hostil	Geografias	2,022	cc-by	8,560	Resumo A exposição diária ao medo da violência e a busca de privacidade têm contribuído para o processo de segregação urbana. Intensificam-se, no Brasil, soluções individuais para problemas coletivos. Pela literatura, observamos que a desertificação do espaço urbano é um dos fatores- chave da percepção da sensação de insegurança, pois o espaço perde a atratividade e pessoas atraem pessoas, o que gera a tão desejada vitalidade urbana. Quando a arquitetura se reveste de formas limitadoras – visuais, físicas e sociais – essa arquitetura é hostil. O objetivo desse artigo é discutir como a arquitetura hostil tem se instituído como elemento ativo na retroalimentação da percepção da sensação de insegurança urbana, na medida em que limita e condiciona a experiência do espaço urbano entre iguais e produz “extramuros”, espaços áridos, impermeáveis, desproporcionais à escala humana. Para pesquisa foi estudado o bairro do Espinheiro, na cidade do Recife, pois foi o bairro que mais verticalizou na cidade dentro do recorte de estudo, sendo modificador do padrão da habitação e da forma de habitar. Percebemos que a arquitetura promovida pelo mercado imobiliário não caminha na direção estabelecer relações que acolham o indivíduo. Cada dia mais edificamos barreira no espaço urbano. Os indivíduos vêm ao longo das últimas décadas buscando uma proteção individualista, o que reflete uma perda de urbanidade, ou seja, as características inerentes da arquitetura levam para uma urbanização privatizada, centrada em homogêneos. Palavras- Chaves: Arquitetura hostil. Violência urbana. Bairro Espinheiro. Palavras- Chaves: Arquitetura hostil. Violência urbana. Bairro Espinheiro. Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X Cidade hostil Shayenne Barbosa Dias Pós-Graduação em Estudos Urbanos e Regionais - UFRN shayennedias@gmail.com Cláudio Roberto de Jesus Pós-Graduação em Estudos Urbanos e Regionais – UFRN claudiorobertojesus@gmail.com ISSN 2237-549X over the last decades, which reflects a loss of urbanity, that is, the inherent characteristics of architecture lead to a homogenized, privatized urbanization. Keywords: Hostile architecture. Urban violence. Espinheiro Neighborhood. Abstract The daily exposition to fear and violence as well as the quest for privacy have meaningfully contributed to human segregation process. In Brazil, individual solutions to collective problems have been fostered. According to scientific literature, it is possible to acknowledge that urban space desertification is a key factor that contributes to the perception of insecurity making urban space lose attractiveness. Urban vitality is affected since people attract other people. It is seen that when architecture presents limited forms, either visual, physical or social it is perceived as hostile. This work aims to discuss the ways hostile architecture has been established as an active element in perception of feedback regarding urban insecurity, as it limits and conditions the experience of urban space between equals and produces “Outliers”, arid, impermeable spaces, disproportionate to human scale. The research considered the Espinheiro neighborhood, in Recife-Brazil. This area is considered the most verticalized in the city, modifying housing patterns and the way of living. It is perceived that that the architecture promoted by the real estate market does not work towards establishing relationships that welcome individuals. Barriers in the urban space are built daily. People have been seeking individualistic protection Página 26 Página 26 GEOgrafias Artigos científicos GEOgrafias Artigos científicos GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X através da arquitetura produzida pelos condomínios residenciais verticais, trazer evidências se ela contribui na forma como percebemos a sensação de insegurança. Lira (2019, p. 121) verifica “que as formas e funções da arquitetura do medo são reciprocamente influenciadas pelos temores relativos à violência contemporânea”. Caldeira (2000, p. 294) afirma que existe uma “estética da segurança”, o que, segundo autora, reforça que elementos de defesa são utilizados não só por razões de segurança e segregação, mas também por razões estéticas e de status. O que nos leva a inferir que alguns elementos são dispensáveis, mas mantêm-se presentes na arquitetura, atuando de maneira repressiva. É possível perceber, como alerta Davis (2009, p.237), que a forma como se percebe a ameaça é dada em função da própria mobilização por segurança, não estando diretamente ligada aos índices de criminalidade, e conclui que “embora os críticos da arquitetura, em geral, não prestem atenção em como um ambiente contribuem para segregação, os grupos de párias, leem o significado imediatamente”. Sem negar que a violência e os índices de criminalidade encontram-se arraigados na percepção da sensação de insegurança e partindo da ideia que a forma urbana, apesar de extrapolar a cidade como forma física delimitada pelo espaço construído é desenvolvida com a articulação dos elementos arquitetônicos, buscaremos identificar a relação entre a forma edificada e a percepção de sensação de estar inseguro. A referida análise será desenvolvida com base na análise do perfil espacial do bairro do Espinheiro, como bairro que primeiro se verticalizou na Zona Norte da cidade do Recife. Sem pretensão de criar uma categorização ou tipificação da arquitetura, a prospecção no bairro buscou entender se há uma relação do aspecto arquitetônico edificado, mesmo que não intencional, com a percepção sobre a sensação de insegurança. Para alcançar o objetivo proposto foi realizada uma contextualização teórica e pesquisa empírica, o que possibilitou a execução de análises em torno das ideias relacionadas ao objeto, permitindo as investigações do objeto empírico. Introdução Com a estagnação econômica nas décadas de 1980 e 90 ocorreu o acirramento das desigualdades sociais e da pobreza na estrutura social brasileira. Nesse período, os indicadores associados à violência urbana sofreram uma alta sensível, assim como a sensação de insegurança. Além disso, os mercados ilícitos, ilegais e informais ganharam nova dinâmica, impulsionados pela globalização. A integração nacional e global, bem como a ampliação no volume de negócios desses mercados, alterou os ganhos e as formas de apropriação do espaço dos grupos marginais. Na década de 1990, o tema da segurança pública emerge de forma central nos debates acadêmicos, na mídia e na agenda governamental, tendo como centro os altos índices de violência urbana. A exposição constante à sensação de insegurança e a ampliação do medo levaram a uma corrida por segurança, que vem alterando as configurações arquitetônicas e as práticas sociais nas cidades. Em um primeiro momento, a arquitetura é influenciada pela violência e insegurança urbana, porém a proliferação da arquitetura hostil passa também a influenciar na percepção da sensação de insegurança, o que se torna perceptível ao longo dos anos 2000. A busca por segurança transforma casas, ruas, praças e conduz a uma perda da vitalidade e urbanidade, na medida em que edifica espaços sem qualidade. A urbanização, com foco de estudo nos condomínios residenciais verticais, reflete uma dissociação entre os espaços públicos e privados e ostenta uma forma de se organizar com base nos padrões de fuga e isolamento, o que é rebatido também na desertificação das ruas e amplia a percepção da vulnerabilidade humana no espaço público agravando a sensação de insegurança urbana. A proposta desta pesquisa é compreender o papel da arquitetura na atmosfera da sensação de insegurança urbana, indo além de aceitar a arquitetura como efeito das cruzadas humanas pela segurança, buscando compreendê-la, também, como um elemento ativo dessa atmosfera. Mais especificamente, o objetivo deste trabalho consiste em investigar o bairro do Espinheiro, na cidade do Recife, Pernambuco e, Página 27 GEOgrafias Artigos científicos Página 27 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Cidade e insegurança Um dos aspectos mais relevante apontado na literatura, da relação entre a arquitetura e a insegurança, é o critério da visibilidade. Os estudos convergem para uma GEOgrafias Artigos científicos Página 28 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X consequente redução da insegurança do desenho arquitetônico e uso de espaços que sejam capazes de contribuir com a vitalidade urbana: presença de vigilância, atividades, manutenção e fluxo de pessoas no espaço (JACOBS, 2000; NEWMAN, 1972; HILLIER, 1988). Lynch (2010) afirma que a compreensão dos componentes e o desenvolvimento da imagem de um lugar e seus constituintes ocorrem pela atuação sobre a forma exterior do ambiente, em conformidade com o processo cognitivo interior. Logo, conceitos como legibilidade e funcionalidade relacionam-se a com a vitalidade do local. Sem pretensão de criar uma categoria, pelo aspecto semiótico da avaliação do espaço edificado, a arquitetura do medo corresponde à arquitetura edificada com propósito de inibir as condutas suspeitas. As características da arquitetura do medo (ELLIN, 1997) e arquitetura da violência (FERRAZ, 2005) são, grosso modo, similares em suas formas. Muros altos, cercas elétricas, falta de permeabilidade física e visual, diminuições de conexões com espaço público, dentre outros artifícios. Essas são algumas das medidas as quais o indivíduo (ou empresa) recorre com propósito de mitigar uma ameaça externa e caracterizam uma arquitetura subordinada ao medo, à violência e a insegurança. Este estudo propõe chamar de “Arquitetura Hostil” esses mesmos aspectos, que edificam um espaço descontínuo e limitam a experiência do viver o espaço, do caminhar, além de trabalhar com aspectos arquitetônicos que atualmente parecem negligenciados. Assim o leitor é convidado a olhar a arquitetura por outro prisma, da ótica de um transeunte circulante no espaço público da cidade. A arquitetura, aqui entendida enquanto forma-conteúdo, pela presença da ação em que a forma se torna capaz de influenciar de volta, o desenvolvimento da totalidade (SANTOS, 1996), que veicula o medo e a insegurança, torna-se ela mesma portadora e/ou catalizadora desses sentimentos. Afirma Santos (1996a) que “[...] o espaço organizado pelo homem é como as demais estruturas sociais, uma estrutura subordinada-subordinante”. Se num primeiro momento a arquitetura é reflexo da sensação de insegurança e medo, que nem sempre se traduz em índices reais de violência, em um segundo momento passa influenciar na percepção da sensação. Uma arquitetura que protege o espaço privado, num segundo momento, pode ser capaz limitar relações, GEOgrafias Artigos científicos Página 29 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X impedir o convívio e romper laços. A experiência do espaço urbano é única para cada indivíduo e é também parte constituinte do ser humano e, assim, do ser urbano. Uma arquitetura disforme para experiência do corpo, ilegível para indivíduo, que não edifica a cidade como abrigo para o homem, é capaz de se tornar hostil, mesmo que, à primeira vista, essa ação arquitetônica não seja percebida. Ao longo da história, as cidades e suas arquiteturas servem como testemunho das sociedades e como importantes sistemas de informação, comunicação e transmissão cultural no decorrer dos anos. Guattari (1992) assinala que o “devir da humanidade parece inseparável do devir urbano”. A cidade é a expressão material máxima do fenômeno urbano, mas a arquitetura se torna responsável para além da materialidade do abrigo humano. Pois, ela media, acolhe e organiza determinadas ações e relações sociais. No espaço da cidade são escritas suas histórias e relações de dominação, formação e conquista. A maneira como são edificadas são fontes históricas que nos permitem entender essas relações existentes. O processo de urbanização, sob a égide da propriedade privada, apresenta transformações nas relações sociais, econômicas e locacionais. A consolidação do processo de urbanização avançou, trazendo consequências danosas para a população, para as relações sociais e de trabalho que se desenvolviam. Bauman (2010) aponta como o capitalismo pode florescer e prosperar durante certo tempo, desde que haja condições favoráveis a ele, “mas não pode fazer isso sem prejudicar o hospedeiro, destruindo cedo ou tarde as condições de sua prosperidade ou mesmo de sua sobrevivência”. Intensifica-se a urbanização, nasce à metrópole. Um espaço onde a luta do indivíduo contra a sociedade ganha claros contornos: no subjetivismo exagerado, no distanciamento um dos outros e no medo do contato com o outro (STECHER, 1995). Para proteger-se das ameaças que o desenraizariam, o homem da metrópole se torna racional. O indivíduo cosmopolita passou a ter uma imagem do estranho como ameaçador. Ele não reage de forma direta e emocional, como faz o indivíduo da cidade pequena. O cidadão metropolitano é obrigado a mascarar seus sentimentos e, muitas vezes, a não reagir aos estímulos sofridos, assumindo um ar blasé no cotidiano, essa capacidade é uma das principais características do homem da metrópole. GEOgrafias Artigos científicos GEOgrafias Artigos científicos Página 30 Página 30 Revista GEOgrafias, v. 15, n. 1, jan./jun. ISSN 2237-549X 2019 ISSN 2237-549X ISSN 2237-549X A atitude blasé resulta, em primeiro lugar, dos estímulos contrastantes que, em rápidas mudanças e compressão concentrada, são impostos aos nervos. Disto também parece, originalmente, jorrar a intensificação da intelectualidade metropolitana. (SIMMEL, 1967) Ainda que Simmel tenha deixado de lado uma série de outras relações pautadas na cooperação e solidariedade que se desenvolvem na metrópole, sua grande contribuição é mostrar que parte dos indivíduos reagem ao cotidiano metropolitano com uma atitude de reserva, de busca da privacidade. Afirma Sennett (1988) que “o silêncio em público se tornou o único modo pelo qual se poderia experimentar a vida pública, especialmente a vida nas ruas, sem se sentir esmagado” todo cidadão seria possuidor de um “escudo invisível, um direito de ser deixado em paz”. Não será objeto de debate desta dissertação a esfera pública ou espaço público versus privado, mas como o urbanismo neoliberal influencia estilos, modos de vida e hábitos, das mais diferentes formas. Reforçando a ideia capitalista de separação e do isolamento, Sennett (1988) faz uma crítica mostrando que o ambiente incita a pensar no domínio público como desprovido de sentido e nesse momento passa a ser materializada no ambiente urbano, da arquitetura toda contradição da vida pública e privada. A transformação do espaço público nos centros urbanos também é a sua popularização. Uma vez que os operários e populares passam a fazer parte da cena pública e ocupam o espaço com suas práticas de lazer e contestação, cria-se a imagem de horror e perigo (JESUS, 2011) Talvez seja este um importante ponto para proliferação da sensação da insegurança. A popularização do espaço urbano impede o controle das massas populares? Segundo Bauman (2009), a proliferação da insegurança urbana está ligada aos medos modernos que tiveram início com a redução do controle estatal, considerando que, desde o princípio, o Estado participava das “redes de proteção”, de uma estratégia de contenção de medos sociais, que foram enfraquecidas. A modernidade sólida, afirma Bauman (2009), administrava o medo da solidariedade, um “laço natural”, que foi substituído pela competição. Nesse momento, os indivíduos despreparados estão entregues à própria sorte e, posteriormente, com a modernidade líquida, estão entregues aos seus próprios recursos que são claramente Página 31 Página 31 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X inadequados. As relações se diluíram, os indivíduos estão fisicamente próximos e socialmente distantes. O indivíduo passa a ter uma necessidade de se proteger da ameaça, todos se tornam suspeitos, os laços solidários se esgarçam, as suspeitas se tornam generalizantes assim como a ansiedade, pois se vive a instabilidade da vida contemporânea. O impulso para se proteger da ameaça redefine as escolhas e fomenta estratégias de evitar “o outro” e de se separar do “perigo potencial”, dando amplitude ao processo de segregação espacial e social. Ponto chave para estudos urbanos contemporâneos, a estreita interação entre as pressões globalizantes e o modo como as identidades locais são negociadas, modeladas e remodeladas (BAUMAN, 2009), acrescentada e manifestada no tecido urbano, apresentam relevância por alterar significativamente a qualidade de vida do homem. Pois a combinação de espaços construídos e vividos constituem lugares de significado, identidade e sentido social e cultural. Na vivência do espaço urbano, baseada na não vivência e no não contato com o desconhecido, “suspeitamos dos outros e de suas intenções, nos recusamos a confiar (ou não conseguimos fazê-lo) na constância e na regularidade da solidariedade humana”. Reproduzindo a ideia de perigo iminente, “os medos nos estimulam a assumir uma ação defensiva. Quando isso ocorre à ação defensiva confere proximidade e tangibilidade ao medo [...] o medo agora se estabeleceu, saturando nossas rotinas cotidianas” (BAUMAN, 2007, p.15). Para Sennett (2002) é bastante natural que a flexibilidade cause ansiedade, as pessoas não sabem se os riscos serão compensados, ou que caminhos eles seguirão. Isso faz com que o indivíduo seja facilmente encorajado a tomar decisões que, aparentemente, são certas em benefício próprio, sem identificar os efeitos de sua ação. Podemos exemplificar apresentando como a locomoção exclusiva por veículo automotor vem contribuindo diretamente para a desertificação das ruas. As duas transformações modernas que contribuem para espalhar uma sensação de insegurança, segundo Bauman (2007), são a valorização do indivíduo e suas decisões e a vulnerabilidade da “rede de vínculos sociais”. Vivemos hoje uma enorme valorização do “eu”, seja por uma sensação de desamparo, seja pela Página 32 GEOgrafias Artigos científicos GEOgrafias Artigos científicos GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X individualidade exacerbada, contudo se faz importante entender os efeitos macro dessa individualização. Na cidade contemporânea, alia-se ao processo de individualização o aumento gradativo dos números da violência. Este é um fenômeno que atinge a sociedade: cotidianamente, as práticas são penetradas pela ameaça da criminalidade urbana e enfraquecimento dos laços sociais. A insegurança se relaciona com a falta de confiança e com a iminência de uma ameaça. Nasce de “um sentimento de impotência: de não estar mais no controle” (BAUMAN, 2007, p. 32), que se assemelha ao o homem como o lobo do próprio homem, possuidor de um poder de violência ilimitado. É um sentimento real que mobiliza as populações e que tem impactos negativos na qualidade de vida e na relação com o outro. Influenciando no cotidiano das pessoas, promovendo, cada vez mais, o distanciamento entre os indivíduos e dos indivíduos com a cidade. Para Bauman, há uma característica que sempre se fará presente nas cidades: ela é um espaço cheio de desconhecidos convivendo em extrema proximidade. Guedes (2004) afirma: “A arquitetura leva à cidade [...] pessoas singulares, de natureza social, em constante e inesperada mutação, como ficou claro na fragilização política a que ficou exposta a classe operária, nas últimas décadas. Pessoas individuais ou coletivas, públicas ou privadas e seus projetos legítimos em conflito permanente por novas configurações sociais, exigindo e conciliando para sobreviver ou, simplesmente, trabalhar, comer, reproduzir e educar, com o pensamento treinado em batalhas violentas, onde o amor assoma como alimento e força”. Como imaginar cidades onde as pessoas busquem se isolar? Bauman (2007) responde que, o ambiente social vivido nas grandes metrópoles é permeado por um movimento ambivalente, o confronto “mixofobia” versus “mixofilia”. Onde a “mixofobia” é o típico medo de se envolver com diferentes, interagir ou conviver de alguma forma. A formação de condomínios, academias, clubes fechados, são exemplos desse processo, pois criam núcleos de sociedade homogênea. Mas da mesma forma a “mixofilia” que é o prazer de estar num ambiente diferente e estimulante, de partilhar uma vida social. Fazem o indivíduo viver nesse espaço. As razões da “mixofobia” podem ser corriqueiras e quando expostas são facilmente entendíveis, mas não necessariamente fáceis de esquecer. Seria manifestada no impulso de construir ilhas de similaridade e identidade em meio a um oceano de Página 33 Página 33 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 A insegurança moderna é caracterizada pelo medo dos crimes e dos criminosos, na constância e na regularidade da solidariedade humana. Relatando o que Castells atribui a culpa por esse estado de coisas ao individualismo moderno. Dever individual de cuidar de si próprio, areia movediça na contingência, o perigo está em toda parte são inerentes a essa sociedade. Com isso, é reforçada a imagem dos espaços privados como refúgio e lugar de segurança, o que acaba não sendo verdade, pois a reprodução dessa ideia eleva o medo de estar sozinho, tendo em vista que o perigo está em todo lugar e isso se traduz no esvaziamento do espaço público e reverbera ainda mais na sensação de insegurança coletiva. ISSN 2237-549X diversidade e diferença. “A concentração maciça de estranhos é, simultaneamente, um repelente e um poderosíssimo ímã [...]” (BAUMAN, 2007). Para exemplificar, podemos nos referir aos espaços exclusivos, tipo camarote. A fragmentação da vida urbana permite o surgimento de grupos de indivíduos unidos por interesses, hábitos, opiniões e condições financeiras iguais. Evitar o “outro”, o “estranho”, o “estrangeiro”, traz uma suposta segurança e uma superficial sensação de “paz social” e isso é possibilitado pela homogeneidade social encontrada nos espaços fechados, efeito que potencializa o que Bauman denominada de autossegregação, que é uma tendência a se separar. A tendência a se isolar aumenta, se espalhando por todo tecido urbano. A importância do debate se dá para entendermos a transformação da arquitetura da cidade e sua influência na forma como as pessoas percebem e se apropriam desse espaço. Entender a mudança da postura arquitetônica, que passa a se fechar para o espaço público, e negar a rua e vida urbana que erguendo barreiras físicas segregam espaços, vidas, relações e interações que o viver urbano permite (BAUMAN, 2003). Caldeira (2000) afirma que é possível descobrir, no entorno do processo de construção física dos enclaves, um processo de construção simbólica de um círculo, ao redor do qual o medo é trabalhado e reproduzido, além de produzir a própria segregação socioespacial e a própria violência. Outro fato interessante nessa busca por se proteger é que, de forma contraditória e aparentemente desconectada, quando o processo civilizatório registra os maiores avanços na direção da democracia e progresso tecnológico, afirma Souza (2008), é quando a violência assume posição de destaque dentre as preocupações urbanas. O aumento real ou percebido da violência criminal tem efeitos no plano material, simbólico e econômico, podendo causar marcas, no indivíduo e na cidade, de difícil remoção. O espaço público contemporâneo perde, na atualidade, sua função precípua de lócus de sociabilidade. As relações sociais de vizinhança não criam laços reais e não se estabelecem. Bauman (2007) faz referência ao individualismo presente na contemporaneidade: Página 34 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X movediça na contingência, o perigo está em toda parte são inerentes a essa sociedade. movediça na contingência, o perigo está em toda parte são inerentes a essa sociedade. ISSN 2237-549X Quando sob justificativa de buscar proteção, evita-se sair de casa, andar nas ruas, sair sozinho, tomar um ônibus ou andar a pé, se desenvolve com a cidade uma relação avessa ao processo de apropriação do espaço urbano. E como se não bastasse, é necessário buscar espaços e relações de sociabilidade desenvolvidas em locais “seguros”. É necessário “estar protegido”. Cada decisão individual e coletiva influencia na estruturação do espaço e essas escolhas redesenham a cidade. Quando a decisão é pautada na individualidade fomenta estratégias de evitar “o outro” e de se separar do “perigo potencial”, dando amplitude ao processo de segregação espacial e social. A sensação de insegurança se traduz na desconfiança e interfere nas formas como as pessoas interagem e se relacionam. A sensação da ameaça tende a desencadear ações defensivas por parte da população que, se sentindo desprotegida, modifica a forma de se relacionar com o outro e com o espaço e alterando as práticas cotidianas, na busca de se proteger de um perigo virtualmente criado. Uma urbanização sem urbanidade No Brasil, da década de 1980, as cidades passam por processos de mudanças relacionados à economia, infraestrutura e política. Entre os vários elementos em transformação cabe aqui nos deter nos aspectos do ambiente construído, mas que não passam desconexos dos processos econômicos e sociais. Na década de 1990 foram implantadas no Brasil as reformas neoliberais que incluíram, entre outras demandas, adequar a economia nacional aos interesses do capital financeiro internacional, a abertura comercial e a diminuição do papel do Estado no planejamento e regulamentação da economia, ou seja, a condição econômica submetida ao controle externo, isto é, subordinada a ele. Uma nova estética de segurança nasce atrelada ao projeto arquitetônico e renasce na cidade, sob a justificativa da violência. O aspecto arquitetônico de defesa, onde os muros são marcantes e se oferecem como alternativa para uma população amedrontada, faz sobreviver uma ameaça, uma incerteza que paira sobre a sociedade inteira: a crise do fordismo. As relações estabelecidas na cidade, e com a cidade, deixam marcas e restabelecem quase diariamente limites, experiências e apropriações. Os lugares são componentes estratégicos do instrumental da arquitetura urbanística e são as expressões das resistências, dos conflitos, que ali se estabelecem. O indivíduo percebe a cidade e, de acordo com suas experiências, define suas estratégias diárias de movimentação de acordo com o espaço apropriado. O espaço arquitetônico, é permeado por feitos culturais e sociais. O homem, como seu par dialético, dá sentido à materialidade na apropriação e uso do espaço, os transformando em lugares e aí os espaços vazios são preenchidos por gestos, convivências e sentido. GEOgrafias Artigos científicos Página 35 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 GEOgrafias Artigos científicos Arquitetura hostil e o Bairro do Espinheiro Conhecido como Sítio Matinha, nome oriundo da densidade de vegetação, onde se localizava, no “beco do espinheiro”, atual Rua do Espinheiro, começa o adensamento residencial do bairro, formado em sua maioria por usineiros e comerciantes portugueses sendo eles os primeiros a fixar suas residências nessa região. Atualmente o bairro vive uma crescente transformação com a modificação intensa de espaços comerciais e edifícios empresariais, além do incremento do mercado de residências. A estrutura espacial do Bairro é uma grelha reticulada com certa regularidade, confluindo para o centro. É caracterizada pela existência de 4 vias longas na direção norte-sul, que são interceptadas transversalmente por vias na direção leste – oeste. Essa configuração faz com que o Espinheiro seja irrigado de transporte, e como peculiaridade apresenta uma ZEIS – Campo do Vila, remanescente da LCM – que se sustentam ali desde 1930, muito antes da construção da Avenida Agamenon Magalhães, e resistem no local desde sua expulsão 1970, para construção da via, quando o solo passou a valorizar de forma intensa. Página 36 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X LE GE ND A: Mapa 1: Mapa de uso e ocupação do solo Residência unifamiliar Residência multifamiliar Convento Atividade que não conferem troca de mercadorias Serviços religiosos Vendas de mercadorias Atividade de comércio ou serviço no pavimento térreo e residência no superior (conforme LUOS) Habitacional Serviço Comércio Misto Fonte: A autora, 2018. Mapa 1: Mapa de uso e ocupação do solo Fonte: A autora, 2018. No mapa é evidenciado e confirmado a uso residencial do bairro. Além das edificações habitacionais o Espinheiro possui uma variedade de comércios e serviços. Os eixos principais localizam-se na franja dos principais corredores viários, alguns serviços de utilidade pública, também estão localizados na região como Tribunal Regional do Trabalho, Gerência Regional Escolar e o Ministério Público. Na parte oeste Página 37 Página 37 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X do mapa temos a concentração de serviços de saúde privados, e o Hospital dos Servidores do Estado. do mapa temos a concentração de serviços de saúde privados, e o Hospital dos Servidores do Estado. Gráfico 1: Uso e ocupação do solo Fonte: A autora, 2018 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Gráfico 1: Uso e ocupação do solo Gráfico 1: Uso e ocupação do solo Gráfico 1: Uso e ocupação do solo Fonte: A autora, 2018 O bairro é composto por 783 lotes, dos quais 54% (422) são residenciais; 19% (150) são comerciais. Os serviços desenvolvidos no bairro contam com 25% (192) do total de lotes. Possui 1% (8) dos lotes ainda vazios (sem registro de atividade na prefeitura), e 1% (11) de uso misto. O levantamento que culminou nos gráficos e mapas foi realizado com “in loco” com base na atividade realizada no momento. O comércio principal conta com dois supermercados, e duas lojas menores de uma rede também de hipermercados. O bairro possui ainda duas escolas particulares. Além de uma Escola Pública Municipal e uma Estadual. Além da creche, que atende prioritariamente a população da ZEIS. Padarias, farmácias, bares e restaurantes, são encontrados facilmente no bairro. O Espinheiro possui um polo gastronômico, atrativo para toda Zona Norte da cidade. Apesar de sua centralidade é possível ainda observar comércios de bairro, com salões de beleza, e mercearias. No tecido urbano do bairro, 21% das ruas não possui nenhuma edificação verticalizada. É importante salientar que como estamos estudando o bairro como divisão político-administrativa, as análises são para o recorte estudado, ou seja, a Avenida Rosa e Silva possui edificações verticais, mas não no segmento dentro do bairro do Espinheiro. Página 38 GEOgrafias Artigos científicos GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X A concentração das edificações verticais de uso habitacional está na franja do corredor viário principal interno do bairro, a Rua do Espinheiro, que foi eixo de ocupação inicial do bairro apresenta conexão relacional com as vias com mais unidade. É importante também ressaltar que as vias são corredores de transporte coletivo conferindo certa capilaridade aos espaços. Se visualmente o bairro não apresenta um adensamento construtivo em altura muito alarmante, é importante fazer a análise de como para estrutura urbana, por exemplo, de e projetada para um terreno que comporta uma casa unifamiliar, passe a comportar dez famílias. Grosso modo é isso que ocorreu em cada mancha vermelha apresentada no mapa. Página 39 GEOgrafias Artigos científicos GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X Legenda Mapa 2: Mapa de verticalização do bairro do espinheiro Fonte: A autora, 2018. Gráfico 1: Uso e ocupação do solo Mapa 2: Mapa de verticalização do bairro do espinheiro Mapa 2: Mapa de verticalização do bairro do espinheiro Fonte: A autora, 2018. Quando o assunto é equipamentos públicos é importante esclarecer que o Espinheiro é um dos 15 bairros da cidade do Recife que não possui nenhum espaço de lazer público. A Praça da FEB, que para muitos está contida no bairro, está situada administrativamente no bairro dos Aflitos, isto também se repete como a Igreja Matriz do Espinheiro, que está administrativamente lotada no mesmo bairro. É importante destacar que apesar da ausência de equipamentos de lazer, há uma extensa massa arbórea, que oferta a população uma média de 24 árvores por rua, sendo uma das áreas mais verdes da cidade. Página 40 GEOgrafias Artigos científicos GEOgrafias Artigos científicos Página 40 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X Ao mesmo tempo em que tal constatação é um ponto positivo, pois proporciona sombreamento e uma caminhada agradável, a falta de cuidados e manutenção, torna difícil o dia a dia. A circulação de pedestres é comprometida, visto que as dimensões da faixa de serviço são irrisórias. Outro ponto é que o tipo do indivíduo arbóreo (oitis) é incompatível com as calçadas aliadas, isso torna qualquer simples deslocamento em uma tarefa difícil. A importância de conhecer e reconhecer os caminhos do bairro do Espinheiro está na possibilidade de vivenciar o bairro no seu sistema viário e na paisagem revelada pelo caminhar. Assim começamos a entender como a influência da arquitetura na percepção individual. Um bairro sem um local de encontro ou descanso não é atrativo para as pessoas, a arquitetura cumpre papel no acolhimento e integração das pessoas e as relações estabelecidas, no espaço da cidade. Mas, o que os levantamentos têm a dizer sobre o espaço físico da arquitetura do Espinheiro? A arquitetura adota como premissa a proteção, é como uma prioridade de preocupação do ato de arquitetar, a cidade foi construída para isso. Porém quando a arquitetura é imbuída de outras funções (manter segurança patrimonial) que não lhe são próprias, passa a haver uma mistura de sentidos e significados, que não permite ao indivíduo a conferir identidade com a cidade e assim se apropriar do espaço. O homem passa a ver e a interagir com a arquitetura como barreira, pois ela sendo portador de significados, ressalta além dos aspectos materiais os aspectos sociais, quem está dentro e quem está fora, e quem pode ou não entrar, o que é público e o espaço que é privado, o que é de dentro e o que é “da rua”. Dirigimo-nos assim, a analisar, no bairro do Espinheiro quais dos elementos que a arquitetura hoje utiliza para se apresentar como barreira entre o espaço público e o espaço privado. Acreditamos que a sobre posição de elementos da arquitetura do medo associado a aspectos da produção capitalista do espaço, assim como elementos de segurança, pode sim gerar uma hostilidade captada pelos indivíduos na sensação de insegurança urbana. Com o instrumento de análise elaborado para pesquisa conseguimos montar um mapeamento do bairro identificando áreas com mais e menos edificações que possuam mais ou menos características hostis. O instrumento nos possibilitou criar dois GEOgrafias Artigos científicos Página 41 Página 41 Revista GEOgrafias, v. 15, n. ISSN 2237-549X 1, jan./jun. 2019 ISSN 2237-549X perfis. Um de características inatas da arquitetura: muros, relação com escala humana e porosidades. E outro de características agregadas à arquitetura, ou seja, dos elementos de segurança: perfis. Um de características inatas da arquitetura: muros, relação com escala humana e porosidades. E outro de características agregadas à arquitetura, ou seja, dos elementos de segurança: Figura 1: Exemplo do instrumento de análise Aspectos Arquitetura Aspectos Segurança 0 a 14 Fonte: A autora, 2018. Figura 1: Exemplo do instrumento de análise Aspectos Arquitetura Fonte: A autora, 2018. Fonte: A autora, 2018. Com base nos levantamentos de campo, que foram agrupados, foi possível estabelecer os perfis das edificações no bairro Espinheiro, e quantificar os elementos dispostos nas edificações verticais no bairro. A planilha foi baseada em uma pontuação de 0 pontos a 14 pontos, com a lógica quando menor melhor. Possui a parte de itens genuinamente arquitetônicos, onde as edificações podem somar até o 8 pontos. E os equipamentos de segurança num total de 6 pontos. Podemos assim saber quantos e quais equipamentos são utilizados, e como eles estão dispostos no tecido urbano do bairro. Podemos facilmente identificar que na Rua da Hora há uma edificação que utiliza todos os elementos de segurança, assim como se observar várias edificações que não fazem uso de nenhum elemento de segurança. Ressaltamos a Avenida Norte onde as edificações analisadas não fazem uso de nenhum elemento de segurança. Essas informações nos possibilitaram um perfil discricionário dos itens e sua pulverização no bairro. Página 42 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X Figura 2: Edificações por elemento de segurança Fonte: A autora, 2018. O d â d lif d b i i Figura 2: Edificações por elemento de segurança Figura 2: Edificações por elemento de segurança Fonte: A autora, 2018. Os usos das câmeras de segurança proliferaram em todo bairro. Na Figura 3 observa-se que por toda parte há câmera de vídeo monitoramento. No bairro 70% (92) das edificações utilizam desse elemento com vista garantir sua segurança, podendo chegar a ser ainda maior. O debate que precisa ser travado é sobre qual a efetividade real desse componente. O mercado da segurança privada é baseado num atendimento Página 43 Página 43 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X remoto e vídeo monitoramento. O crescimento desse nicho se dá em progressão geométrica e suscita debates sobre a efetividade do recurso. Porém já tem um efeito real, a perda da individualidade e privacidade urbana. ágina 44 GEOgraf 2 3 2 4 1 5 , p p Figura 3: Câmeras de monitoramento Fonte: A autora, 2018. As cercas elétricas são elementos que podem variar de forma, tamanho e altura, bairro se pode identificar cercas com 3, 4, ou 6 fios. E a cerca ainda é utiliza conjugada com concertinas e/ou grampos. Esse item circula quase que a totalidade d 2 3 4 5 1 Figura 3: Câmeras de monitoramento Figura 3: Câmeras de monitoramento 2 3 2 4 1 5 Figura 3: Câmeras de monitoramento Fonte: A autora, 2018. 2 3 4 5 1 4 5 1 Fonte: A autora, 2018. As cercas elétricas são elementos que podem variar de forma, tamanho e altura, no bairro se pode identificar cercas com 3, 4, ou 6 fios. E a cerca ainda é utilizada conjugada com concertinas e/ou grampos. Esse item circula quase que a totalidade dos GEOgrafias Artigos científicos Página 44 Página 44 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 empreendimentos, e quando não é utilizado pelo prédio, no geral está entre as fronteiras eletrificadas dos edifícios a sua volta, como pode ser observado na Figura 4. 1 3 2 1 3 2 3 2 1 Figura 4: Cercas elétricas Fonte: A autora, 2018. No gráfico 2 abaixo percebemos de uma forma geral, os principais componentes de segurança visíveis no bairro. 2 1 3 2 Figura 4: Cercas elétricas Figura 4: Cercas elétricas 1 3 3 2 1 Fonte: A autora, 2018. 3 3 1 Fonte: A autora, 2018. No gráfico 2 abaixo percebemos de uma forma geral, os principais componentes de segurança visíveis no bairro. No gráfico 2 abaixo percebemos de uma forma geral, os principais componentes de segurança visíveis no bairro. No gráfico 2 abaixo percebemos de uma forma geral, os principais componentes de segurança visíveis no bairro. GEOgrafias Artigos científicos Página 45 Página 45 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Gráfico 2 – Total dos elementos de segurança no bairro Gráfico 2 – Total dos elementos de segurança no bairro Fonte: A autora, 2018. Fonte: A autora, 2018. Fonte: A autora, 2018. Fonte: A autora, 2018. De acordo com o Gráfico 2, do total dos itens analisados a presença de câmeras de segurança e cercas elétricas sobressaem a utilização dos demais elementos. É ainda pior quando há o uso articulado de vários elementos É perceptível que o uso indiscriminado dessas estratégias, ao contrário de trazer soluções coerentes para segurança nutrem uma sociedade de espetáculos, potencializando o pânico, banalizando a violência. As extensas áreas margeadas por muros cegos são desencorajadoras ao caminhar. Áreas com algum tipo de relação física e visual geram uma sensação de segurança. Uma edificação vertical aprovada pela Nº. 16.176/1996, no caso do Espinheiro entre os anos 1980 e 2000, ou seja, antes da aprovação da LDB, tem a possibilidade de assumir as características das imagens, um artificio que sugere a forma final do volume da imagem (Figura 5). Isso ocorre em grade parte dos edifícios do Recife, pois possibilita máximo aproveitamento do solo. No artigo 78, parágrafo 6º: § 6º - As edificações com mais de 2 (dois) pavimentos poderão colar em 2 (duas) das divisas laterais e/ou de fundos os 2 (dois) primeiros pavimentos, desde que: I - quando colarem em 2 (duas) divisas laterais, mantenham um afastamento mínimo de 3m (três metros) da divisa de fundos; I - quando colarem em 1 (uma) divisa lateral e 1(uma) divisa de fundo, deverão manter um afastamento mínimo de 1.50 m (um metro e cinquenta centímetros) da outra divisa lateral; III - a altura total das edificações coladas nas divisas laterais e/ou de fundos não poderá exceder à cota de 7.50 m (sete metros e cinquenta III - a altura total das edificações coladas nas divisas laterais e/ou de fundos não poderá exceder à cota de 7.50 m (sete metros e cinquenta Página 46 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X centímetros), cota esta medida a partir do nível do meio-fio, admitindo-se um peitoril relativo ao piso do 2º pavimento com altura máxima de 1,10 m (um metro e dez centímetros) e afastado 1,50 m (um metro e cinquenta centímetros) das respectivas divisas. Figura 5: Exemplo de forma arquitetônica gerada pela legislação Figura 5: Exemplo de forma arquitetônica gerada pela legislação Fonte: A autora, 2018. Fonte: A autora, 2018. Fonte: A autora, 2018. Diante do perfil espacial que se coloca, partimos da hipótese de que a arquitetura também é capaz de influenciar na percepção da sensação de insegurança e ao longo de todo o caminho teórico e metodológico aqui apresentado se encerra acreditando que a sensação de insegurança pode sim ser retroalimentada pela arquitetura hostil. E que através da urbanidade, inerente a forma urbana, a cidade é capaz aproximar os laços pessoais, é necessário que essa urbanização se torne urbanidade e a população seja convidada a utilizar o espaço público. Considerações finais Percebemos que mais caminhos se abrem para prospecção do espaço. Procuramos investigar como a arquitetura hostil, que nega a vivência do espaço urbano, se distribui pelo bairro do Espinheiro em Recife. De fato, a arquitetura promovida pelo mercado imobiliário não caminha na direção estabelecer relações que acolham o indivíduo. Cada dia mais edificamos barreira no espaço urbano. GEOgrafias Artigos científicos Página 47 Página 47 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ISSN 2237-549X ISSN 2237-549X pela mídia, e afins a arquitetura hostil como anunciadora de que a rua é m espaço ruim e por isso nos fechamos entre iguais, afastando do público e promove a perda de urbanidade e vitalidade, causando efeitos na sensação de insegurança. A sobreposição de elementos de segurança se tornou um aspecto gritante na paisagem. Mas talvez o costume com uma imagem da cidade já formada no inconsciente, impeça que o homem perceba a alteração diária da paisagem. Vários estudos apontam que os aspectos visíveis da arquitetura podem estar ligados a algum aspecto de violência, criminalidade ou da percepção da sensação de estar inseguro. Porém o fato é que forma a arquitetura da cidade como materialidade visível, desse medo ou da sensação de estar inseguro, traz consigo aspectos que reverberam no espaço público e que alteram as percepções dos indivíduos citadinos, às vezes mesmo sem que eles percebam. ISSN 2237-549X Os espaços seguros propostos por Newman (1972), que são dotados de autocontrole (presença de muros, guaritas recuadas), com definições claras entre espaço público e privado etc, podem ser características individualmente importantes para segurança objetiva, mas o homem como ser coletivo busca a coletividade e espaços vazios e desertos não são atrativos, pessoas atraem pessoas. Cada elemento precisa ser pensado em sua escala de atuação, parques, praças, rua, caçada, pois todos os elementos são importantes pra constituição da cidade. A verticalização é importante para criar uma cidade mais compacta e com possibilidade de moradia e deslocamento para todos. Porém a forma com ocorre a inserção desse componente precisa ser alvo de estudos e políticas públicas que visem uma distribuição igualitária dos serviços e infraestruturas. No caso do Espinheiro houve uma mudança nos padrões do habitat e do habitar. A população mais abastada trocou a casa unifamiliar pelos condomínios verticais, pelo luxo e moda das “novas coletividades” – Condomínios verticais-, acentuando a segregação e auto segregação, agravando ainda mais as relações com o espaço urbano. A sensação de insegurança aumenta à medida que não há mecanismo capaz de proteger a integridade do homem. A solução passa a ser uma busca individual por proteção, que reflete na arquitetura da cidade, que atrelada às contradições e hierarquizações sócio espaciais ajudam na desertificação do espaço público. As formas arquiteturais predominantes no bairro do Espinheiro, em larga escala, são sustentadas pela alimentação da cultura do medo, mesmo as residências térreas, onde ainda se pode observar uma boa relação da edificação com a escala humana, o recado está estampado: “não entre risco de morte” - cercas elétricas, concertinas entre outros anunciam que o outro não é bem-vindo. O indivíduo que ainda assim buscar percorrer o espaço urbano se depara com infraestrutura urbana deficitária. Sem espaço de lazer e área de convívio. Calçadas ocupadas por árvores, desniveladas áridas, ou seja, você está sozinho em um espaço desconfortável e que não oferece nenhum atrativo. Então o que fazer nas ruas? A autossegregação tem contribuído para o agravamento dos processos de desqualificação e deterioração dos espaços públicos. A insegurança, o medo são sim processos ligados à violência, mas assim como a sensação de insegurança é ampliada GEOgrafias Artigos científicos Página 48 Página 48 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 BAUMAN, Z. Confiança e Medo na Cidade. Zahar, 2009 BAUMAN, Z. Confiança e Medo na Cidade. Zahar, 2009 BAUMAN, Z. Modernidade líquida. Rio de Janeiro: Zahar, 2001. BAUMAN, Z. Modernidade líquida. Rio de Janeiro: Zahar, 2001. BAUMAN, Z. Tempos líquidos. Rio de Janeiro: Zahar, 2007. BAUMAN, Z. Tempos líquidos. Rio de Janeiro: Zahar, 2007. CALDEIRA, T. P. R. Cidade de Muros. São Paulo: Edusp, 2000. DAVIS, M. Cidade de Quartzo. São Paulo: Boitempo, 2009. DAVIS, M. Cidade de Quartzo. São Paulo: Boitempo, 2009. ELLIN, Nan (Editor). Architecture of Fear. Princeton: Princeton Architectural Press, 1997 FERRAZ, S. M. T. et al. Arquitetura da violência: a arquitetura antimendigo como eureca da regeneração urbana. Movimento revista de educação, n. 3, 2016. Disponível em:http://www.revistamovimento.uff.br/index.php/revistamovimento/article/view/264/2 22. Acesso em: 02 fev 2018. GUATTARI, F. Caosmose: um novo paradigma estético. São Paulo: Ed. 34, 1992. GUEDES, J. Cité et espace politique. Psicologia USP, v. 14, n. 3, p. 73-78, 2004. HILLIER, B.; HANSON, J. The Social Logic of Space. Cambridge: Cambridge University Press, 1984. JACOBS, Jane. Morte e vida de grandes cidades. São Paulo: Martins Fontes, 2000. Página 49 Página 49 GEOgrafias Artigos científicos Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 Revista GEOgrafias, v. 15, n. 1, jan./jun. 2019 ERRATA - Na Capa da revista Geografias, v. 15, n. 1, jan./jun. 2019: Onde se lia: “Janeiro - Junho de 2019 vol. 27 - nº 1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - Na Ficha catalográfica da revista Geografias, v. 15, n. 1, jan./jun. 2019: Onde se lia: “Geografias: Revista do Departamento de Geografia/Programa de Pós-graduação em Geografia, Departamento de Geografia do Institutode Geociências, UFMG - Vol. 27 nº 1 (Jan-Jun) 2019 - Belo Horizonte: UFMG, Departamento de Geografia, 2019” Leia-se: “Geografias: Revista do Departamento de Geografia/Programa de Pós-graduação em Geografia, Departamento de Geografia do Institutode Geociências, UFMG - Vol. 15 nº 1 (Jan-Jun) 2019 - Belo Horizonte: UFMG, Departamento de Geografia, 2019” - No artigo A proteção de mananciais na Região Metropolitana de Belo Horizonte (MG)os desafios de compatibilização entre usos da terra e da água na Área de Proteção Especial (APE) Taboões, de autoria de Felipe Gabriel Silva Alves, Catharina de Oliveira Maia, Gabriel Lucas Vieira Lazaro, Guilherme Eduardo Macedo Cota e Antônio Pereira Magalhães Júnior, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - No artigo Cidade hostil, de autoria de Shayenne Barbosa Dias e Cláudio Roberto de Jesus, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: - No artigo Cidade hostil, de autoria de Shayenne Barbosa Dias e Cláudio Roberto de Jesus, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias v 27 n 1 2019” - No artigo Cidade hostil, de autoria de Shayenne Barbosa Dias e Cláudio Roberto de Jesus, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “B l H i t 15 1 j /j 2019” - No artigo Cidade hostil, de autoria de Shayenne Barbosa Dias e Cláudio Roberto de Jesus, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. ISSN 2237-549X Revista Geografias, Edição Especial 2022 - Erratas ERRATA 2019” “Revista GEOgrafias, v.27, n.1, 2019” - No artigo Índice de vulnerabilidade das políticas sociais para o município de Belo Horizonte/MG, de autoria de Pedro Henrique de Souza Oliveira, Plínio da Costa Temba, Marcelo Antonio Nero, Karla Albuquerque de Vasconcelos Borges, Marcos Antonio Timbó Elmiro e Sandra Cristina Deodoro, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: Revista Geografias, Edição Especial 2022 - Erratas ISSN 2237-549X JESUS, A. Geografia urbana do camelô belo-horizontino. 2011. 229 p. Tese (Doutorado em Geografia) Instituto de Geociências Universidade Federal de Minas Gerais, Belo Horizonte, 2011. LIRA, P. S. Instâncias urbanas e violência: uma análise dialética. 2009. Dissertação (Mestrado) UFES/PPGAU, Vitória, 2009. LYNCH, Kevin. A imagem da cidade. 2. ed. São Paulo: WMF Martins Fontes, 2010. NEWMAN, O. Defensible space:cCrime prevention through urban design. New York: Macmillan, 1972. SANTOS, M. A natureza do espaço: técnica e tempo, razão e emoção. São Paulo: Hucitec, 1996 SANTOS, M. Metamorfoses do espaço habitado. 4 ed. São Paulo: Hucitec, 1996a. SENNETT, Richard. A corrosão do caráter. Rio de Janeiro São Paulo: Record, 2002. SENNETT, Richard. O declínio do homem público: as tiranias da intimidade. São Paulo: Companhia das Letras, 1988. SIMMEL. A metrópole e a vida mental. In: VELHO, O. G. O fenômeno urbano. Rio de Janeiro: Zahar, 1967. p. 13-28. SOUZA, M. L. Fobópole: o medo generalizado e a militarização da questão urbana. Rio de Janeiro: Bertrand Brasil, 2008. STECHER, H. Georg Simmel: Dinheiro, a solidez do efêmero. Cad. CRH, Salvador, n.22, p. 185-191, jan./jun, 1995. Página 50 GEOgrafias Artigos científicos GEOgrafias Artigos científicos ISSN 2237-549X Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” “Revista GEOgrafias, v.27, n.1, 2019” - No artigo Ferramenta de pesquisas sobre estabelecimentos baseada na geolocalização: uma proposta para aprimorar a experiência do turista em Tiradentes – MG, de autoria de Rodrigo Otávio Passos Ferreira e Guilherme Augusto Pereira Malta, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: ISSN 2237-549X Revista Geografias, Edição Especial 2022 - Erratas - No artigo Análise da relação e da destinação dos resíduos sólidos e dos restos numa comunidade rural, de autoria de Mônica Martins de Godoy Fonseca e Sergio Donizete Faria, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019 Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - No artigo Situação do trabalho análogo à escravidão: análise estatística espacial quanto à vulnerabilidade socioeconômica e à presença de territórios quilombolas em Minas Gerais (2003- 2013), de autoria de Bernardo Freitas Fantato Gonçalves e Diego Rodrigues Macedo, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - No artigo Situação do trabalho análogo à escravidão: análise estatística espacial quanto à vulnerabilidade socioeconômica e à presença de territórios quilombolas em Minas Gerais (2003- 2013), de autoria de Bernardo Freitas Fantato Gonçalves e Diego Rodrigues Macedo, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - No documento Teses e Dissertações defendidas no Programa de Pós - Graduação em Geografia, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019” - No documento Teses e Dissertações defendidas no Programa de Pós - Graduação em Geografia, publicado na revista Geografias, v. 15, n. 1, jan./jun. 2019, no cabeçalho de todas as páginas: Onde se lia: “Revista GEOgrafias, v.27, n.1, 2019” Leia-se: “Belo Horizonte, v. 15, n. 1, jan./jun. 2019”
https://openalex.org/W4205183391	https://www.matec-conferences.org/10.1051/matecconf/202235503038/pdf	English	null	Computational intelligence model based on GA-BP neural network	MATEC web of conferences	2,022	cc-by	4,298	Corresponding author: 2595643823@qq.com Computational intelligence model based on GA- BP neural network Chengzhao Zhang Chengdu Polytechnic, Chengdu, PR China Abstract. Since the birth of the secondary stock market, the prediction of the stock price trend has become a research direction concerned by many people. Aiming at the problem of non-stationary and non-linear stock price forecasting, this paper builds a computational intelligence model to improve the neural network with genetic algorithm. The results show that, compared with other models, the GA-BP neural network model proposed in this article can effectively improve the prediction of the rise and fall of the HS300 index, and the withdrawal range is small when the market falls. The research of this paper enriches the method of financial time series data analysis, which can not only provide decision-making reference for investors, but also help to enhance the cognition of financial market rules. The model can significantly reduce the forecast error and improve the model fitting ability. Keywords: Stock market, Genetic algorithm, BP neural network, Prediction error. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/matecconf/202235503038 https://doi.org/10.1051/matecconf/202235503038 MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 1 Introduction Stock market forecasting is a very demanding problem. Many factors influence stock market prices, for example, company news and results, sector performance, consumer sentiment, social media sentiment and financial factors. The characteristics used for prediction are diverse: price data, technical indicators of historical data, other market price fluctuations related to individual stocks, currency exchange rate, oil prices, etc. Automatic feature extraction and accurate market prediction have attracted more and more attention. The existing financial market analysis methods are generally divided into two categories. One is fundamental analysis and the other is technical analysis. In the technical analysis, the historical data of the target market and some other technical indicators are important factors in the prediction. According to the efficient market hypothesis, stock prices can reflect all their information. Technical analysis predicts the future trend of market price by analyzing the previous price data and technical indicators. The fundamental analysis of the intrinsic value of securities investment can be achieved by obtaining the detailed information of the financial statements of related assets, MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 such as cash flow, growth rate, discount factor, annual discount and capitalization rate, so as to deeply understand the future development of the company. Fundamental analysis is often used to decide which stock to choose for trading. such as cash flow, growth rate, discount factor, annual discount and capitalization rate, so as to deeply understand the future development of the company. Fundamental analysis is often used to decide which stock to choose for trading. Deep learning is a kind of method suitable for automatic feature extraction and prediction. In fields like machine vision, natural language processing, and speech signal processing, deep learning methods can gradually construct useful and complex features from raw data or simple features. Since the actions of stock markets are complex and nonlinear, and the stock data has noise, it is facing a great challenge to extract accurate features to predict. The stock market is one of the most important parts of the investment market. Compared with other industries, the stock market has a higher rate of return on investment, but there are also higher risks. Stock market forecasting has always been a problem that investors pay close attention to. 1 Introduction Therefore, the research on the stock market prediction methods and how to reduce the error of stock price prediction has become a research hotspot of many scholars at home and abroad. At present, the common stock forecasting methods include econometric method and machine learning method. Econometric method is based on statistical theory, the application of statistical analysis model to predict the stock price. The common models are ARMA, ARIMA, and GARCH. Although the econometric model is objective and has good theoretical support, its advantages lie in the processing of linear data and the need for strict basic assumptions. The stock price series is a complex nonlinear time series, so the traditional econometric model can not achieve the best prediction effect. Compared with the econometric model, machine learning method can directly mine valuable information from the data without making assumptions in advance, and machine learning method can better deal with nonlinear data, so it is widely used in stock price forecasting. Common machine learning methods include decision tree, SVM, SVR, neural network and so on. Among them, the learning mechanism of neural network makes it have more powerful learning ability and nonlinear fitting ability, and has more advantages in the field of stock price prediction. The latest literature related to GA-BP neural network is as follows. The GA-BP neural network model proposed by Wang[1] et al. provide a new practical method for knowledge fusion risk assessment of Creative ecosystem. Li[2] et al. construct an evolutionary neural network model by combines GA with BP neural network to solve the global optimization problem. Song[3] et al. point out that GA-NN model has a good application prospect in ionosphere research. A BP neural network prediction model optimized on the basis of genetic algorithm is proposed by Cao[4] et al. and Wang[5] et al. The experimental results achieved by Zou[6] et al. show that the GA-BP algorithm is accurate and effective for online identification. The experimental results of Peng[7] et al. show that GA-BP model is superior to other competitors. The BP neural network based on PEC and GA proposed by Wang[8] et al. has been proved to detect cracks in ferromagnetic steel quantitatively. An optimized GA-BP neural network model is established by Zheng[9] et al. to predict the long-term skid resistance. A wind speed prediction method based on EEMD and GA-BP neural network is proposed by Wang[10] et al.. 2 Basic knowledge of GA Genetic algorithm is a stochastic worldwide search and optimization method that simulates biological world based on the natural genetic mechanism of natural selection and species evolution. Optimization of BP neural network using genetic algorithm can obtain better initial weights and thresholds, avoid network training into partial minima and improve convergence speed. Genetic algorithms were first proposed by John Hollander in the USA in the 1970s. The algorithm was designed and proposed based on the evolutionary laws of organisms in nature. Genetic algorithm is a kind of random search algorithm. It is a process based on Darwin's theory of biological evolution and genetic mechanism to obtain an optimal solution by simulating the process of natural selection and biological evolution. It is widely used in areas like function optimization and picture processing production scheduling, machine learning and combinatorial optimization. Different from the traditional search algorithm, genetic algorithm is an optimization method based on probability search. It does not need to determine the search rules in advance, and can adjust the search direction adaptively to achieve automatic search space and optimization guidance. The genetic algorithm used in this paper starts from the randomly generated partial solution set of the problem to be solved, which contains a certain number of individuals. These individuals generally use a string of data or array to represent the coding of the solution, which is called chromosome. Then the quality of these chromosomes is measured using the fitness function. On this basis, crossover and mutation operations are performed on the chromosomes in the initial population using genetic operators to form a new population. According to the evolutionary principle of survival, this evolutionary process will make the fitness value of chromosomes in the population gradually increase after several generations, that is, the algorithm will converge to the best chromosome, which is the optimal solution we seek. Therefore, the main contents of genetic algorithm include: initial coding design, determination of fitness function, selection, crossover, mutation, setting termination conditions. 1 Introduction Other chapters are summarized as shown below. Section 2 introduces the fundamental knowledge of genetic algorithms. Moreover, Section 3 presents the introduction of BP neural network and Section 4 introduces BP neural network model optimized by GA. Furthermore, Section 5 presents the empirical result analysis and comparative analysis. Finally, Section 6 gives the conclusions. 2 2 MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 2.2 Crossover Crossover means that two individuals selected from the parents exchange partial genes to form new offspring, which is used to guarantee the diversity of individuals in the population. This paper uses a two point crossover method to select sires from the mating pool, and then two gene positions are randomly assigned in each pair to cross, so as to produce a new pair of individuals until the end of the crossover, forming a new population. For example, individuals y and z are selected for crossover, and the chromosome codes before and after crossover are as follows. Before crossover: 1 2 3 , , , , y n R y y y y (3) 1 2 3 , , , , z n R z z z z (4) (3) (4) After crossover: 1 2 3 , , , , , , , , y i j n R y y y z z y (5) 1 2 3 , , , , , , , , z i j n R z z z y y z (6) (5) (6) 2.3 Mutation and setting termination conditions Mutation is to select some genes in some chromosomes to make changes, which enhances the diversity of the population and effectively prevents the algorithm from entering the convergence state early. After selection, crossover and mutation, we have a new population. In the case of successive choices, crossing and variation, if the termination condition is not set, the genetic algorithm will continue to calculate, so we need to set a reliable termination condition. The termination conditions set in this paper are as follows: When the iteration count hits its maximum value, the algorithm stops running. When the algorithm matures and converges, it stops running when there is only one chromosome type left in the population. 2.1 Selection Selection refers to the selection of several chromosomes from the population according to a certain probability, which is a process of converging the population to excellent individuals based on fitness value. In this paper, the strategy of combining fitness proportion method and best individual retention method is used to select. The main idea of fitness proportion method is that the probability of selecting an individual from a group is related to its fitness evaluation. The higher fitness of the individual's corresponding solution, the larger value of objective function and the greater probability of selection. Suppose that the initial population size is H, that is, the chromosome number is 1, 2,..., h. The fitness value of individual r is rf , the probability of individual r being selected is set to rp , and the cumulative probability is set to rq , then: 1 r r h r r f p f (1) 1 r r s s q p (2) (1) (1) (2) MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 After selecting chromosomes from male parents for cross mutation, the offspring population will be formed. According to the fitness, 50% of the individuals with the highest fitness will be selected from the parents and offspring to form a new population, which can not only make the excellent individuals participate in the cross mutation operation, but also keep the excellent individuals in the male parents and avoid missing the better solution. At the same time, the convergence speed is accelerated. 3 BP neural network Artificial neural network model has strong nonlinear mapping power, and can process plenty of data at one time. The idea of neural network model is: the information input, the parameters are adjusted according to the set error expectation, and the output conforms to the expected error result. Neural network data is a classical machine learning method when dealing with large data. The model has strong nonlinear mapping capability, self-adaptive capability and self- 4 4 MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 learning capability. It has good generalization ability and fault tolerance when dealing with multi-source heterogeneous data prediction because of its wide applicability and good fitting effect, multilayer feedforward neural network realizes the feedforward learning and training of model threshold through the feedback learning mechanism of hidden layer, which has been widely used in classification, prediction and other fields. learning capability. It has good generalization ability and fault tolerance when dealing with multi-source heterogeneous data prediction because of its wide applicability and good fitting effect, multilayer feedforward neural network realizes the feedforward learning and training of model threshold through the feedback learning mechanism of hidden layer, which has been widely used in classification, prediction and other fields. BP neural network is a kind of feedforward neural network model. Its core idea is to analyze the error according to the comparison between the output of each training and the actual result, and to correct the weight and bias of BP neural network depending on the error. Finally, the output of the model is consistent with the expected result step by step. Generally, A BP neural network is composed of an input layer, a hidden layer and an output layer. BP neural network is a kind of multi-layer perceptron, which is used to solve the linear indivisible problem in prediction. Neural network is a kind of model similar to black box, which includes several hidden layers besides input layer and output layer. A neural network model, on the other hand, has three or more layers of neurons. Among them, the neurons between the upper and lower layers of the adjacent BP neural network in the fully connected layer achieve full connectivity, while the neurons in the same layer are not connected to each other. 3 BP neural network In this case, the connection between the input layer and the hidden layer is based on the weights of the network, i.e., the strength of the connection between two neurons. Input layer transfers information contained in data into hidden layer, while hidden layer integrates all information from all neurons in the previous layer and continues to pass down until it is passed to output layer. Although adding the number of network layers can reduce the error, it also complicates the neural network and increases the network weights' learning time. Actually, improving the error accuracy can be achieved by increasing the number of neurons in the hidden layer, and the training effect is better than increasing the number of network layers. Meanwhile, in accordance with Kosmogorov's theorem, the three-layer neural network can approximate any continuous function under the condition that the neural network structure is reasonable and the node weights are appropriate. Therefore, based on the above analysis, the number of hidden layers is set to 1, that is to build a three-layer neural network. 4 Genetic algorithm for optimizing BP neural network models BP neural network is a multi-layer forward neural network trained using the error back propagation algorithm. BP neural network can achieve arbitrary non-linear mapping of input and output, self-learning and simple structure, but the speed of training of BP neural network is slow and easy to fall into local minima. Genetic algorithm is a biological natural genetic mechanism based on imitation genetic algorithm. It is a randomised global search optimization method based on natural biological genetic mechanisms, mimicking natural selection and species evolution. Genetic algorithms are a stochastic global search and optimization method based on biological natural genetic mechanisms that emulate natural selection and species evolution. Optimizing BP neural networks with genetic algorithms can obtain better initial weights and thresholds, avoid network construction falling into local minima and improve convergence speed. The neural network model divides the samples as training samples and testing samples. The training samples are used to train the optimal simulation network model, and the test samples are employed to test the prediction results of the model. In this paper, a 3-layer neural network structure is constructed, in which the input layer, hidden layer and output layer are all one layer, and the structure is shown in Figure 1. The weights and thresholds of the BP neural network are used as the initial populations of the genetic algorithm. The next generation population is obtained by genetic action, and 5 https://doi.org/10.1051/matecconf/202235503038 MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 the individual fitness value is obtained. The weight and threshold are adjusted and optimized by selection, crossover and mutation until the individual learning error of fitness value is less than the specified value. the individual fitness value is obtained. The weight and threshold are adjusted and optimized by selection, crossover and mutation until the individual learning error of fitness value is less than the specified value. 1 Input Layer Hidden Layer Output Layer 2 i 1 2 j 1 2 k 1x 2x ix 1y 2 y jy 1z 2z kz ij w jk w Fig. 1. Architecture of BP Neural Networks. 1 Input Layer Hidden Layer Output Layer 2 i 1 2 j 1 2 k 1x 2x ix 1y 2 y jy 1z 2z kz ij w jk w Input Layer Fig. 1. Architecture of BP Neural Networks. Fig. 1. Architecture of BP Neural Networks. Fig. 1. Architecture of BP Neural Networks. Finally, training the neural network and calculating the mean square error after each iteration. The iteration is stopped when the error between the outcome and the desired output reaches a predetermined level of error convergence. The sequence of optimal network weights is obtained. The trained simulation network is used to simulate and predict the test samples corresponding to the trained simulation network, and the prediction results are obtained. The specific implementation process of the GA optimized BP neural network is shown in Figure 2. Fig. 2. Flow chart of BP neural network optimized by GA. Genetic algorithm section BP neural network section Initial population Initial data coding based on GA The error of BP is regarded as the initial fitness value of GA Selection operation Crossover operation Mutation operation Calculate the fitness value Is it satisfied Closing request Determine the structure of BP neural network Initialization of BP neural network The optimal weights and thresholds are obtained calculation error Weight and threshold correction The trained simulation network is obtained to simulate the prediction Is it satisfied Error requirements Y N Initialization of BP neural network The optimal weights and thresholds are obtained Is it satisfied Error requirements Fig. 2. Flow chart of BP neural network optimized by GA. 6 MATEC Web of Conferences 355, 03038 (2022) ICPCM2021 https://doi.org/10.1051/matecconf/202235503038 GA-BP neural network model has stronger simulation ability. The output value of the network is nearer to the actual value. The prediction error, convergence speed and accuracy are better than BP neural network and GARCH model, which can be used as the basis for the determination of the trend of HS300 index. 5 Empirical result analysis and comparative analysis This paper uses the 975 data points of HS300 close price index from June 9, 2017 to June 9, 2021 as the sample set. 725 data points from June 9, 2017 to May 29, 2020 were used as training sets, and 250 data points from June 1, 2020 to June 9, 2021 were used as test sets to implement the test. GA-BP neural network, BP neural network and GARCH model are used for prediction, and root mean square error (MSE) and mean relative error (MAPE) are used for comparative analysis. where ny is the real value, ny is the simulation predicted value, and 1,2, , n N is the number of test samples. 1 n n N n n y y y MAPE N (7) 2 1 1 N n n n MSE y y N (8) 1 n n N n n y y y MAPE N (7) 2 1 1 N n n n MSE y y N (8) (7) 2 1 1 N n n n MSE y y N (8) (8) 6 Conclusion This paper uses three models including GA-BP neural network model to forecast the HS300 index and compare the results based on 975 data points from 2017 to 2021. The empirical results show the following conclusion. On the one hand, GA-BP neural network inherits the self-learning and nonlinear mapping ability of BP neural network, and on the other hand, it solves the problems of slow convergence and easy to fall into local optimum of BP neural network. It has the benefits of stable output, fast speed of convergence and high prediction accuracy. The GA-BP neural network outperforms the BP neural network in terms of simulation capability, error level, convergence accuracy and number of iterations. It has high prediction accuracy, strong simulation ability and good data fitting ability. We developed a GA-BP neural network tool to test the HS300 index and use the daily data for analysis in this paper. The results show that the MAPE of the simulation prediction is 23.48 when the daily close price of the HS300 index is used as the input layer variable of the neural network. It shows that the GA-BP neural network model based on daily data can accurately predict the trend of daily close price data of HS300 index. In the future research, we will consider the trend of the highest price and the lowest price. Financial support for this work was provided by a grant from Chengdu Polytechnic No.16CZYTD951 and Chengdu Polytechnic under grant No.20KYTD05 and Chengdu Polytechnic under grant No.19KYPT07. Financial support for this work was provided by a grant from Chengdu Polytechnic No.16CZYTD951 and Chengdu Polytechnic under grant No.20KYTD05 and Chengdu Polytechnic under grant No.19KYPT07. The flow chart of HS3000 close price index sequence is shown in Figure 3. The flow chart of HS3000 close price index sequence is shown in Figure 3. The flow chart of HS3000 close price index sequence is shown in Figure 3. p q g Fig. 3. Process flow diagram HS3000 close price index sequence. Fig. 3. Process flow diagram HS3000 close price index sequence. The simulation errors of the three models are calculated by formula (7) and formula (8), and the results are shown in Table 1. The MAPE of GA-BP neural network model is 23.48, the MAPE of BP neural network is 69.78 and the MAPE of GARCH model is 75.84. The empirical findings show that the simulation prediction effect of GA-BP neural network is better and can reveal the trend of HS300 index better. Comparing the results of the three models, the mean square error and MAPE error of the GA-BP neural network model are smaller than those of the BP neural network model and the GARCH model, indicating that the simulation accuracy of the GA-BP neural network model is higher. Table 1. Simulation performance comparison of three models. Table 1. Simulation performance comparison of three models. Table 1. Simulation performance comparison of three models. Model MSE MAPE GA-BP 3806.54 23.48 BP 23854.36 69.78 GARCH 24567.23 75.84 GA-BP neural network model has stronger simulation ability. The output value of the network is nearer to the actual value. 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https://openalex.org/W4200182063	https://stemcellres.biomedcentral.com/track/pdf/10.1186/s13287-022-02831-1	English	null	Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy	Research Square (Research Square)	2,021	cc-by	11,182	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Various methods have been developed to generate hepatic cells from human pluripotent stem cells (hPSCs) that rely on the combined use of multiple expensive growth factors, limiting industrial-scale production and widespread applications. Small molecules offer an attractive alternative to growth factors for producing hepatic cells since they are more economical and relatively stable. Methods: We dissect small-molecule combinations and identify the ideal cocktails to achieve an optimally efficient and cost-effective strategy for hepatic cells differentiation, expansion, and maturation. Results: We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling. Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification. The obtained hepatoblasts (HBs) could subsequently expand and mature into functional hepatocyte- like cells (HLCs) by the established chemical formulas. Thus, we established a stepwise strategy with complete small molecules for efficiently producing scalable HBs and functionally matured HLCs. The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo. Conclusion: Our current small-molecule-based hepatic generation protocol presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery using. Keywords: Hepatic differentiation, Small molecule, Human pluripotent stem cells, Hepatoblasts, Hepatocyte-like cells Pan et al. Stem Cell Research & Therapy (2022) 13:159 https://doi.org/10.1186/s13287-022-02831-1 Open Access Backgroundfi Developing efficient and cost-effective technology to gen- erate large quantities and high-quality functional human hepatic cells is essential for applying hepatic cells to cell- based therapy and drug toxicity screening. To date, pro- tocols have been developed to generate hepatic cells from hPSCs, while most of the reported protocols relied on expensive growth factors, such as Activin A, BMPs, FGFs, and HGF [1–5]. This leads to critical challenges such as high cost and low reproducibility. In contrast, small mol- ecules offer an attractive alternative to growth factors Correspondence: li_yinxiong_iph@gibh.ac.cn; gaoyi@smu.edu.cn †Tingcai Pan, Ning Wang and Jiaye Zhang contributed equally to this work 1 General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou , Guangdong, China 2 Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Full list of author information is available at the end of the article Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small‑molecule strategy Tingcai Pan1,2†, Ning Wang2†, Jiaye Zhang2†, Fan Yang3, Yan Chen2, Yuanqi Zhuang2, Yingying Xu2, Ji Fang2, Kai You2, Xianhua Lin2, Yang Li1, Shao Li1, Kangyan Liang1, Yin‑xiong Li2 and Yi Gao1,4* © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 2 of 14 Page 2 of 14 The small-molecule-derived HLCs showed typical mor- phologic and functional features of mature hepatocytes, with hepatocyte-specific markers expression and func- tionalities in vitro and in vivo. In summary, our current chemical-based hepatic specification, expansion, and maturation method presents an efficient and robust plat- form for the cost-effective and large-scale production of functional human hepatic cells for clinical regenerative medicine and drug discovery applications. since they are usually more economical, relatively more stable, and possibly more efficient for generating hepatic cells from hPSCs [6–8]. Thus, substituting growth factors with small molecules and optimizing the hepatic genera- tion method could contribute to scalable and integrated producing functional hepatic cells, improving cost-effec- tiveness and reproducibility.f Previous efforts have been made to identify small mole- cules that support the differentiation and maintenance of hepatic cells, and impressive advances have been under- taken to identify numerous chemical candidates [9–11]. Our previous study established a synergistic cocktail to regulate BMP, Wnt, Hedgehog, and other signaling pathways, creating a delicate balance for HBs expan- sion and bipotency maintenance [12]. Based on this, we further created a robust and cost-effective culture con- dition for the generation of self-renewing HBs and func- tional mature HLCs, using a small-molecule cocktail in a defined culture system [13]. Further use of a small- molecule protocol to direct hPSCs differentiation into HBs and establish a complete small-molecule formula to generate expanded HBs and functional mature HLCs effi- ciently would benefit many hepatic cell potential clinical applications, including cell transplantation, bio-artificial liver, drug development, and disease modeling. Human PSCs culture and hepatic differentiation Human PSC lines, including iPSC line (UC15) and ESC line (H1), were cultured in mTeSR1 medium (Stem Cell Technologies) on 100-fold-diluted Matrigel matrix (Growth Factor Reduced, BD Bioscience). The hPSCs col- onies were passaged using Accutase (life). For hepatic differentiation, we started DE induction first. Briefly, while hPSCs reached nearly 70% conflu- ence, the medium was replaced with RPMI1640 (Gibco) supply with 1 × B27 (minus insulin, Invitrogen), contain- ing CHIR, IDE1, 2 µM Ly294002 (Ly), and 0.75 µM PD as indicated in Figs. 1A and 2A for 3 days. Further DE specifying to HBs, DE cells were cultured in differen- tiation medium (RPMI1640 supply with 1 × B27 supple- ment [Invitrogen]), containing growth factors (20 ng/mL BMP2 and 30 ng/mL FGF4) or small molecules (10 μg/ mL Vitamin C, 0.1 µM Dihexa, and 10 μM Forskolin) as indicated in Fig. 4A. So far, complete small-molecule-derived HLCs pro- tocols have been reported in several works of the litera- ture, with a lower hepatic differentiated efficiency than growth-factor-based approaches [6, 14–16]. This may be due to the indistinct contribution of exogenous and endogenous signals involved in hepatic differentiation. More importantly, these studies are generally character- ized by hepatic marker genes and proteins expression and albumin secretion and glycogen uptake while lack- ing hepatocyte-specific functional analyses in vitro and in vivo. Thus, a more detailed understanding of the pre- cise regulatory mechanisms of hepatic differentiation is vital to establishing a small-molecule-based induction protocol. Also, further efforts are needed to optimize the current chemical differentiation procedure for efficient and robust differentiation hPSCs into functional hepatic cells. Human HBs culture and hepatic maturation HBs were maintained in expansion medium as described previously [12]. Briefly, the purified HBs were cultured in expansion basal medium (RPMI1640, 1 × B27 supple- ment, 1 × ITS) supplemented with chemical cocktails (ACDFSV), including 5 µM A8301, 3 µM CHIR, 0.1 µM Dihexa, 10 μM Forskolin, 0.5 µM SAG, and 10 μg/mL Vc. Used chemical compounds were purchased from Sell- eck except indicated. For cell replating in our current method, the expanding HBs were dissociated by Accutase (life) and replated on Matrigel Matrix pre-coated plates. For hepatocyte maturation, HBs were cultured in hepatoZYME-SFM (Gibco) medium supply with 1X GlutaMAX, containing small molecules, including 5 µM A8301, 0.1 µM dexamethasone (Dex), 0.1 µM Dihexa, and 0.5 mM NH4Cl for 5 days. The medium was changed daily during the differentiation period. Used small mol- ecules were purchased from Selleck, and growth factors were purchased from PeproTech except that indicate. In the present study, we focused on improving chemi- cal strategy to generate functional hepatic cells from hPSCs. We demonstrated that stepwise treatment with small molecules, including CHIR99021 (CHIR), IDE1, and PD0332991 (PD), could elevate endogenous TGF-β/ Nodal signaling activity and direct hPSCs toward defini- tive endoderm (DE) efficiently. We further screened small molecules to identify an optimal chemical condition VDF for HBs specification. Moreover, the small-molecule- derived HBs could subsequently expand and mature into HLCs via our previously established chemical cocktail. Functional analyses of differentiated hepatocyte in vitro For the urea secretion assay, the culture media of 24 h incubated in differentiated cells were collected and stored Material and methods Human PSCs culture and hepatic differentiation Animal model and hepatic cells transplantationi p p Immune-deficient NOD-SCID-IL2RG−/− mice (NSI mice, GIBH) were used as recipients of human hepatic cells. Before hepatic cells transplantation, 8-week-old NSI mice received DMN intraperitoneal injections (7 mg/kg, Sigma, 1.0% dissolved in saline) for 2 consecutive days for inducing acute liver injury. Two days later, 1 × 106 hepatic cells were intrasplenically transplanted into the DMN-treated NSI mice. To monitor the transplantation state, recipient mice livers were harvested at different time points after hepatic cells transplantation. All ani- mal experiments were approved by the Animal Welfare Committee of GIBH. All protocols were approved by the appropriate Institutional Animal Care and Use Commit- tee (IACUC). To evaluate the glycogen production and storage abil- ity, periodic acid-Schiff (PAS) staining was performed. According to the manufacturer’s instructions, the cul- tured cells were fixed with 4% PFA for 30 min, and intracellular glycogen was stained using a PAS staining solution (Muto Pure Chemicals). For cellular indocyanine green (ICG) uptake assay, differentiated cells were incubated in media supple- mented with ICG (final concentration of 1 mg/ml, Sigma- Aldrich) for 1 h at 37 °C. Cells were then washed with PBS and imaged using a phase-contrast microscope (X71, Olympus). Functional analyses of differentiated hepatocyte in vitro E Immunostaining analyzed SOX17 and SOX7 expression in the differentiated cells. Scale bars 200 μm. F Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean±SEM n=3 For cellular LDL uptake assay, the HLCs were cultured with a medium containing Alexa-Flour 488-ac-LDL for 1 h, and immunohistochemistry was performed. Nuclei were counterstained with DAPI. For cellular LDL uptake assay, the HLCs were cultured with a medium containing Alexa-Flour 488-ac-LDL for 1 h, and immunohistochemistry was performed. Nuclei were counterstained with DAPI. at − 80 °C. Urea concentration was analyzed by LC/ MS/MS API3000 and normalized with total cell protein concentration. To evaluate the CYP450 activity, cultures were incu- bated with conventional probe substrates (CYP3A4: 6 μM midazolam, CYP2C9: 10 μM diclofenan, CYP2D6: 10 μM dextromethorphan), respectively, for quantifying metabolite production. After 2 h of exposure, the cul- ture medium was collected and stored at − 80 °C subse- quently, and CYP450 activity was analyzed by LC/MS/ MS API3000. Metabolite products were normalized to total cell protein. Functional analyses of differentiated hepatocyte in vitro yf y For the urea secretion assay, the culture media of 24 h incubated in differentiated cells were collected and stored Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 3 of 14 Fig. 1 CHIR and IDE1 inefficiently induced DE differentiation from hPSCs. A Schematic of the two-step chemical strategy to differentiate hPSCs into DE cells. CHIR3: 3 µM CHIR99021, CHIR1: 1 µM CHIR99021. B Immunostaining analyzed SOX17, OCT4, and BRA expression in the differentiated cells. Scale bars 200 μm. C Overview of lineage relationships during DE differentiation from hPSCs. PS: primitive streak, PrE: primitive endoderm, MD: mesoderm, DE: definitive endoderm. D Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction. Data are presented as mean ± SEM, n = 3. E Immunostaining analyzed SOX17 and SOX7 expression in the differentiated cells. Scale bars 200 μm. F Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3 Fig. 1 CHIR and IDE1 inefficiently induced DE differentiation from hPSCs. A Schematic of the two-step chemical strategy to differentiate hPSCs into DE cells. CHIR3: 3 µM CHIR99021, CHIR1: 1 µM CHIR99021. B Immunostaining analyzed SOX17, OCT4, and BRA expression in the differentiated cells. Scale bars 200 μm. C Overview of lineage relationships during DE differentiation from hPSCs. PS: primitive streak, PrE: primitive endoderm, MD: mesoderm, DE: definitive endoderm. D Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction. Data are presented as mean ± SEM, n = 3. E Immunostaining analyzed SOX17 and SOX7 expression in the differentiated cells. Scale bars 200 μm. F Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3 Fig. 1 CHIR and IDE1 inefficiently induced DE differentiation from hPSCs. A Schematic of the two-step chemical strategy to differentiate hPSCs into DE cells. CHIR3: 3 µM CHIR99021, CHIR1: 1 µM CHIR99021. B Immunostaining analyzed SOX17, OCT4, and BRA expression in the differentiated cells. Scale bars 200 μm. C Overview of lineage relationships during DE differentiation from hPSCs. PS: primitive streak, PrE: primitive endoderm, MD: mesoderm, DE: definitive endoderm. D Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction. Data are presented as mean ± SEM, n = 3. Quantitative RT‑PCR analysis According to the manufacturer’s protocol, total RNA was extracted using TRIzol reagent (Invitrogen) and Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 4 of 14 Fig. 2 Manipulating small-molecule cocktails to improve DE differentiation. A Schematic of the strategy to differentiate hPSCs into DE cells with small molecules. B Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction with different protocols. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of SOX17 and FOXA2 expression in DE cells derived from different protocols. Scale bars 200 μm. D The efficiency of SOX17 and FOXA2 expression after different cocktails treatment, determined by the FACS examination in differentiated cells Fig. 2 Manipulating small-molecule cocktails to improve DE differentiation. A Schematic of the strategy to differentiate hPSCs into DE cells with small molecules. B Quantitative RT-PCR analysis lineage relative genes expression of differentiated cells during DE induction with different protocols. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of SOX17 and FOXA2 expression in DE cells derived from different protocols. Scale bars 200 μm. D The efficiency of SOX17 and FOXA2 expression after different cocktails treatment, determined by the FACS examination in differentiated cells quantified with NanoDrop 2000 (Thermo Fisher). cDNA was reverse transcribed from 2 μg RNA using Rever- Tra Ace (Toyobo) and oligo-dT (Takara). Quantitative RT-PCR was performed with the CFX96 machine (Bio- Rad) and SYBR Green Premix (Bio-Rad) following the manufactures’ manual. The GAPDH was used for quan- titative RT-PCR normalization, and the experiments have repeated a minimum of three times to confirm the results. Primer sequences are listed in the Additional file 2: Table S1. confocal microscope. The primary antibodies and sec- ondary antibodies are described in the Additional file 2: Table S2. Fluorescence‑activated cell sorting (FACS) and flow cytometry analysis Cells were dissociated with Accutase and then resus- pended in PBS containing 3% BSA. The collected cell suspensions were stained with FITC-conjugated human Ep-CAM antibody (Milteny) and Alexa Fluor APC-con- jugated antibody against human C-Kit (B56; BD Bio- sciences) for 30 min on ice. EpCAM+/C-Kit− cells were sorted using a MoFloTM fluorescence-activated cell sorter. Sorted cells were seeded on Matrigel pre-coated plates and maintained in expansion basal medium con- taining small-molecule cocktails. CHIR and IDE1 inefficiently induced DE differentiation from hPSCs Our previous studies and other reports have demon- strated that CHIR, a GSK-3β inhibitor that acts as a Wnt agonist, combined with Activin A could induce hPSCs efficient differentiation into DE [12, 17–19]. However, Activin A is an expensive growth factor, limiting its use in large-scale cell production. In order to replace Activin A, researchers have tried to screen available small molecules and found that IDE1 and IDE2 are the only two mol- ecules that could partly replace Activin A for DE induc- tion [7, 20–22]. Thus, we examined if IDE1 could replace Activin A, combined with CHIR, to direct DE differen- tiation efficiently, according to the Activin A plus CHIR- treated pattern (CA) (Fig. 1A). Immunohistochemistry staining y g Cells were washed with PBS and fixed with 4% PFA (Sigma-Aldrich) for 30 min at room temperature. After washing the cells with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min and blocked with PBS containing 5% normal goat or donkey serum for 30 min at room temperature. The primary antibod- ies were diluted with blocking solution and incubated at 4 °C for overnight. After washing the cells with PBS, cells were then stained with compatible Alexa Fluor-conju- gated secondary antibodies in blocking solution for 1 h at room temperature. Nucleus stained with 5 μg/mL DAPI (Invitrogen). Imaging was performed on a Zeiss LSM 710 To analyze the cell proliferation, the expanded HBs were dissociated and then resuspended in PBS containing 10% FBS. The collected cell suspensions were fixed with 4% PFA (Sigma-Aldrich) and incubated in blocking and permeabilizing buffer, containing 0.1% Triton X-100, and 5% normal donkey serum in PBS for 30 min at room tem- perature. The cells were then incubated with APC-con- jugated Ki67 (B56; BD Biosciences) and other indicated Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 5 of 14 antibody for 30 min on ice. Corresponding isotype anti- bodies were used as controls. Flow cytometry analyses were performed using a FACS Aria II flow cytometer (BD Biosciences). increased lower in cells treated with CI-I or CI condition, compared with CA-treated group (Fig. 1D). Notably, the expression of PrE markers SOX7 and GATA6 has been highly upregulated in the process of CI-I or CI induction, indicating that PrE lineage was induced (Fig. 1D). Addi- tionally, immunostaining showed the SOX7-positive cells induced by CI-I or CI condition further confirmed the genes expression profile (Fig. 1E). These results suggest that the combination of CHIR and IDE1 cannot induce DE efficiently, but derived an MD, PrE, DE lineages, and undifferentiated hPSCs mixed population. Statistical analysish The data were analyzed with Sigma Plot 10.0. Statisti- cal differences between two groups were tested with a two-tailed Student’s t test. Data are represented as mean ± SEM. Survival data were analyzed with the Kaplan–Meier test. For all tests, p < 0.05 was considered significant. f During the differentiation process, the dynamic expres- sion of crucial pathway-related genes was analyzed by RT-PCR. Results showed that the expression pattern of Wnt3A and FGF8 has no significant difference among the groups. In contrast, BMP2 expression was increased progressively after day 1, while NODAL remains a low- level expression in cells treated with CI-I and CI condi- tion, compared with the CA treatment (Fig. 1F). These results suggested that lower endogenous Nodal signal and high level of BMP signal activities might be the rea- son why CI-I and CI treatment yielded a mixed popula- tion. Hence, increasing the endogenous TGF-β/Nodal signaling activity to improve current differentiation pro- tocols potentially could be a reliable strategy for efficient DE differentiation. Manipulating small‑molecule cocktails to improve DE differentiation p g Our results showed that this small-molecule cocktail (CI-I) failed to induce homogeneous DE-like cells mor- phology as CA (Additional file 2: Fig. S1). OCT4 and NANOG are essential transcription factors required to maintain the pluripotency and self-renewal of undif- ferentiated hPSCs. And SOX17 and Brachyury (BRA) are the markers of DE and MD, respectively. Thus, we analyzed the expression of these proteins to identify the cell fate determination and lineage differentiation. The immunostaining showed that the CI-I merely yielded 70% SOX17-positive cells, and plenty of cells remained OCT4 positive or appeared to express Brachyury (BRA), indicating mixed undifferentiated hPSCs and mesoderm (MD) cells (Fig. 1B). Therefore, we further optimized the small-molecule cocktail by supplying 1 μM CHIR on day 2 and day 3 (CI). The results showed that the optimized condition improved DE induction slightly, but still tricky to derive DE efficiently as CA formula (Fig. 1B). f Inhibition of PI3K signaling pathway or cell-cycle regu- lators CDK4/6 have been reported to promote the phosphorylation of Smad2/3 and regulate the signal transduction of the Nodal pathway while inducing DE differentiation [23, 24]. Accordingly, we combined CHIR and IDE1 with the above candidate small molecules, including PI3K inhibitor LY294002 (Ly) and CDK4/6 inhibitor PD, to induce DE differentiation (Fig. 2A). Dur- ing the differentiation process, morphological changes of the differentiated cells were monitored by microscopy, and phase-contrast images showed sequential morpho- logic changes of hPSCs differentiation into DE-like cells (Fig. S1). RT-PCR analyzed the dynamic expression of related genes, and results showed that pluripotency markers OCT4 and NANOG downregulated significantly. In contrast, CILy and CIP induced higher DE markers SOX17 and FOXA2 expression levels than the CI treated alone. Notably, the highly upregulated expression pat- tern of PrE markers SOX7 and GATA6 was induced by CI condition not seen in CILy- and CIP-treated cultures (Fig. 2B). These genes expression patterns suggested that added Ly or PD could improve DE differentiation versus PrE differentiation. fi Moreover, genes expressions of pluripotency, primi- tive streak (PS), primitive endoderm (PrE), and DE were dynamically traced daily during the differentiation pro- cess (Fig. 1C). The results showed a minor decrease in the pluripotency markers OCT4 and NANOG expres- sion levels. In contrast, DE markers SOX17 and FOXA2 Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 6 of 14 Pan et al. Manipulating small‑molecule cocktails to improve DE differentiation Stem Cell Research & Therapy (2022) 13:159 Immunostaining with DE markers SOX17 and FOXA2 further confirmed that a higher proportion of DE popula- tion in CILy and CIP condition, especially CIP, efficiently generated a near homogenous SOX17 and FOXA2 co- expressed DE population, which comparable with CA treatment as a control (Fig. 2C). Further FACS analy- sis showed that only 68.32% of the cells co-expressing SOX17 and FOXA2 were induced by CI, while up to 85.12%, 98.15%, and 98.5% in the cells induced by CILy, CIP, and CA, respectively (Fig. 2D). These results indicate that the combination of small-molecule CILy and CIP can efficiently induce DE differentiation. CIP, comparable with the CA control group but signifi- cantly higher than CI and CILy treatment. By contrast, the expressions of BMP2 and BMP4 were relatively lower in CIP and CA condition on the last two days of the dif- ferentiation process (Fig. 3A). These results indicate that the addition of PD could increase the endogenous TGF-β/Nodal signal while relatively downregulate BMP signal activity. On the other hand, adding Ly increased significantly and accelerated the expression of WNT3A, with the expression peak appearing two days earlier and twice higher than other induction groups (Fig. 3A). As well known that Wnt is crucial to direct hPSCs differentia- tion and also induce MD formation. Thus, these results aligned with the above results that CILy rapidly reduced expression of pluripotent markers OCT4 and NANOG within 1-day induction while inducing the highest level of PS/MD marker gene BRA during the differentiation and accelerated Mixl1 expression to transiently peak at day 1 (Fig. 2B). Further immunostaining showed that many BRA-positive cells were induced in CILy treatment at day Expansion and maintain of small‑molecule‑derived HBs Our previous study has established a robust and cost- effective culture condition for generating self-renewing HBs and functional mature HLCs, using a small-mole- cule cocktail in a defined culture system [13]. Based on this, we attempted to set up a complete small-molecule formula to efficiently generate expanded HBs and func- tional mature HLCs (Fig. 5A). ( g ) Firstly, we purified the HBs by cell sorting with EpCAM antibody, which is well known as a specific surface marker to isolate HBs (Fig. 5B). As expected, HBs popu- lations were isolated from the differentiated cells (day 8) with a proportion of 95.2%, which is consistent with the ratio of small-molecule VDF-derived AFP and HNF4α double-positive HBs showing above. The purified HBs successfully expanded in the small-molecule culture condition (ACDFSV) with a reliable self-renewal capac- ity. Additionally, the proliferative HBs could be cultured stably for at least 20 passages, without significant mor- phologic change and an apparent decrease in prolifera- tive capacity (Fig. 5C).FACS analysis showed that 54.7% EpCAM positive HBs (at passage 10) co-positive with proliferative marker Ki67 (Fig. 5D). Immunostaining fur- ther confirmed that expanding HBs almost co-expressed typical hepatic marker AFP and HNF4α. Moreover, considerable AFP-positive cells co-expressed with Ki67 (Fig. 5E), indicating the preferable proliferative capac- ity of HBs. Further results exhibited bipotency features in the proliferative HBs, with hepatic markers and early cholangiocyte markers SOX9 and Ep-CAM co-expres- sion (Fig. 5E). During expansion, these cells could be fro- zen and thawed repeatedly. Small‑molecule cocktail VDF directed hepatic specification Further endodermal specifying a hepatic fate generally utilize growth factors including BMPs, FGFs, and HGF. To identify effective small molecules substitute growth factors for hepatic specification, we specifically explored the availability of Vc (previously used to replace BMPs in HBs expansion), the HGF agonist Dihexa, and Forskolin (FSK, an adenylate cyclase activator and thus activating cAMP/PKA signal transduction) in inducing a hepatic fate, since these small molecules have been shown to pro- mote differentiation and proliferation of HBs in previous studies [6, 13, 25] (Fig. 4A). We firstly induced CIP-derived DE cells into HBs, using the combination of BMP2 and FGF4 (BF). As expected, genes expression and immunostaining analyses showed the expression of HBs marker AFP and EpCAM after five days of induction (Fig. 4B, C), indicating that CIP- derived DE cells could be differentiated into HBs effi- ciently. Elevating endogenic TGF‑β/Nodal signal to promote DE differentiationf To test whether PD and Ly promoted the DE differentia- tion by regulating the transcriptional activity of TGF-β/ Nodal-Smad2/3 signaling, we examined the expression profiles of genes related to TGF-β/Nodal, BMP, and Wnt signal pathways. Gene expression analysis showed a pro- gressive induction of NODAL, TGF-β1, and TGF-β2 by Fig. 3 Elevating endogenic TGF-β/Nodal signal to promote DE differentiation. A Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3. B Immunostaining analyzed SOX17 and BRA expression in the CILy and CIP-derived differentiated cells. Scale bars 100 μm. C Representative immunoblots of NODAL, TGF-β1, and the phosphorylation of Smad2/3 at day 2 during DE induction with different protocols. D Quantification of the protein expression levels. Data were expressed as mean ± SD, n = 3 for each group, P < 0.05, ** P < 0.01 vs control (CI) group Fig. 3 Elevating endogenic TGF-β/Nodal signal to promote DE differentiation. A Quantitative RT-PCR analyzed signaling pathway-related genes expression patterns in DE induction with different protocols. Data are presented as mean ± SEM, n = 3. B Immunostaining analyzed SOX17 and BRA expression in the CILy and CIP-derived differentiated cells. Scale bars 100 μm. C Representative immunoblots of NODAL, TGF-β1, and the phosphorylation of Smad2/3 at day 2 during DE induction with different protocols. D Quantification of the protein expression levels. Data were expressed as mean ± SD, n = 3 for each group, * P < 0.05, ** P < 0.01 vs control (CI) group Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 7 of 14 3, while few in CIP-treated condition (Fig. 3B). Moreover, these CILy-derived MDs could develop into contracting cardiomyocytes in the later phase of the hepatic differen- tiation process (Additional file 1: Movie.1). These results indicated that blocking the PI3K pathway with Ly ampli- fies the Wnt signaling, which accelerated hPSCs differen- tiation and MD induction. immunostaining with AFP and HNF4α confirmed that cells treated with the combination of VDF highly co- expressed the hepatic markers (Fig. 4E, Additional file 2: Fig. S2), and the efficiency (95.5%) was comparable with the BF control group (96.2%) (Fig. 4F). To further confirm the HBs differentiation methodol- ogy, we tested another hPSC line (iPSC line UC15) using the optimized small-molecule condition. Expansion and maintain of small‑molecule‑derived HBs Furthermore, we observed that exposure to either Dihexa or FSK in the presence of Vc resulted in induction of the hepatic marker genes AFP, HNF4α, and Albumin (ALB), with an expression rate slightly lower than the BF control group. In comparison, the triple combination of Vc, Dihexa, and FSK (VDF) results in the best overall induction of the three hepatic markers (Fig. 4D). Further Elevating endogenic TGF‑β/Nodal signal to promote DE differentiationf The results showed that this iPSC line also could sequentially dif- ferentiate into DE cells and HBs, with high efficiency and expressed stages-specific markers (Fig. S3). These results indicated that the combination of small-molecule VDF could substitute growth factors to induce hepatic specification. Further analysis of protein expression by Western blot showed the effect of the different protocols on TGF-β/ Nodal signaling molecules and downstream phospho- rylation level of Smad2/3. Results showed that adding PD (CIP) increased the amounts of NODAL and TGF-β1 proteins significantly, while little change was observed in CILy-treated cells compared with CI treatment. In con- trast, protein bands upon CA-treated cells revealed the highest expression level. Similarly, Smad2/3 and phos- phorylated Smad2/3 (p-Smad2 and p-Smad2) showed a low protein expression in CI-treated cells while increased upon treatment with CIP and CA (Fig. 3C). Quantifica- tion of the protein bands verified the higher NODAL, TGF-β1 proteins, and stronger phosphorylation Smad2/3 of CIP-treated samples compared to the CI and CILy groups (Fig. 3D). Thus, we concluded that the addition of PD to the combination of CI could upregulate the endog- enous TGF-β/Nodal signal and the downstream Smad2/3 transduction activities to promote the differentiation of DE. Differentiation of proliferative HBs into functional mature hepatocytes For hepatic maturation, the proliferative HBs then cul- tured in previously established small-molecule culture conditions, including A8301, Dex, Dihexa, and NH4Cl Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 8 of 14 Fig. 4 Small-molecule cocktail VDF directed hepatic specification. A Schematic of the strategy to identify chemical culture cocktails for HB specification. B RT-PCR analyzed AFP and HNF4α expression during hepatic specification. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of AFP and HNF4α expansion after 5 days of BF induction. Scale bars 100 μm. D RT-PCR results show hepatic genes expression in differentiated cells induced by different small molecule cocktails. Data are presented as mean ± SEM, n = 3. E Immunostaining analyses of AFP and HNF4α expression after different small molecule cocktails induced. F The efficiency of AFP and HNF4α expression after different chemical cocktails treatment, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted Fig. 4 Small-molecule cocktail VDF directed hepatic specification. A Schematic of the strategy to identify chemical culture cocktails for HB specification. B RT-PCR analyzed AFP and HNF4α expression during hepatic specification. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of AFP and HNF4α expansion after 5 days of BF induction. Scale bars 100 μm. D RT-PCR results show hepatic genes expression in differentiated cells induced by different small molecule cocktails. Data are presented as mean ± SEM, n = 3. E Immunostaining analyses of AFP and HNF4α expression after different small molecule cocktails induced. F The efficiency of AFP and HNF4α expression after different chemical cocktails treatment, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted Fig. 4 Small-molecule cocktail VDF directed hepatic specification. A Schematic of the strategy to identify chemical culture cocktails for HB specification. B RT-PCR analyzed AFP and HNF4α expression during hepatic specification. Data are presented as mean ± SEM, n = 3. C Immunostaining analyses of AFP and HNF4α expansion after 5 days of BF induction. Scale bars 100 μm. D RT-PCR results show hepatic genes expression in differentiated cells induced by different small molecule cocktails. Data are presented as mean ± SEM, n = 3. Differentiation of proliferative HBs into functional mature hepatocytes C Phase-contrast images of expanding HBs. Scale bar 100 μm. D Flow cytometric analyses of E-pCAM and Ki67 expression in expanded HBs (passage 20). E Immunostaining analyses of HBs marker proteins on expanded HBs. Scale bars 100 μm Repopulation of mice injured liver by HBs transplantation To determine whether the HBs could differentiate into functional hepatocytes and repopulate the injured liver in vivo, we transplanted HBs into DMN-induced acute liver failure of NSI mice (Fig. 7A). The Kaplan–Meier survival estimates were determined for ten days after cell transplantation. In the sham control group, two-thirds (6 of 9) of animals died within five days after the DMN injection while inducing acute liver failure. For the HBs transplanted groups, the survival rate was nearly 80% (7 of 9) throughout the examination period (Fig. 7B). Hema- toxylin and eosin (H&E) staining revealed massive necro- sis loci associated with inflammatory cell infiltration in the DMN-treated mouse liver, suggesting DMN-induced acute liver failure. In contrast, the necrosis loci dramati- cally decreased and morphologically nearly restored to normal control in the HBs transplanted mice liver (Fig. 7C). cells were detected in the sham control group (Fig. 7E). These results indicated that the small-molecule protocol- derived HBs could home and differentiate into mature hepatocytes after being transplanted in vivo. The levels of AST and ALT were significantly decreased in mice trans- planted with HBs compared to the sham-operated mice (Fig. 7F). These results suggest that HBs transplantation benefits the parenchymal tissue and the damaged recipi- ent liver’s functional recovery. Four weeks post-transplantation, Di1 and hALB co- positive cells were observed more widespread in HBs transplanted mice liver (Fig. 7G). We further analyzed the cell engraftment by the FACS examination of hALB-pos- itive hepatocytes in the recipient’s liver. Results showed that around 15% (14.8% and 17.6% in two mice liver, respectively) of the hepatocyte mass mightily derived from the transplanted human HBs, judging by the hALB labeled cells (Fig. 7H). Examination of human ALB in the mice’s serum showed increasing human ALB secretion from 1 week to four weeks after hepatic cell transplanta- tion (Fig. 7I). Moreover, to trace the homing cells and analyze the repopulation efficiency, HBs were labeled with Di1 dye before transplantation. After one week of transplantation, immunostaining with a human ALB (hALB) antibody was conducted. Representative patterns of Di1-positive and hALB-positive cells in the HBs transplanted mice liver were shown (Fig. 7D). Differentiation of proliferative HBs into functional mature hepatocytes E Immunostaining analyses of AFP and HNF4α expression after different small molecule cocktails induced. F The efficiency of AFP and HNF4α expression after different chemical cocktails treatment, determined by counting positive cells. Efficiencies are presented as the percentage of positive cells plus or minus the SD of all fields counted typical CYP isoforms (CYP3A4, CYP2C9, and CYP 2D6) were examined by measuring the increase in CYP iso- form metabolites in response to exposure to their respec- tive probe substrates. Results released considerable levels of CYP enzyme metabolic activities compared with that of human PHs (Fig. 6D). The HLCs also showed a high urea secretion pattern, corresponding to approximately 50% urea secreted by the human PHs (Fig. 6E). Moreo- ver, periodic acid-Schiff staining (PAS), indocyanine green (ICG), and LDL uptake analyses showed that HLCs displayed cytoplasmic glycogen storage, ICG, and LDL uptake abilities (Fig. 6F-H). Taken together, these results indicated that the current small-molecule protocol could direct hPSCs to efficiently differentiate, expand, and mat- urate into metabolically functioning hepatocytes in vitro. (DNAD) [13]. After five days of induction, differenti- ated cells displayed a homogenous and typical mature hepatocyte-like polygonal morphology (Fig. 6A). Genes expression of hepatic matured and relative functional biomarkers were analyzed to identify the derived HLCs. The results indicated that the ALB and cytochrome P450 (CYP) and urea cycle enzymes transcription were induced in the derived HLCs (Fig. 6B). Human PHs used as a control in the experiments. Immunostaining analy- ses further confirmed that the derived HLCs typically co- stained with mature hepatocyte-specific proteins ALB, A1AT, CYP3A4, and CYP2C9 (Fig. 6C). Furthermore, we assessed the metabolism and detoxi- fication functions of the small-molecule-derived HLCs by characterizing the activities of CYP enzymes. Three Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 9 of 14 Fig. 5 Expansion and maintain of small-molecule-derived HBs. A A schematic description of stepwise differentiates hPSCs into HBs and HLCs. B Flow cytometric sorting of E-pCAM+ HBs from day 8 differentiated cells. C Phase-contrast images of expanding HBs. Scale bar 100 μm. D Flow cytometric analyses of E-pCAM and Ki67 expression in expanded HBs (passage 20). E Immunostaining analyses of HBs marker proteins on expanded HBs. Scale bars 100 μm Fig. 5 Expansion and maintain of small-molecule-derived HBs. A A schematic description of stepwise differentiates hPSCs into HBs and HLCs. B Flow cytometric sorting of E-pCAM+ HBs from day 8 differentiated cells. Differentiation of proliferative HBs into functional mature hepatocytes In contrast, no hALB-positive No teratomas or other tumor types were found in any transplanted recipients during a 4-week period. There- fore, we concluded that the current small-molecule pro- tocol-derived HBs could engraft and differentiate into Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 10 of 14 Fig. 6 Differentiation of proliferative HBs into functional hepatocytes. A The morphology of small-molecule protocol-derived HLCs. HLCs exhibited polygonal shapes and distinct round nuclei; even some had two nuclei. Scale bar 100 μm. B Quantitative RT-PCR analyzed the genes expression levels of ALB, the CYP and urea cycle enzymes. Gene expression was normalized to HBs. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. C Immunostaining analyses of mature hepatocyte markers (ALB, A1AT, CYP3A4, and CYP2C9) on small-molecule protocol-derived HLCs. Scale bars 100 μm. D CYP450 activity assay of HLCs. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. E Urea secretion of HLCs was analyzed. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. F PAS staining on HLCs. Scale bar represents 200 μm. G ICG uptake analyses in HLCs. Scale bar represents 200 μm. H The HLCs were stained by Alexa-Flour 488-ac-LDL. Scale bar 200 μM Fig. 6 Differentiation of proliferative HBs into functional hepatocytes. A The morphology of small-molecule protocol-derived HLCs. HLCs exhibited polygonal shapes and distinct round nuclei; even some had two nuclei. Scale bar 100 μm. B Quantitative RT-PCR analyzed the genes expression levels of ALB, the CYP and urea cycle enzymes. Gene expression was normalized to HBs. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. C Immunostaining analyses of mature hepatocyte markers (ALB, A1AT, CYP3A4, and CYP2C9) on small-molecule protocol-derived HLCs. Scale bars 100 μm. D CYP450 activity assay of HLCs. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. E Urea secretion of HLCs was analyzed. Data are presented as mean ± SEM, n = 3. * P < 0.05, ** P < 0.01. F PAS staining on HLCs. Scale bar represents 200 μm. G ICG uptake analyses in HLCs. Scale bar represents 200 μm. H The HLCs were stained by Alexa-Flour 488-ac-LDL. Differentiation of proliferative HBs into functional mature hepatocytes Scale bar 200 μM HBs and functional mature hepatocytes for cell-based therapy and drug discovery use. functional hepatocytes, which further attains injured parenchyma tissue repopulation in the recipient’s liver after transplantation. y g y DE induction is the first stage also a critical stage of hPSCs differentiation into hepatic cells. The efficacy of DE induction and their heterogeneity influenced the fol- lowing hepatic differentiation trajectory and outcome significantly [23, 26]. Although applying a high concen- tration of Activin A combined with Wnt3a or Wnt ago- nist CHIR could efficiently differentiate hPSCs into DE [17, 19, 27], while Activin A is an expensive growth factor, limiting its use in large-scale cell production. Small-mol- ecule methods yielded low efficiency and reproducibil- ity differentiation [8, 15, 22, 28], which may be due to no available small molecule that can efficiently substi- tute Activin A to induce DE differentiation. Melton et al. have reported that IDE1/2 can phosphorylate Smad2/3 and activate Activin A /Nodal signaling to induce hPSCs into the endodermal lineage [20]. However, IDE1 treated alone failed to efficiently generate DE in our preliminary studies (data not shown), and similar results (40–80% SOX17-positive cells) were also reported in other studies [7, 8, 22, 28]. Discussionh F Detection of AST and ALT levels in mice serum at one week after HBs transplantation. Data were analyzed by 2-tailed t tests. G After 4 weeks of cell transplantation, the reproduction of mice liver is indicated by immunostaining of human ALB (green) and Di1 (red). Scale bar 100 μM. H Flow cytometric analyses human ALB-positive hepatocytes in mice liver transplanted with human HBs. I Human ALB secretion in the mice serum. Data are presented as mean ± SEM, n = 4 the phosphorylation of Smad2/3 and control the sig- nal transduction of the Activin A/Nodal pathway [24]. Moreover, inhibiting PI3K or CDK4/6 with small mole- cules promoted Smad2/3 to bind and activate DE genes while inducing DE differentiation. Hence, we combined CI with candidate small molecules, including PI3K inhibitor LY and CDK4/6 inhibitor PD, to induce DE differentiation. still yielded a mixture population, including DE, unde- sired MD and PrE lineage, and also with undifferenti- ated hPSCs. Both CI-I and CI treatment induced PrE marker SOX7 expression while barely expressed in CA- induced DE differentiation. Previously reported that low Nodal and high BMP signals would lead to the upregu- lation of SOX7 and PrE differentiation [29–31]. Thus, inefficient differentiation outcome may be due to insuf- ficient endogenous Nodal signal while a high level of BMP signal activities stimulated by CI-I or CI, compared with CA treatment. That highlights the critical roles of endogenous signals in DE differentiation. Therefore, we attempted to increase endogenous TGF-b/Nodal signal- ing activity by manipulating the small-molecule cocktails to improve DE differentiation. f Our finding demonstrated that adding PD to CI treat- ment (CIP) improved DE gene expression while dis- missing SOX7 upregulation, resulting in efficient DE formation. Further results released that adding PD increased endogenous TGF-β superfamily genes and pro- teins (Nodal and TGF-β1) expression significantly while downregulating the transcription of BMP2 and BMP4. Furthermore, the addition of PD also increased the phos- phorylation of Smad2/3 (p-Smad2 and p-Smad2), which is comparable with CA treatment. These results aligned with previous reports that PD could induce endogenous Nodal signaling in hPSCs differentiation [24]. There- fore, we concluded that adding PD to CI can upregulate f PI3K signaling pathway acted as a threshold at the initial stage of hPSCs differentiation [23, 32, 33]. The activation and transduction of Nodal and Wnt signal- ing pathways require decreased PI3K activity to trig- ger hPSCs differentiation [33]. Discussionh This study established an efficient protocol for the speci- fication, expansion, and maturation of hepatic cells from hPSCs, using small molecules in a chemical defined cul- ture system. In developing the protocol, we first dem- onstrated that the combination of CIP could efficiently induce DE differentiation via cell cycle inhibition by PD to increase endogenous TGF-β/Nodal signaling. Fur- thermore, we identified that combining Vc, Dihexa, and FSK (VDF) could substitute growth factor BMPs, FGFs, and HGF to induce hepatic specification. In addition, the small-molecule-derived HBs subsequently expanded and matured into functional HLCs by the strategy established in our previous study. The generated HLCs displayed adult hepatocyte’s typical functional characteristics in vitro and the ability to repopulate injured liver in vivo. Thus, the current full small-molecule-based hepatic gen- eration protocol presents an efficient and cost-effective strategy for the large-scale production of self-renewal Further combining CHIR with IDE1 (CI-I and CI methods) improved the differentiation significantly but Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 11 of 14 Fig. 7 Repopulation of mice injured liver by HBs transplantation. A Schematic diagram depicting HBs transplantation experimental schedule in immune-deficiency mice B Survival curve of mice. C Hematoxylin and eosin staining in mice liver. Scale bar 200 μM. D, E Engraftment of transplanted human HBs in mice liver after 1 week transplantation, as indicated by immunostaining of human ALB (green) and Di1 (red). Sham mice’s liver as the negative control. Scale bar 100 μM. F Detection of AST and ALT levels in mice serum at one week after HBs transplantation. Data were analyzed by 2-tailed t tests. G After 4 weeks of cell transplantation, the reproduction of mice liver is indicated by immunostaining of human ALB (green) and Di1 (red). Scale bar 100 μM. H Flow cytometric analyses human ALB-positive hepatocytes in mice liver transplanted with human HBs. I Human ALB secretion in the mice serum. Data are presented as mean ± SEM, n = 4 Fig. 7 Repopulation of mice injured liver by HBs transplantation. A Schematic diagram depicting HBs transplantation experimental schedule in immune-deficiency mice B Survival curve of mice. C Hematoxylin and eosin staining in mice liver. Scale bar 200 μM. D, E Engraftment of transplanted human HBs in mice liver after 1 week transplantation, as indicated by immunostaining of human ALB (green) and Di1 (red). Sham mice’s liver as the negative control. Scale bar 100 μM. Discussionh Similarly, cell-cycle regulators CDK4/6 also have been reported to regulate Pan et al. Stem Cell Research & Therapy (2022) 13:159 Page 12 of 14 Page 12 of 14 VDF could substitute BMPs, FGFs, and HGF to induce hepatic specification efficiently. endogenous TGF-β/Nodal signaling and downstream Smad2/3 transduction activities while relatively down- regulating BMP signal activity, which leads to the promo- tion of DE fate versus MD/PrE differentiation. ifi HBs have capable of self-renewal and potency to dif- ferentiate into hepatocytes. Hence expanding HBs, especially in small-molecule culture conditions to scal- able expand HBs derived from hPSCs would be an ideal strategy for efficient, large-scale, and cost-effective gen- erating hepatic cells for the potential clinical application [12, 42]. According to this view, our previous study has established a robust and cost-effective small-molecule culture condition for the generation of self-renewing HBs and functional mature HLCs [13]. Thus, we then adopted the previously established culture condition to expand the small-molecule-derived HBs. Our finding released that the purified HBs could successfully expand in the proliferative culture condition (ACDFSV), with a reliable self-renewal capacity and hepatic properties maintained. Additionally, the proliferative HBs could undergo long- term culture for at least 20 passages without significant morphologic change and an obvious decrease in their proliferative capacity. f Moreover, we found that the addition of Ly to CI treat- ment (CILy) led to exceeding amplification of Wnt signal, consistent with previously reported that PI3K negatively regulates the activity of the Wnt signal pathway [33, 34]. However, high Wnt signal activity tends to specify a ME formation [35–37], thereby affecting the differentiation of DE. This might account for why CILy treatment led to the highest MD marker gene BRA level and induced BRA- positive MD lineages during the differentiation process while yielding lower efficiency DE formation.i fi Small molecules have been identified to promote DE induction, while few chemicals have been reported to support hepatic specification effectively. Further speci- fies DE to a hepatic fate in previous small-molecule- based protocols mainly relied on sequential treatment with sodium butyrate and DMSO, which played a part in epigenetic modifications and were associated with ambiguous hepatic induction [15, 17, 38]. However, vari- able studies reported whether these nonspecific agents are sufficient for hepatic differentiation. Rambhatla et al. indicated that DMSO was insufficient to differentiate DE cells into hepatic lineage [39]. Discussionh In addition, both DMSO and sodium butyrate inhibit cell growth, which might compromise the yield of hESCs-derived hepatocytes for applications. Yet, no report indicated effective small mol- ecules substitute for BMPs and FGFs to direct hepatic specification efficiently. Thus, identifying novel small molecules with hepatic-specific induction would contrib- ute to the strategy of functional hepatic cells production.i Moreover, the proliferative HBs could be further effi- ciently differentiated into functional mature HLCs after being induced with mature conditions (DNAD). The small-molecule-derived HLCs expressed various repre- sentative markers and exhibited typical function activi- ties. Remarkably, the metabolic activity of CYP enzymes was comparable to PHs. In addition, after being trans- planted into NSI mice with acute liver failure, these hepatic cells could engraft and differentiate into hepat- ocytes, resulting in considerable liver repopulation and functional recovery. These results indicated that our small-molecule-based hepatic induction method induces acquired hepatocytes to function in vitro and in vivo. Our recent study has identified that Vc, FSK, and Dihexa could replace BMP4, EGF, and HGF, respectively, supporting HBs expansion and maintenance [13]. Also, we demonstrated that Vc could phosphorylate BMP downstream Smad1/5/8, consistent with the previous report that Vc serves a similar effect in cardiomyogen- esis [40]. Besides, FSK has been demonstrated essential for downregulating EMT marker gene expression and sustaining PHs gene expression [25]. Moreover, Dihexa was identified in a screen that assessed the capacity to potentiate the biological activity of HGF and can act as a potent HGF agonist [6, 41], which could mimic the func- tion of HGF in HBs differentiation and expansion. These results suggested the potential of Vc, Dihexa, and FSK to substitute growth factors for hepatic specification. In the present study, we showed that treatment with Vc and FSK combined with Dihexa (VDF) resulted in high expression of hepatic genes and proteins (AFP and HNF4α), compa- rable with growth factors-treated group as control. These results indicated that the combination of small-molecule Conclusions In summary, we have developed a robust and cost-effec- tive strategy for inducing hPSCs to proliferative HBs and functional hepatocytes using the small-molecule pro- tocol, which could serve as a powerful foundation for future studies to define culture conditions using GMP- compatible products. Furthermore, this work provides a novel, cost-effective way for generating large-scale and functional human hepatic cells, which would result in an invaluable impact in the cell-based therapy industry. Abbreviations hPSCs: Human pluripotent stem cells; HBs: Hepatoblasts; HLCs: Hepatocyte- like cells; PHs: Primary hepatocytes; DE: Definitive endoderm; PrE: Primitive endoderm; NSI mice: NOD-SCID-IL2RG−/− mice; CHIR: CHIR99021; CYP: Cytochrome P450; ICG: Indocyanine green; PAS: Periodic acid-schiff staining. Acknowledgements h h h k h The authors thank the National Basic Research Program of China and Guang‑ dong Province Science and Technology Plan for the funding support. 4. Chen S, Wang J, Ren H, Liu Y, Xiang C, Li C, Lu S, Shi Y, Deng H, Shi X. Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure. Cell Res. 2020;30(1):95–7. 4. Chen S, Wang J, Ren H, Liu Y, Xiang C, Li C, Lu S, Shi Y, Deng H, Shi X. Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure. Cell Res. 2020;30(1):95–7. Availability of data and material The datasets supporting the conclusions of this article are included within the article. 10. Korostylev A, Mahaddalkar PU, Keminer O, Hadian K, Schorpp K, Gribbon P, Lickert H. A high-content small molecule screen identifies novel induc‑ ers of definitive endoderm. Mol Metabolism. 2017;6(7):640–50. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. 13. Pan T, Tao J, Chen Y, Zhang J, Getachew A, Zhuang Y, Wang N, Xu Y, Tan S, Fang J, Yang F, Lin X, You K, Gao Y, Li YX. Robust expansion and functional maturation of human hepatoblasts by chemical strategy. Stem Cell Res Ther. 2021;12(1):151. Author contributions TP, NW, and JZ contributed equally to this work. TP wrote the manuscript; JZ, NW, YC, YZ, YX, and JF revised the manuscript; TP performed the experiments and analyzed the data; YC performed the animal experiments and function assays of hepatocytes; FY, XL, YL, SL, KL, and KY assisted with the experiments and analyzed the data; YL and YG directed the study, critically revised, and contributed to final approval of the manuscript. 5. Feng S, Wu J, Qiu WL, Yang L, Deng X, Zhou Y, Chen Y, Li X, Yu L, Li H, Xu ZR, Xiao Y, Ren X, Zhang L, Wang C, Sun Z, Wang J, Ding X, Chen Y, Gadue P, Pan G, Ogawa M, Ogawa S, Na J, Zhang P, Hui L, Yin H, Chen L, Xu CR, Cheng X. Large-scale generation of functional and transplantable hepatocytes and cholangiocytes from human endoderm stem cells. Cell Rep. 2020;33(10):108455. Received: 14 December 2021 Accepted: 20 March 2022 Received: 14 December 2021 Accepted: 20 March 2022 Additional file 1: CILy-derived MDs developed into contracting cardio‑ myocytes during hepatic differentiation. Additional file 1: CILy-derived MDs developed into contracting cardio‑ myocytes during hepatic differentiation. Additional file 1: CILy-derived MDs developed into contracting cardio‑ myocytes during hepatic differentiation. Additional file 2: Figure S1. Small-molecule cocktails induced DE differ‑ entiation from hPSCs. Sequential morphologic changes in the differen‑ tiation of hPSCs into DE. Scale bars 100 μm. Figure S2. Small-molecule cocktails directed hepatic specification. Immunostaining analyses of AFP and HNF4α expression after different small molecule cocktails induced. Figure S3. Generation of HBs from human iPSC line. Immunostaining analyses results showed that human iPSC line (UC15) could sequentially differentiate into DE cells and HBs with high efficiency similarity as previ‑ ous hESC line H1, and expressed stage specific markers. Declarations 11. Kim Y, Kang K, Lee SB, Seo D, Yoon S, Kim SJ, Jang K, Jung YK, Lee KG, Factor VM, Jeong J, Choi D. Small molecule-mediated reprogram‑ ming of human hepatocytes into bipotent progenitor cells. J Hepatol. 2019;70(1):97–107. Competing interests The authors declare that they have no competing interests. 14. Asumda FZ, Hatzistergos KE, Dykxhoorn DM, Jakubski S, Edwards J, Thomas E, Schiff ER. Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Differentiation. 2018;101:16–24. Funding h d 6. Siller R, Greenhough S, Naumovska E, Sullivan GJ. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Rep. 2015;4(5):939–52. 6. Siller R, Greenhough S, Naumovska E, Sullivan GJ. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Rep. 2015;4(5):939–52. This study was supported by the National Key R&D Program of China (20 18YFA0108200, 2019YFA0111300, 2018YFC1106400), the Sino-German rapid response funding call for COVID-19 related research (C-0031), Frontier Research Program of Bioland Laboratory (Guangzhou Regenerative Medi‑ cine and Health Guangdong Laboratory) (2018GZR110105011), National Natural Science Foundation of China (31871379, 22075127, 31972926), the Natural Science Foundation of Guangdong Province (2018A030313128, 2018A030313214), and the Guangdong Key Research and Development Plan (2019B020234003), Guangzhou Science and technology planning project (202102020988). 7. Tasnim F, Phan D, Toh YC, Yu H. Cost-effective differentiation of hepato‑ cyte-like cells from human pluripotent stem cells using small molecules. Biomaterials. 2015;70:115–25. 8. Bogacheva MS, Khan S, Kanninen LK, Yliperttula M, Leung AW, Lou YR. Differences in definitive endoderm induction approaches using growth factors and small molecules. 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https://openalex.org/W2561122543	https://europepmc.org/articles/pmc5297705?pdf=render	English	null	Brain Vascular Imaging Techniques	International journal of molecular sciences	2,016	cc-by	14,688	Bàrbara Laviña Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 75185 Uppsala, Sweden; barbara.lavina-siemsen@igp.uu.se; Tel.: +46-018-471-4504 Academic Editor: Michael Henein Received: 24 October 2016; Accepted: 26 December 2016; Published: 30 December 2016 Abstract: Recent major improvements in a number of imaging techniques now allow for the study of the brain in ways that could not be considered previously. Researchers today have well-developed tools to speciﬁcally examine the dynamic nature of the blood vessels in the brain during development and adulthood; as well as to observe the vascular responses in disease situations in vivo. This review offers a concise summary and brief historical reference of different imaging techniques and how these tools can be applied to study the brain vasculature and the blood-brain barrier integrity in both healthy and disease states. Moreover, it offers an overview on available transgenic animal models to study vascular biology and a description of useful online brain atlases. Keywords: brain vascular disorders; imaging; vascular biology; brain atlas; animal models; molecular imaging; computed tomography (CT); positron emission tomography (PET); magnetic resonance angiography (MRI); photoacoustic imaging (PAI); magnetic resonance angiography (MRA) International Journal of Molecular Sciences International Journal of Molecular Sciences Review 1. Introduction The brain is one of the most sophisticated and complex organs generated by evolution and its impressive anatomical composition and its entangled functionality has been admired for thousands of years. Although early civilizations lacked adequate means to obtain knowledge about the nervous system, the ancient Egyptians in the 17th century Before Common Era (BCE) wrote the earliest recorded reference to the brain in the Edwin Smith papyrus [1]. It was not until the 5th century BCE that the concept of the nervous system appeared [2]. In the 1600s, William Harvey proved the theory of blood circulation in De Motu Cordis [3], although the ﬁrst descriptions of the pulmonary circulation by Ibn al-Naﬁs date back to the 16th century BCE [4]. The interest in dynamics stimulated the study of angiology and neuroanatomy and, in 1664, Thomas Willis published Cerebri anatome, a text on the brain that was a groundbreaking work for neuroscience and remained very inﬂuential for the next two centuries [5,6]. In 1882, Angelo Mosso invented the ﬁrst neuroimaging technique, called “human circulated balance” that could non-invasively measure the redistribution of blood during emotional and intellectual activity [7]. Nevertheless, the origin of structural imaging was the X-ray, discovered by Roentgen in 1895 [8]. Shortly after, Haschek and Lindenthal produced radiographs of blood vessels by injecting opaque solution into cadavers; however, it was not until 1927 that Egas Moniz performed the ﬁrst cerebral angiography in humans [9]. Since then, key events and major technological innovations in physics, mathematics, computing and clinical imaging have promoted the development of at least the following techniques: (1) computed tomography (CT), for which Hounsﬁeld and Cormack were awarded the Nobel Prize in 1979; (2) positron emission tomography (PET) [10]; and (3) magnetic resonance imaging (MRI), for which Lauterbur and Mansﬁeld were awarded the Nobel Prize in 2003 and magnetic resonance angiography (MRA). The abovementioned techniques together with other imaging modalities, including digital subtraction angiography (DSA), photoacoustic imaging (PAI) and trans-cranial doppler (TCD), have contributed to the knowledge of the brain vasculature, promoting and improving our understanding of the complexity of the central nervous system (CNS). Int. J. Mol. Sci. 2017, 18, 70; doi:10.3390/ijms18010070 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 70 2 of 23 The strategies that scientists have adopted for studying the brain have varied over the years as new techniques and methods have been developed. 1. Introduction Elucidating the composition and functions of the brain is one of the most challenging areas of research. Characterizing the structure of the brain at high resolution is crucial for understanding its functions and dysfunction. Neuroimaging, the process of producing images of the structure or activity of the brain, is becoming an increasingly important tool in both research and clinical care, tremendously helping our understanding of brain morphology and physiology in healthy and disease states. The aim of this review is to give a short overview of the most important recent scientiﬁc advances achieved using imaging techniques, with particular focus on their relevance to the ﬁeld of brain vascular imaging and including a section on molecular imaging of the blood-brain barrier (BBB). A special focus on animal models is also included, as their use is motivated by a desire to better understand human diseases. Moreover, a summary of available online brain atlases for both human and animal models and the way these approaches contribute to a better understanding of the brain on multiple levels are also highlighted. 2. Computed Tomography (CT) Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) The development of CT soon led to other imaging techniques, including PET [37,38], which involves injecting rapidly decaying radioactive substances into the bloodstream. The ﬁrst simple PET scanner able to detect brain tumors was made in the early 1950s by Bromwell and Sweet and by 1975, more sophisticated types of PET, which could measure blood ﬂow, were developed [39–41]. The ﬁrst PET image of a rat brain using a clinical PET scanner was performed in 1991 [42] and the initial dedicated small PET scanner was introduced several years later in 1995 [43]. The recent development of a high-resolution small animal PET scanner suitable for imaging the mouse brain will quickly improve the imaging quality of brain analysis [44]. PET functional imaging is most useful in combination with anatomical imaging and hybrid imaging systems such as PET/MRI [45,46] and PET/CT [47,48]. Multimodality imaging PET/CT and PET/MRI provide advantages in the imaging evaluation of patients with a variety of diseases [49]. A PET/CT scanner has the ability to improve image quality and image accuracy of PET images, enhancing lesion identiﬁcation and localization, which affects clinical decision making and thereby improves patient management [50–52]. PET/MRI combination offers functional structural and metabolic data, which can potentially contribute to more accurate diagnoses and ultimately affect patient survival. The advantages and drawbacks of PET/CT are discussed in [53,54]. Multimodality imaging is nowadays available in the clinical practice and for small animals [55]. Rodent SPECT/CT has been used to assess in vivo CBF and blood-brain barrier (BBB) disruption after focal cerebral ischemia [56] and the recently developed trimodal PET/SPECT/CT scanner for small animals [57] has allowed for new insights into brain function and the visualization of cerebral ischemia in living rats [58]. PET and single photon emission computed tomography (SPECT) [59,60] are molecular imaging modalities which by the use of speciﬁc radioactive tracers, allow visualization and measurement of physiological processes in intact living brains [61]. Recently PET has been used to study hypoxia and inﬂammation in ischemic stroke [62–65], to explore the possible relationship between an acute ischemic stroke and Aβ deposition in patients [66], and to noninvasively image VEGFR expression kinetics to analyze post stroke angiogenesis in rats [67]. Moreover, 11C-methionine is the most popular tracer used in PET imaging of brain tumors [68], and can predict prognosis in gliomas [69]. 2. Computed Tomography (CT) On October 1, 1971, in London, England, CT imaging performed by Godfrey Hounsﬁeld and James Ambrose produced the ﬁrst scan of a patient with a cerebral cyst. The image proved it was possible to produce non-superimposed images of an object slice [11]. Since that time, CT has been improved by a number of important technological advancements, leading to the current ability to acquire thousands of thin-slice images with voxel isotropy in a few seconds with a reduced radiation dose [12,13]. CT angiography (CTA) has gained the most beneﬁt from such evolution in terms of improved diagnostic performance and broadened clinical indications [14,15]. Color-coded CT angiography, a new method of displaying dynamic cerebral CT angiography, provides important additional information on cerebral hemodynamics, including speciﬁcally differentiation between antegrade and retrograde ﬂow [16]. g g Micro-scale computed tomography (microCT) [17,18] and nano-computed tomography (nanoCT) [19] are high-resolution cross-sectional imaging techniques and are essential tools for phenotyping and for elucidating diseases and their therapies. Compared to other imaging methods, the strengths of microCT and nanoCT lay with their high-resolution scanning efﬁciency, velocity and relatively low cost. Additionally, it is a structural imaging modality that provides a high-resolution volumetric representation of vascular structures in brains of rodents [20–24] as well as measurements of cerebral blood volume (CBV) [25]. Recent applications of microCT to the mouse brain vasculature include the in situ analysis of adult brains using iodine-based contrast [26], the evaluation of animal models of cerebral cavernous malformations (CCM) [27] and the imaging of brain tumors in live mice [28]. Perfusion CT is a relatively new imaging technique that allows rapid qualitative and quantitative evaluation of cerebral vascular physiology and hemodynamics including measurement of cerebral blood ﬂow (CBF) and CBV. It involves the sequential acquisition of cerebral CT images performed during the intravenous administration of contrast material [29]. It is an alternative imaging modality with several clinical indications including stroke [30], head trauma [31] and brain tumors [32]. Published data have suggested that perfusion CT might be comparable to MRI [33,34]. Moreover, perfusion CT has been used to evaluate possible clinical beneﬁts of pharmacology therapy in early stroke onset [35], as well as to predict survival in in high-grade gliomas [36]. Int. J. Mol. Sci. 2017, 18, 70 3 of 23 3. 2. Computed Tomography (CT) Other recent examples of brain vascular disorders imaged utilizing PET or SPECT include thrombosis [70], Alzheimer’s [71] and Moyamoya disease [72]. 4.2. Susceptibility-Weighted Imaging (SWI) SWI is an MRI technique that enhances image contrast by using the susceptibility differences between tissues and has become a part of routine brain MRI protocols [111]. The clinical success of SWI arises from its superior sensitivity for detecting small quantities of blood product, its ability to differentiate between arterial and venous vessels, and its ability to differentiate between calciﬁcation and blood product. Thus, SWI is nowadays utilized to obtain images of diverse brain vascular disorders including: hemorrhages, traumatic brain injury, stroke, tumors and multiple sclerosis [112–114]. The fact that this technique does not provide quantitative measurements, which is an important limitation, is currently overcome by the advancement of new technology such as quantitative susceptibility mapping (QSM) [115] and susceptibility tensor imaging (STI) [116]. 4.1. Diffusion and Perfusion Weighted MRI. Diffusion (DWI) and perfusion (PWI) weighted MRI have an increasingly important clinical role (see [97] for a detailed description of the basic principles). The combination of both techniques is especially promising for the early detection and assessment of stroke [98,99]; and for brain tumor characterization [100], as they provide complementary information. DWI is based on the random movement of water molecules caused by their kinetic energy dissipation, known as Brownian motion, in the presence of magnetic pulses. The apparent diffusion coefﬁcient is a measure that displays the magnitude of diffusion of the water molecules within tissue [101,102]. In the ﬁeld of brain imaging, DWI has been applied to diagnose and monitor stroke [103,104] and characterize brain tumors [105]. PWI refers to methods that make use of the effect of endogenous or exogenous tracers on the MR images for deriving various hemodynamic parameters offering the potential for measuring brain perfusion in several pathological conditions including stroke [106] and brain tumors [107,108]. Perfusion MRI techniques can be used for quantitative assessment of speciﬁc pathophysiologic parameters, more accurate grading of intracranial tumors and may predict survival and patient outcome [109,110]. 4. Magnetic Resonance Imaging (MRI) and Other Similar Techniques The basic idea behind the MR phenomenon ﬁrst appeared in 1946 from discoveries by Bloch and Purcell. It took another 25 years before MRI was applied to medical diagnosis, when the ﬁrst discoveries concerning the development of the technique to visualize different structures were published [73–75]. A great advantage with MRI is that it uses magnetic forces rather than potentially harmful ionizing radiation. Moreover, image quality has advanced to a remarkable extent with scan times decreasing by factors of 10 to 100 since the early 1980s [76,77]. The main concepts required to understand MRI include the classiﬁcation of the image contrast following its sensitivity to three different parameters: proton density (ρ), the spin-lattice or longitudinal relaxation times T1, and the spin-spin or transverse relaxation times T2 or T2*. A proton-density-weighted image is a sequence that is mainly sensitive to ρ and T1- or T2-weighted images are, respectively, sensitive to T1 or T2 relaxation times [78]. Additionally, different contrast agents, which enrich MRI, can be used in the so-called Contrast-Enhanced MRI [79]. Several reviews have recently focused on cerebrovascular MRI, compared to other imaging techniques and described the key developments in the last years [12,80–83]. These new advances in neuroimaging methods could also be applied as biomarkers [84]. Biomarkers can be used as a diagnostic tool, a prognostic tool, a predictive tool (for predicting Int. J. Mol. Sci. 2017, 18, 70 4 of 23 response to an intervention), or a substitute for a clinical outcome to measure the response to an intervention (surrogate end point). Some examples of biomarkers based on MR imaging used in acute ischemic stroke are summarized in [85]. Biomarkers based on brain imaging may relate to prognosis in high-grade gliomas [86] and recently, vessel caliber analysis has been proposed as a possible biomarker of tumor response in clinical trials, revised in [87]. The study of hemodynamic alterations in patients with cerebrovascular disease is important to understand the disease, potentially improving diagnostic capabilities and therapeutic planning. There is a demand for noninvasive imaging of cerebrovascular territories; therefore, new emerging techniques for evaluation cerebrovascular hemodynamics and CBF [88], including four-dimensional (4D) ﬂow MRI [89–92]; 2D phase contrast MRI (PC-MRI) [93–95] and magnetic resonance black-blood thrombus imaging technique (MRBTI) [96] are being developed. 4.4. Intracranial Vessel Wall Imaging (IVW) Recently available IVW methods provide the possibility of directly assessing the vessel wall [124–127], providing a useful diagnostic tool, which may improve patient outcomes by helping treatment choice, in comparison to other invasive and non-invasive methods currently available. IVM is especially challenging due to the small caliber and tortuosity of the intracranial vessels and is an upcoming ﬁeld of interest to assess intracranial atherosclerotic lesions [128–130], intracranial vasculopathies [131,132], cerebrovascular inﬂammation [133], CNS vasculitis [134], brain arteriovenous malformations [135], Moyamoya disease [136,137], Cerebral aneurysms [138–140] and intracranial arterial dissection [141]. 4.5. MR Angiography (MRA) MRA is a group of techniques based on MRI to image blood vessels [142,143]. MRA techniques can be divided into two categories: contrast-enhanced and non-contrast enhanced MRA. Since its introduction in 1994 by Prince [144], ﬁrst-pass contrast-enhanced MRA has seen widespread acceptance and details about techniques and contrast agents are reported in [145–149]. A detailed description of non-contrast MRA techniques and the physical mechanisms underlying each method including their clinical applications can be found in [150] and [151]. In the brain, MRA is used to visualize cerebrovascular territories [152,153], and to evaluate stenosis and occlusions [154], aneurysms [155] and other cerebral malformations [156]. A complete description of MRA in brain vascular disorders is reviewed in [157]. 4.3. Quantitative Susceptibility Mapping (QSM) QSM [117] is expected to play an increasing role in the clinic as it permits to unambiguously differentiate between calciﬁed and hemorrhagic lesions which permits a differential diagnosis and simultaneously reveals brain anatomy. This technique allows investigating and obtaining valuable information not only on compositional changes in aging brain, but also in numerous neurodegenerative disorders, providing valuable guidance to clinicians during diagnosis. QSM has recently been applied to monitor CCM disease progression and iron deposition [118,119], intracranial hemorrhages [120], hematoma volume [121] and to differentiate hemorrhages from calciﬁcations [122]. The usefulness of QSM in visualizing the microstructure of the mouse brain at a 10 µm resolution, has been shown by the revealing of detailed structures [123]. Int. J. Mol. Sci. 2017, 18, 70 5 of 23 6. Trans-Cranial Doppler (TCD) TCD ultrasound is a specialized technique introduced in 1982 by Rune Aaslid for detecting blood ﬂow in the basal intracerebral arteries [170]. It is a noninvasive technique that involves the use of a low-frequency (≤2 MHz) transducer probe to insonate speciﬁc areas of the cranium that are relatively thin. TCD enables users to acquire images of some of the major intracranial vessels through the intact skull and monitor cerebral blood ﬂow (CBF) velocity and vessel pulsatility over extended time periods with a high temporal resolution. [171]. Advanced applications of TCD in neurovascular diseases have been extensively revised in [172–175]. 5. Digital Subtraction Angiography (DSA) DSA is an imaging method that permits a distinct visualization of the vasculature in a skeletal or dense soft tissue environment. The introduction of the technique in 1980 provided a method for real-time 2D subtraction imaging, spawning a steady progression of related methods, including 3D [158] and 4D DSA [159] (an outline of some historical milestones and future directions are nicely reviewed in [160,161]). An overview of the technical principles of DSA can be found in [162,163]. Brieﬂy, the target tissue is initially exposed to X-ray or MRI, to obtain the ﬁrst set of images; then a contrast agent is administered into the vasculature and additional X-ray or MRI are obtained. The mask, which is the ﬁrst set of images, is then subtracted from the latter or contrast enhanced images, allowing the visualization of the vascular structure free of the surrounding tissue. g DSA can be utilized to visualize intracranial vascular structures [164], vascular abnormalities such as arteriovenous malformations [165], aneurysms [166], carotid stenosis [167], as well as grading Moyamoya disease [168] and collateral ﬂow in acute middle cerebral artery occlusion [169]. 7. Photoacoustic Imaging (PAI) Recently, a wearable system has been developed that is capable of providing images of cerebral blood vessels noninvasively [199]. Moreover, PAI is a new strategy to visualize and study several brain disorders [200] including stroke [201–203], brain tumors [204–206]; cerebral edema [207], epilepsy [208]; traumatic brain injury [209] and inﬂammation [210]. PAT imaging in patients with brain pathologies is currently not used in clinical practice and is still under development. The main challenge and limitation in humans is the thickness of the skull, but promising recent advances suggest that this technology could be implemented as a clinical device for noninvasive functional brain imaging [184,211–213]. 7. Photoacoustic Imaging (PAI) Photoacoustic imaging (PAI) is also called optoacoustic imaging and is an emerging imaging modality that shows great potential for preclinical research and clinical practice [176]. The method is based on the photoacoustic effect; brieﬂy, the tissue of interest is excited by a pulsed laser and part Int. J. Mol. Sci. 2017, 18, 70 6 of 23 of the locally absorbed light produces thermal excitation, leading to an expansion of the tissue and subsequent generation of ultrasonic waves. Ultrasonic transducers detect the emitted ultrasonic waves, which are ﬁnally converted into images. The applications in biomedicine of the photoacoustic effect, ﬁrst reported by Alexander Graham Bell in 1880, began in the 1970s [177] but progressed slowly until the last decade of the 20th century, when many pioneering works demonstrated the photoacoustic effect in optically scattering media and biological tissue, extensively reviewed in [178–180]. As a hybrid technique, PAI is based on the acoustic detection of optical absorption from either endogenous chromophores or exogenous contrast agents [181], such as chemical dyes [182], nanoparticles [183,184] and reporter genes [185,186]. PAI is especially useful in visualizing blood vessels in vivo due to the fact that blood hemoglobin and deoxyhemoglobin, have a substantially higher absorption than surrounding tissues and therefore create sufﬁcient endogenous contrast. Over the past decade, the photoacoustic technique has been evolving rapidly, leading to a variety of exciting discoveries and applications such as photoacoustic tomography (PAT) that is cross-sectional or three-dimensional (3D) PAI [187–189]. PAT allows visualizing and studying a diverse range of structures: from organelles to whole organs [190] and based on the spatial resolution of the method, PAT is classified into optical-resolution photoacoustic microscopy (OR-PAM) [191], acoustic-resolution photoacoustic microscopy (AR-PAM) [192], photoacoustic computed tomography (PACT) [193,194], and photoacoustic endoscopy (PAE) [195]. PAI has been successfully used in the past few years in small animal models to image the brain [196,197] and determine CBF though the intact skull [198]. Recently, a wearable system has been developed that is capable of providing images of cerebral blood vessels noninvasively [199]. Moreover, PAI is a new strategy to visualize and study several brain disorders [200] including stroke [201–203], brain tumors [204–206]; cerebral edema [207], epilepsy [208]; traumatic brain injury [209] and inﬂammation [210]. PAI has been successfully used in the past few years in small animal models to image the brain [196,197] and determine CBF though the intact skull [198]. 9. Transgenic Animal Models for Vascular Biology Recent advances in imaging techniques [236,237] together with the development of tissue-clearing methods, facilitating volumetric imaging without sectioning [238–240], do now provide the possibility, with adequate resolution, to obtain live imaging data from the vasculature in organs and even whole experimental animals during development and disease conditions. The development of transgenic animal models with ﬂuorescent markers for speciﬁc proteins or speciﬁc vascular cell types are necessary to fully beneﬁt from the newly available microscope techniques. So far, zebraﬁsh has been the most suitable animal model to perform in vivo live imaging experiments of the vascular system. Therefore, several transgenic lines, which express different kinds of ﬂuorescent probes in vascular cell types, are currently available and have extensively contributed to improving our knowledge of key vascular processes such as lumen formation, angiogenic sprouting, remodeling, cell proliferation and circulation of blood cells, reviewed in [241,242]. Although the establishment of ﬂuorescent vascular reporters in mice models to study vascular biology has been slower, it is in fact becoming more relevant. Some transgenic mouse lines are already available and label different cell types including: EC [243–252]; lymphatic endothelial cells (LEC) [253–257], reviewed in [258] and pericytes [259–261]. While the above referenced transgenic lines are useful reporters for the major vascular cell types, other transgenic lines have been developed to visualize various organelles or subcellular structures [262–265]. The effort to develop new tools such as ﬂorescent reporter mice is not only performed by single scientiﬁc groups but also by joined projects. One example is the Gene Expression Nervous System Atlas (GENSAT) project, which is mapping the expression pattern of genes in the CNS and has created several mouse reporters, expressing enhanced green ﬂuorescent protein (EGFP) and td-Tomato, to investigate the distinct gene expression patterns [266] (www.gensat.com). Moreover, other interesting ﬂuorescent reporter mice are also available in the Jackson Laboratory (www.jax.org). 8. Molecular Imaging of the Blood-Brain Barrier (BBB) Multiple cell types, which coordinately form the neurovascular unit (NVU), include neurons; vascular cells (endothelial cells (EC), smooth muscle cells and pericytes); and glia (astrocytes, microglia and oligodendroglia), which are key factors to maintaining CNS functions. Within the NVU, the EC form the BBB that limits entry of substances into the brain and maintains the ideal environment for the brain to properly function [214,215]. A detailed molecular atlas of the BBB transport systems and cellular functions, based on available data on protein and RNA expression, as well as physiological measurements from different published investigations, is meticulously provided in this recent review [216]. Dysfunction of the BBB has been shown to be a common denominator in many CNS disease pathogeneses observed in stroke, cerebral edema, Parkinson’s disease, Alzheimer’s disease, seizures, microcephaly and CCMs, among others [214,217,218]. Work in key molecular components of the ECs composing the BBB—including macromolecule transporters such as the glucose transporter GLUT1 [219]; the major facilitator superfamily domain-containing protein 2a (MFSD2a) [220,221]; and tight junction complexes [222], such as Occuldin [223]; and the junctional adhesion molecule C (JAM-C) [224]—and their implication in several brain diseases with BBB dysfunction and altered cerebrovascular integrity have raised the interest in molecular imaging of the BBB. Dynamic contrast-enhanced MRI (DCE-MRI) is the most widely used imaging method for assessing BBB integrity [225–228]. It has been used to asses abnormal BBB permeability in several Int. J. Mol. Sci. 2017, 18, 70 7 of 23 pathological conditions including: traumatic brain injury [229], vascular cognitive impairment [230], multiple sclerosis [231], Alzheimer’s disease [232], brain tumors [233] and stroke [234]. The development of novel state-of-the art neuroimaging and molecular biomarker approaches are key aspects of future investigations addressing whether molecular and imaging biomarkers of BBB dysfunction can serve as reliable prognostic and/or diagnostic tools to predict the development of several CNS disorders [235]. 10. Brain Atlases Another useful tool generated using some of the imaging methods mentioned in this review are brain atlases [267]. Brain atlases are applicable in in all areas of neuroscience including research, education and clinical applications. An enormous amount of data is produced every day in laboratories worldwide regarding brain mapping, and there are many initiatives to make these data available through public databases. At present, brain atlases are printed, electronic, web-based and some are even available on mobile platforms [268]. The recent technical advances in areas such as sample preparation, optical techniques, quantitative 2D and 3D imaging analysis and high-performance computing, have relevantly contributed to the development of new brain mapping approaches (reviewed in [269]). The latest news about the worldwide brain research initiatives can be found in [270]. Following the publication of the mouse [271], adult [272] and prenatal [273] human brain gene expression atlases in recent years, a high-resolution transcriptional atlas of pre- and post-natal brain development for the rhesus monkey has now become available [274]. Table 1 gives examples of some of the most relevant web-based brain atlases in human, rodents and other species [271,272,274–293]. 8 of 23 Int. J. Mol. Sci. 2017, 18, 70 Table 1. Examples of available online brain atlases. Table 1. Examples of available online brain atlases. Specie Atlas Name Comments Link Selected References Human The Human Brain Atlas at Michigan State University MRI from one specimen + myelin and cell bodies staining https://msu.edu/~brains/brains/human/index.html Tool developed by Sudheimer, K.D.; Winn, B.M.; Kerndt, G.M.; Shoaps, J.M.; Davis, K.K.; Fobbs Jr., A.J.; Johnson; J.I. 10. Brain Atlases Allen Brain Atlas MRI, in situ hybridization, microarray http://human.brain-map.org/ [272,275,276] Scalable Brain Atlas Brain atlas templates for different species https://scalablebrainatlas.incf.org/main/index.php [277] Atlas of the Human Brain Macroscopic and microscopic levels http://www.thehumanbrain.info/ [293] Human Connectome Project MRI-based imaging modalities to measure brain architecture, function and connectivity http://www.humanconnectomeproject.org/ [278–281] BigBrain Cell bodies’ staining https://bigbrain.loris.ca/main.php [282] Jubrain Probabilistic maps based on post mortem brains https://www.jubrain.fz-juelich.de/apps/cytoviewer/cytoviewer-main.php [283] The Whole Brain Atlas Contains atlas from different cerebrovascular diseases http://www.med.harvard.edu/aanlib/home.html [284] Human Brainnetome Atlas Structural and connectivity features http://atlas.brainnetome.org/ [285] Brain Development Atlases (Imperial College London) Different atlas datasets are available (adult, pediatric and neonatal) http://brain-development.org/brain-atlases/ [286–288] FSL Atlas Library of analysis tools for FMRI, MRI and DTI brain imaging data http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/ [289] Rodents Allen Brain Atlas Includes developing brain, spinal cord and connectivity brain atlas http://mouse.brain-map.org/ [271,276] Mouse Atlas Project Contains also a neonatal brain atlas http://map.loni.usc.edu/ [290] The Mouse Brain Library Includes developing mouse brain atlas http://www.mbl.org/ Tool developed by Williams, R.W. and colleagues Others Zebraﬁsh Brain Atlas Includes information about the neuroanatomy of the developing zebraﬁsh brain http://zebraﬁshbrain.org/ [291] Blueprint Atlas (Rhesus macaque) Gene expression data, neuroanatomical data http://www.blueprintnhpatlas.org/ [274] Virtual Fly Brain (Drosophila melanogaster) Neural anatomy and imaging data http://www.virtualﬂybrain.org/ [292] Int. J. Mol. Sci. 2017, 18, 70 9 of 23 Abbreviations BCE Before Common Era CNS central nervous system CT computed tomography MRI magnetic resonance imaging PET positron emission tomography SPECT single photon emission computed tomography CTA computed tomography angiography MicroCT micro-computed tomography NanoCT nano-computed tomography CCM cerebral cavernous malformations QSM quantitative susceptibility mapping STI susceptibility tensor imaging SWI susceptibility-weighted imaging 4D four-dimensional IVW intracranial vessel wall imaging PAI photoacoustic imaging PAT photoacoustic tomography 3D three-dimensional 2D two-dimensional EC endothelial cells LEC lymphatic endothelial cells MRBTI magnetic resonance black-blood thrombus imaging technique NVU neurovascular unit DCE-MRI dynamic contrast-enhanced MRI BBB blood-brain barrier CBV cerebral blood volume TCD trans-cranial doppler PC-MRI phase contrast MRI MRA magnetic resonance angiography DSA digital subtraction angiography DWI diffusion weighted MRI PWI perfusion weighted MRI EGFP Enhanced green ﬂuorescent protein 11. Conclusions Imaging is becoming an increasingly important tool in research and clinical care. A range of imaging techniques now provides unprecedented capacity to visualize the structure of the brain, including the vasculature, from the level of individual molecules and encompassing the whole brain. Most imaging methods are noninvasive and allow monitoring dynamic processes over time. Imaging enables researchers to identify brain vascular abnormalities, understand disease pathways, evaluate blood-brain barrier (BBB) integrity, recognize and diagnose pathologies and determine efﬁcacy of different therapies and treatments. Each technique has strengths and weaknesses relating to cost, availability, temporal and spatial resolution and risk factors, thus improving our knowledge and ensuring the innovation of such tools will be beneﬁcial in the near future. The imaging techniques described in this review, alone and in combination, contribute to transforming and improving our understanding of how the brain functions in health and disease. Acknowledgments: I acknowledge Christer Betsholtz for scientiﬁc support and Jennifer Hofmann for textual suggestions and corrections. Bàrbara Laviña was supported by the Spanish Ministry of Education through a Fundación Española para la ciencia y la tecnología (FECYT) grant, via the postdoctoral mobility contract EDU/2934/2009. Conﬂicts of Interest: The author declares no conﬂict of interest. 1. Breasted, J.H. (Ed.) The Edwin Smith Surgical Papyrus. Published in Faesimile and Hieroglyphie Transliteration, with Translation and Commentary; The Univesity of Chicago Press: Chicago, IL, USA, 1930. 2. Wickens, A.P. A History of the Brain: From Stone Age surgery to Modern Neuroscience; Psychology Press: London, UK; New York, NY, USA, 2015; p. 27. References 1. Breasted, J.H. (Ed.) 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the author; 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/W3099778837	https://www.researchsquare.com/article/rs-14374/latest.pdf	English	null	Effects of contact stress on patellarfemoral joint and quadriceps force in fixed and mobile-bearing medial unicompartmental knee arthroplasty	Journal of orthopaedic surgery and research	2,020	cc-by	5,472	Effects of contact stress on patellarfemoral joint and quadriceps force in fixed and mobile-bearing medial unicompartmental knee arthroplasty Effects of contact stress on patellarfemoral joint and quadriceps force in fixed and mobile-bearing medial unicompartmental knee arthroplasty Hyuck Min Kwon Yonsei University Jin-Ah Lee Yonsei University Yong-Gon Koh yonsei sarang hospital Kwan Kyu Park Yonsei University Kyoung-Tak Kang (  tagi1024@gm Yonsei University Research article Keywords: finite-element method, unicompartmental knee arthroplasty, patellofemoral joint Posted Date: September 30th, 2020 DOI: https://doi.org/10.21203/rs.2.23811/v3 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 10th, 2020. See the published version at https://doi.org/10.1186/s13018-020-02047-0. Page 1/15 Abstract Background Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage, symptomatic unicompartmental osteoarthritis of the knee joint. However, patellofemoral joint degeneration is a contraindication to medial UKA. Therefore, the objective of this study was to evaluate the biomechanical effect of medial UKA using fixed-bearing (FB) and mobile-bearing (MB) design prostheses on the patellofemoral joint. Methods A three-dimensional finite-element model of a normal knee joint was developed using medical image data. We performed statistical analysis for each model. The differences in contact stress on the patellofemoral joint and the quadriceps force between the FB and MB designs were evaluated under a deep-knee-bend condition. Results At an early flexion angle, the results of contact stress showed no significant difference between the FB and MB medial UKA models and the intact model. However, at large flexion angles, we observed a significant increase in contact stress with the FB models. On the contrary, in the case of the MB models, we found no statistically significant increment compared with the intact model. Our results indicate that with medial UKA, the contact stress increased and greater quadriceps force was applied to the patellofemoral joint. However, performing UKA on a patellofemoral joint with osteoarthritis should not be difficult, unless anterior knee pain is present, because the increase in contact stress is negligible. Conclusions Our results show no significant difference in contact stress on the patellofemoral joint between the medial UKA and intact knee joint models. Such a mechanism was easily found in MB medial UKA. Therefore, this study biomechanically showed that degenerative changes in the patellofemoral joint should not be considered an absolute contraindication to treatment with medial UKA. Background Unicompartmental knee arthroplasty (UKA) is a surgical treatment alternative to total knee arthroplasty for isolated medial compartmental arthritis of the knee joint. The benefits of UKA include fewer complications, faster recovery, improved functional outcomes, and cost-effectiveness (1-4). Therefore, medial UKA had been increasingly used for the treatment of medial compartmental osteoarthritis (OA) over the past two decades (5). Historically, patellofemoral (PF) joint degeneration, and more specifically, advanced lateral PF joint facet degeneration, along with anterior knee pain, has been considered as an exclusion criterion for medial UKA (6, 7). However, PF joint degeneration has been reported recently to have no influence on the clinical outcomes after UKA (8, 9). In addition, whether preexisting PF joint degeneration is a contraindication to UKA is controversial. Thein et al. recently performed a study to determine the effect of medial fixed-bearing (FB) UKA on postoperative PF joint congruence and analyzed the effect of preoperative PF joint degeneration on the clinical outcome (10). No correlation was observed between preoperative PF joint congruence or degeneration severity and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores at 2-year follow-up (10). Preoperative PF Page 2/15 Page 2/15 joint congruence and degenerative changes do not affect the clinical outcomes after UKA (10). However, multiple studies that used the Oxford knee system indicated that neither preoperative anterior knee pain nor moderate radiological PF osteoarthritic changes affected the long-term clinical outcomes and survivorship of patients after mobile-bearing (MB) UKA (9, 11, 12). One study suggested that MB UKA provides better restoration of normal knee kinematics, which theoretically translates to better patellar tracking and long-term outcomes (13). Although several studies have revealed no significant differences in clinical outcomes and complication rates between the FB and MB UKA designs, the mode of failure often differs (14). In addition, research on the biomechanical effect of medial UKA on the PF joint is lacking. The biomechanical effect on the PF joint can be investigated by performing a finite-element (FE) analysis to evaluate the contact stress and quadriceps force after medial UKA (15). Accurate in silico evaluations of knee joint replacements are useful for clinical assessment (15). Therefore, the objective of this study was to evaluate the biomechanical effects of medial UKA using FB and MB design prostheses on the PF joint. Background The differences in contact stress on the PF joint and quadriceps force between the FB and MB designs were evaluated under a deep-knee-bend condition. We hypothesized that applying medial UKA would not be difficult even with OA of the PF joint (unless accompanied by anterior knee pain) because the differences in biomechanical effect on the PF joint are negligible between the UKA and normal knee joint models. Normal knee joint model In this study, an existing three-dimensional nonlinear FE model of the knee joint based on data from four male subjects (Subject 1: age, 36 years; height, 178 cm; mass, 75 kg; Subject 2: age, 34 years; height, 173 cm; mass, 83 kg; Subject 3: age, 32 years; height, 182 cm; mass, 79 kg; Subject 4: age, 34 years; height, 173 cm; mass, 71 kg) and one female subject (Subject 5: age, 26 years; height, 163 cm; mass, 65 kg) was used. The FE model was developed using computed tomography and magnetic resonance imaging data with a slice thikness of 0.1mm and 0.4mm slice, respectively (16, 17) and included the bony structures of the knee joint and the soft tissues of the PF and tibiofemoral (TF) joint anatomies. The articular cartilage and menisci were defined as isotropic linearly elastic materials and transversely isotropic and linearly elastic materials, respectively (18). The material properties of the articular cartilage and menisci are presented in Table 1. Table 1. Material properties of the articular cartilage and menisci. Cartilage Linearly elastic, isotropic E = 15 MPa v = 0.475 Menisci Linearly elastic, transversely isotropic Eθ = 150 MPa, Er = Ez = 20 MPa vrz = 0.2, vrθ = vzθ = 0.3, Grθ = Gzθ = 57.7 MPa ble 1. Material properties of the articular cartilage and menisci. able 1. Material properties of the articular cartilage and menisci. Page 3/15 Page 3/15 All the ligaments were modeled with nonlinear and tension-only spring elements (19, 20). Mesh convergence tests were performed to complete the simulation. Convergence was obtained if the relative change between two adjacent meshes was < 5%. The average element sizes were 0.8 mm for the cartilage and menisci, respectively. The details of the meshes used in the FE model were described in table 2. The interfaces between the cartilage and bones were modeled to be fully bonded. Contact was applied between the femoral cartilage and meniscus, meniscus and tibial cartilage, and femoral cartilage and tibial cartilage for both the medial and lateral sides (16). Table 2. Details of meshes used in the FE model. Table 2. Details of meshes used in the FE model. Table 2. Details of meshes used in the FE model. Normal knee joint model Set Nomal knee FB UKA MB UKA Femur bone (Quad) 18,817 17,948 17,899 Tibia bone (Quad) 13,286 12,303 12,289 Fibula bone (Quad) 5,456 5,456 5,456 Patella bone (Hexa) 1,411 1,411 1,411 Femur cartilage (Hexa) 9,629 4,586 4,586 Meniscus (Hexa) 2,978 1,475 1,475 Femoral componennt (Hexa) - 7,335 7,523 PE insert (Hexa) - 2,872 3,125 Tibia tray (Hexa) - 2,421 3,094 Total 51,577 55,807 56,858 Set Nomal knee FB UKA MB UKA Table 3. Material properties of implant. Medial UKA model A FB UKA prosthesis (Zimmer, Inc., Warsaw, IN, USA) and a MB UKA prosthesis of the Oxford knee system (Biomet, Warsaw, IN) were virtually implanted in the medial compartment of the normal knee joint model. (Fig. 1) The bone models were imported and appropriately positioned, trimmed, and meshed with rigid elements according to the surgical techniques (16). The tibial component was defined as a square (0°) inclination in the coronal plane, with a 5° posterior slope. The rotating axis was defined as a line parallel to the lateral edge of the tibial component passing through the center of the femoral component peg. A femoral component distal cut perpendicular to the mechanical axis of the femur and parallel to the tibial cut was reproduced. The height of the PE insert was identical to the anatomy in a sagittal plane aligned with the mechanical axis of the tibia and positioned at the medial edge of the tibia. The materials used for the femoral component, tibial insert, tibial baseplate, and bone cement were cobalt chromium molybdenum alloy, ultrahigh-molecular-weight polyethylene, titanium alloy, and polymethyl methacrylate, respectively (Table 3) (17, 21, 22). Table 3. Material properties of implant. Table 3. Material properties of implant. Page 4/15 Young’s modulus (MPa) Poisson’s ratio CoCr alloy 220,000 0.30 UHMWPE 685 0.47 Ti6AI4V alloy 110,000 0.30 PMMA 1,940 0.4 Young’s modulus (MPa) Poisson’s ratio The femoral component and tibial insert were in contact with a coefficient of friction of 0.04 (21). The FE simulation involved two types of loading conditions corresponding to the loads used in the model validation experiment and to predictions of loading scenarios in daily activities. An axial loading of 1150 N was applied to the model to evaluate the contact stresses and compare them with those reported in previous studies (23). (Fig. 2) The second loading condition corresponded to a deep knee bend, and squat loading was applied to evaluate the knee joint mechanics. A computational analysis was performed using an anteroposterior force applied to the femur that was based on the compressive load applied to the hip with constrained femoral internal-external (IE) rotation, free medial-lateral translation, and knee flexion, for a combination of vertical hip and quadriceps loads. Therefore, a 6-degrees-of-freedom (df) TF joint was developed (24, 25). A proportional-integral-derivative controller was incorporated into the computational model to control the quadriceps in a manner similar to that in previous experiments (26). Medial UKA model A control system was used to calculate the instantaneous displacement of the quadricep muscles to match the target flexion profile used in the experiment. Furthermore, IE and varus-valgus torques were applied to the tibia, while the remaining tibial df were constrained (24, 25). The FE models were analyzed using Abaqus version 6.11 software (Simulia, Providence, RI, USA). The contact stress and quadriceps force on the PF joint were evaluated for the FB and MB medial UKA designs. Statistical analyses We performed the test at 11 time points (0.0 to 1.0 phases) for single cycles of deep-knee-bend loading conditions. To assess the two models, FB and MB, the condition of each model was compared with that of the normal knee in a pairwise manner by using non-parametric repeated-measures Friedman tests at each phase of the cycle. In this study, we used Wilcoxon rank test with Holm correction for post hoc comparisons to control the familywise error rate for the tests conducted within each phase of the cycle. Statistical analyses were performed using SPSS version 20.0.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical significance was set at P < .05. Results The results of the five subject-specific FE models were compared with previous results of the same model for model validation (23). The mean contact stresses on the medial and lateral menisci in the present and previous studies are presented in Table 4. Page 5/15 Table 4. Comparison of the average contact stresses on the menisci for the validation of the model under an axial loading condition. Previous study [23] Present study Standard deviation Medial meniscus (MPa) 2.9 3.1 0.5 Lateral meniscus (MPa) 1.4 1.5 0.4 The minor differences may be due to the variations in geometry, such as the thicknesses of the cartilage and meniscus, between the studies. However, the consistency between the results confirms the ability of the FE model to produce reasonable results (23). Figure 3 shows the contact stresses on the PF joint with the FB and MB medial UKA designs under the deep-knee-bend condition. No significant difference in contact stress on the PF joint was observed between the FB and MB medial UKA models and the intact model at an early flexion angle. At a larger flexion angle, the contact stress showed a significant increase of 7% (on average), which is a small but significant increase, for the FB model compared with the intact model. For the MB models, we observed an increase of 4% (on average) in contact stress. However, we found no statistically significant increment. The quadriceps forces exerted on the PF joint with the FB and MB medial UKA designs under the deep- knee-bend condition are shown in Figure 4. A larger quadriceps force was needed to produce an identical flexion angle for both the FB and MB UKA designs than for the intact model. The quadriceps force rapidly increased the flexion of the knee joint in all the models. On average, the maximum quadriceps force significantly ranged from 2710 N for the MB UKA design to 2830 N for the FB UKA design. At a mid- flexion angle, the quadriceps forces were smaller for the FB and MB UKA designs than for the intact model. In addition, a lower quadriceps force was needed to produce identical flexion angles with the MB UKA design than with the FB UKA design. The FB and MB UKA designs required 12% and 8% (on average) more quadriceps force, respectively, than the intact model. Discussion The most important finding of this study was that the contact stress on the PF joint increased less with the MB UKA design than with the FB UKA design; however, no significant difference in contact stress on the PF joint was found between the medial UKA and intact knee joints. The quadriceps force needed to produce the same flexion angle with the MB UKA design was lower than that with the FB UKA design. UKA can be performed with either an FB or MB design. In a prospective study that involved 48 patients, who were randomly assigned to either FB or MB UKA prostheses, Li et al. observed better knee kinematics and a lower incidence of radiolucency with the MB design, but the Knee Society, WOMAC, and SF-36 scores were equivalent between the two designs (27). In another study, the range of motion, limb alignment, patient-reported outcomes, incidence of aseptic loosening, and reoperation rate were identical between the FB and MB UKA designs (28). However, the time to reoperation and failure mode differed. Early failure due to bearing dislocation occurred with the MB design, whereas late failure due to polyethylene wear Page 6/15 Page 6/15 occurred with the FB design. A previous study indicated that during a ≥15-year follow-up period, some type of revision arthroplasty was required for 12 (15%) of 77 knees in the case of FB UKA (Miller-Galante; Zimmer) and for 10 (12%) of 79 knees in the case of MB UKA (Oxford; Biomet) (29). No significant differences were observed in the number of knees with progressive lateral OA that required revision arthroplasty between the FB and MB UKA designs [33]. Thus, many arguments have emerged regarding the biomechanical issues of the FB and MB UKA designs. In previous studies on the progression of OA after UKA, the radiological assessment was neither blinded nor randomized (27). The advantage of FE analysis is that the impact of the UKA design can be determined without external variables [34]. Most in vitro biomechanical studies have involved evaluations using aged cadaveric subjects with loosening between the specimen and the device, and tissue attenuation, which can occur owing to the successive loading in mechanical testing (26). An intact joint model was the foundation of this study and involved FEM validation steps. The results exhibited good agreement with those of previous computational studies (23, 30). Discussion In addition, the MB UKA design requires a lower quadriceps force and makes it easier for recipients to kneel, squat, or rise from a chair. FB UKA design. In addition, the MB UKA design requires a lower quadriceps force and makes it easier for recipients to kneel, squat, or rise from a chair. The three strengths of our study should be highlighted. First, a well-validated setup that accounted for numerous previous results was used. Second, in contrast to previous UKA studies, the present study included the tibia, femur, and related soft tissues in the FE model. Third, in contrast to the current biomechanical UKA models, the model used in this study included the deep-knee-bend and squat loading conditions, rather than the simple vertical static loading condition. Despite these strengths, this study had certain limitations. First, the results did not predict clinical results or patient satisfaction. Second, the computational model was developed using data from four male subjects and one female subject. Using data from subjects of various ages would improve the validity of the results, as it would increase the diversity of the knee joint geometry. However, in this study, our objective was to evaluate the biomechanical effect of UKA in young individuals. Third, the bony structures were assumed to be rigid. In reality, the bone is composed of cortical and cancellous tissues. However, the main purpose of the study was not to evaluate the effects of different prostheses on bone. In addition, this assumption had a minimal influence on the results of the study because the bone stiffness exceeds that of the relevant soft tissues (23). Finally, the simulation only involved a deep knee bend; thus, simulations involving rising from or sitting on chairs, climbing/descending stairs, and squatting should be performed in future investigations. In conclusion, this study provides biomechanical evidence that degenerative changes in the PF joint should not be considered an absolute contraindication to treatment with medial UKA. In addition, UKA is not problematic even in PF joints with OA, unless accompanied by anterior knee pain, because no significant difference in contact stress was observed. List Of Abbreviations UKA: Unicompartmental knee arthroplasty; OA: Osteoarthritis; PF: Patellofemoral; FB: Fixed-bearing; MB: Mobile-bearing; TF: Tibiofemoral; IE: Internal-external; df: Degree of freedom. Ethics approval and consent to participate Approval was not required, as neither human participants nor animals were involved in this study. Consent for publication Approval was not required, as neither human participants nor animals were involved in this study. Discussion Therefore, the UKA models used in the present study and related analyses are considered reliable. Kozinn and Scott proposed that UKA should not be offered to patients with PF joint arthritis for optimal results (31). This sparked a contentious debate on PF joint disease because other authors demonstrated only a weak correlation between PF degenerative changes and anterior knee pain (11, 32). In addition, owing to the differences in the design and biomechanics of the FB UKA model, damage to the PF joint has traditionally been a contraindication. Lim et al. recently showed that the presence of significant preoperative radiological PF disease does not affect long-term implant success, and patients had excellent postoperative functional outcomes for 10 years (33). In the present study, the MB UKA design produced a smaller increase in contact stress on the PF joint than the intact joint and FB UKA design models. The results of previous studies on MB UKA indicated that the presence of PF degeneration does not compromise clinical outcomes because the implant is believed to be more patella friendly owing to better kinematics, which supports our results (33, 34). Moreover, although the contact stress on the PF joint increased with both the FB and MB UKA designs, the increase was not statistically significant. Biomechanical studies have indicated that the progression of arthritis of the PF joint typically does not necessitate revision. The quadriceps force needed to produce a squatting motion was greater for the FB design than for the MB design (by as much as 120 N for knee flexion angles >100°). Thus, increased quadriceps strength leads to improved functional performance (35). As patients who have undergone OA and knee arthroplasty experience significant quadriceps weakness, the FB UKA design, which increases the required quadriceps force, can result in more difficulty for patients to walk, kneel, or perform a deep knee bend (36). This agrees with the results of a previous in vitro study in which a UKA model required less quadriceps force at a mid-flexion angle than an intact model (27). Page 7/15 From a biomechanical viewpoint, our results indicate that the risk of progressive OA of the PF joint can be reduced with the MB UKA design because it preserves the normal biomechanical effect, in contrast to the Page 7/15 FB UKA design. Declarations Ethics approval and consent to participate Consent for publication Not applicable. Availability of data and materials Page 8/15 Page 8/15 Page 8/15 Not applicable. Funding There is no funding source. Competing interests The authors declare that they have no competing interests. Authors’ contributions HMK designed the study and drafted the paper; JAL developed the three-dimensional model; YGK evaluated the FEA results; KKP validated the data; and KTK supervised the study and analyzed the data. Competing interests The authors declare that they have no competing interests. References 1. Siman H, Kamath AF, Carrillo N, Harmsen WS, Pagnano MW, Sierra RJ. Unicompartmental Knee Arthroplasty vs Total Knee Arthroplasty for Medial Compartment Arthritis in Patients Older Than 75 Years: Comparable Reoperation, Revision, and Complication Rates. Journal of Arthroplasty. 2017;32(6):1792-7. 1. Siman H, Kamath AF, Carrillo N, Harmsen WS, Pagnano MW, Sierra RJ. Unicompartmental Knee Arthroplasty vs Total Knee Arthroplasty for Medial Compartment Arthritis in Patients Older Than 75 Years: Comparable Reoperation, Revision, and Complication Rates. Journal of Arthroplasty. 2017;32(6):1792-7. 2. Liddle AD, Judge A, Pandit H, Murray DW. 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The Journal of bone and joint surgery American volume. 1989;71(1):145-50. 32. Song EK, Park JK, Park CH, Kim MC, Agrawal PR, Seon JK. No difference in anterior knee pain after medial unicompartmental knee arthroplasty in patients with or without patellofemoral osteoarthritis. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA. 2016;24(1):208-13. 33. Lim JW, Chen JY, Chong HC, Pang HN, Tay DKJ, Chia SL, et al. Pre-existing patellofemoral disease does not affect 10-year survivorship in fixed bearing unicompartmental knee arthroplasty. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA. 2019;27(6):2030-6. Page 11/15 Page 11/15 34. Argenson JN, O'Connor JJ. Polyethylene wear in meniscal knee replacement. A one to nine-year retrieval analysis of the Oxford knee. The Journal of bone and joint surgery British volume. 1992;74(2):228-32. 35. Hurley MV, Scott DL. Improvements in quadriceps sensorimotor function and disability of patients with knee osteoarthritis following a clinically practicable exercise regime. British journal of rheumatology. 1998;37(11):1181-7. 35. Hurley MV, Scott DL. Improvements in quadriceps sensorimotor function and disability of patients with knee osteoarthritis following a clinically practicable exercise regime. British journal of rheumatology. 1998;37(11):1181-7. 36. Mizner RL, Petterson SC, Stevens JE, Vandenborne K, Snyder-Mackler L. Early quadriceps strength loss after total knee arthroplasty. Figures Figure 1 Figure 1 Figure 1 FE model used in this study for (a) UKA model, (b) FB UKA componenet design and (c) MB UKA componenet design. FE model used in this study for (a) UKA model, (b) FB UKA componenet design and (c) MB UKA componenet design. FE model used in this study for (a) UKA model, (b) FB UKA componenet design and (c) MB UKA componenet design. FE model used in this study for (a) UKA model, (b) FB UKA componenet design and (c) MB UKA componenet design. Page 12/15 Page 12/15 ure 2 Figure 2 Loading condition of the UKA FE model Figure 2 Loading condition of the UKA FE model Loading condition of the UKA FE model Page 13/15 Differences in contact stress on the PF joint with intact, FB and MB design medial UKA under the deep- knee-bend condition (p<.05). Differences in the quadriceps muscle force for intact, FB and MB tibial insert materials under the deep- knee-bend condition (p<.05). Figure 3 Differences in contact stress on the PF joint with intact, FB and MB design medial UKA under the deep- knee-bend condition (p<.05). Differences in contact stress on the PF joint with intact, FB and MB design medial UKA under the deep- knee-bend condition (p<.05). Page 14/15 Page 14/15 Figure 4 Differences in the quadriceps muscle force for intact, FB and MB tibial insert materials under the deep- Figure 4 Figure 4 Differences in the quadriceps muscle force for intact, FB and MB tibial insert materials under the deep- knee-bend condition (p<.05). Differences in the quadriceps muscle force for intact, FB and MB tibial insert materials under the deep- knee-bend condition (p<.05). Page 15/15 Page 15/15
https://openalex.org/W2285538757	https://europepmc.org/articles/pmc4739454?pdf=render	English	null	New Horizons in Diabetology	Journal of diabetes research	2,016	cc-by	1,131	Correspondence should be addressed to Bagher Larijani; emrc@sina.tums.ac.ir Received 18 June 2015; Accepted 18 June 2015 Received 18 June 2015; Accepted 18 June 2015 Copyright © 2016 Ali Tootee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The prevalence of diabetes has alarmingly increased in both developed and developing countries all across the world in the recent years. The prevalence of different complications and comorbid conditions associated with diabetes is also rampantly increasing, thereby negatively affecting lives of many people. In fact, obesity which is associated with diabetes is considered as a major public health concern in many countries. Nevertheless, it can be argued that still more progress is to be made in different fields of prevention, diagnosis, and treatment of diabetes. This gap in the science of Diabetology, arguably, stems from insufficient synchronized interdisciplinary basic and clinical research in the field. This deficiency may be addressed through finding the gaps in the published literature by means of plotting different diabetes maps and focusing research on them to promote the concept of translational medicine. of the RAS can modestly influence the risk of type 2 diabetes. P. Haghvirdizadeh et al., in a different study, reported that R219K polymorphism of ABCA1 gene could be considered as a genetic risk factor for type 2 diabetes among certain populations. In another article, M. Khodaeian et al. assert that genome wide association studies on large number of samples can be helpful in the identification of diabetes susceptible genes and may be used as an alternative to studying individual candidate genes which is a time-consuming and expensive method. In a different article, P. Haghvirdizadeh et al. findings provide evidence for the contribution of common KCNJ11 genetic variants to the development of DM. In regard to the underlying mechanisms of diabetes and its complications, M. K´apl´ar et al., in an original research arti- cle, demonstrated a dual role for mannose binding lecithin as a risk factor for carotid intima media thickness in patients with type 2 diabetes mellitus. N. Bertoncello et al., in another article, shed some light on the complex underlying mecha- nisms involved in the development of diabetic nephropathy. Hindawi Publishing Corporation Journal of Diabetes Research Volume 2016, Article ID 9075924, 2 pages http://dx.doi.org/10.1155/2016/9075924 Hindawi Publishing Corporation Journal of Diabetes Research Volume 2016, Article ID 9075924, 2 pages http://dx.doi.org/10.1155/2016/9075924 Hindawi Publishing Corporation Journal of Diabetes Research Volume 2016, Article ID 9075924, 2 pages http://dx.doi.org/10.1155/2016/9075924 Editorial New Horizons in Diabetology Ali Tootee,1 Garth Warnock,2 Aziz Ghahari,3 and Bagher Larijani4 1Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Scien Iran 2University of British Columbia, Vancouver, Canada 3Vancouver General Hospital, Vancouver, Canada 4Endocrinology and Metabolism Research Center (EMRC), Tehran University Medical Sciences, Tehran, Iran Correspondence should be addressed to Bagher Larijani; emrc@sina.tums.ac.ir Received 18 June 2015; Accepted 18 June 2015 Copyright © 2016 Ali Tootee et al. This is an open access article distributed under the Creative Commons Attributi which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly Ali Tootee,1 Garth Warnock,2 Aziz Ghahari,3 and Bagher Larijani4 Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medica Iran 2University of British Columbia, Vancouver, Canada 3Vancouver General Hospital, Vancouver, Canada 4Endocrinology and Metabolism Research Center (EMRC), Tehran University Medical Sciences, Tehran, Iran Correspondence should be addressed to Bagher Larijani; emrc@sina.tums.ac.ir Correspondence should be addressed to Bagher Larijani; emrc@sina.tums.ac.ir Lack of comprehensive diabetes research maps and the scarcity of the literature in the related interdisciplinary fields can be considered as major hindrance to the future advancement of the science of Diabetology. With this view, we conceived the concept of publication of a special issue on the subject and invited authors to submit any original research or review paper with the potential of opening new horizons to prevention, screening, and treatment of diabetes. Out of approximately 50 manuscripts submitted, in this special issue, 15 articles have been approved by reviewers to be published on a wide range of relevant basic and clinical subjects.ii As for novel diagnostic, preventive, and therapeutic options for diabetes, T. Mizushige showed that urinary angiotensinogen could be a prognostic marker of renopro- tective effects of alogliptin in patients with type 2 diabetes. Findings of a study by R. D´ıez-L´aiz demonstrate significant therapeutic potentials for Plantago ovata husk as an oral antihyperglycemic agent for treatment of type 2 diabetes. An article by M. S. Klein and J. Shearer argues that metabolomics has the potential to enable informed decision-making in the realm of personalized medicine. S. J. Hashemian et al., in In the field of screening for diabetes, findings of a study by S. M. Joyce-Tan et al. demonstrated that genetic variants Journal of Diabetes Research 2 a comprehensive review article, claim that despite all unre- solved concerns about clinical applications of mesenchymal stem cells, this group of stem cells still remains a promising therapeutic modality for treatment of diabetes. A study by K. Kurek et al. demonstrates that myriocin can find potential future application as a therapeutic agent for the reduction of insulin resistance and its serious consequences in obese patients. Findings of S. Wang et al. study show that certain renoprotective therapeutic agents which can specifically abol- ish CTGF CCN2 expression, or nonspecifically inhibit CTGF CCN2 expression, may be protective against the development and progression of diabetes nephropathy. P. Senesi et al., in their study, demonstrate that metformin treatment can prevent sedentariness related damage in mice. In this regard, in another study by M. Bo et al., it is asserted that an inappropriate and aggressive glucose lowering therapeutic approach in frail and vulnerable elderly residents of long- term care facilities can adversely affect their health. Correspondence should be addressed to Bagher Larijani; emrc@sina.tums.ac.ir f It can be concluded that this special issue provides a series of original and review articles on a wide range of diabetes- related subjects which can assist researchers to design and carry out research projects in similar directions in the future. Moreover, this issue may be helpful in spotting current gaps in the science of Diabetology and facilitate focusing more research on them. We hope that the present special issue provides useful information for a superior understanding of the underlying mechanisms involved in the pathogenesis of diabetes as well as novel diagnostic and therapeutic techniques for the diseases. Acknowledgments Finally, we would like to sincerely acknowledge the efforts of the reviewers who carried out the task of critical appraisal of the articles and we sincerely wish that our readers enjoy the issue and find its content informative and intellectually motivating for similar research in the future. Ali Tootee Garth Warnock Aziz Ghahari Bagher Larijani
https://openalex.org/W2011619479	https://www.scielo.br/j/ramb/a/sQfK9Bzp4Xczcjmb9tFJH9M/?lang=pt&format=pdf	Portuguese	null	Indicaria operação com base apenas em exame genético?	Revista da Associação Médica Brasileira	2,002	cc-by	3,543	VICENTE RENATO BAGNOLI ANGELA MAGGIO DA FONSECA PAULO AUGUSTO DE ALMEIDA JUNQUEIRA WILSON MAÇA YUKI ARIÊ VICENTE RENATO BAGNOLI ANGELA MAGGIO DA FONSECA PAULO AUGUSTO DE ALMEIDA JUNQUEIRA WILSON MAÇA YUKI ARIÊ No climatério, principalmente na pós- menopausa, são freqüentes as queixas envol- vendo os aparelhos genital e urinário, tais como: corrimento, prurido, sangramento, dispareunia, incontinência urinária, tenesmo vesical, infecções urinárias de repetição, pois o hipoestrogenismo desencadeia mudanças significativas no ambiente vaginal e nos órgãos gênito-urinários, gerando dúvidas e nem sempre conduzidas de forma satisfatória. Existem diferenças nas mulheres submetidas à reposição hormonal (TRH) e naquelas não medicadas. Assim, nas não usuárias de hor- mônios, as queixas não infecciosas represen- tam cerca de 60% dos casos, decorrentes da atrofia urogenital e os demais casos por vaginose bacteriana, vaginite por trichomo- nas, infecções urinárias e outras infecções menos comuns. Por outro lado, as usuárias de TRH costumam apresentar melhor tro- fismo gênitourinário, maior incidência de vaginite fúngica ,vaginite mista, e maior inci- dência de outras infecções devido as caracte- rísticas do ambiente vaginal e das demais estruturas conferidas pela TRH. Deve-se ain- da salientar que a Síndrome da Imuno- deficiência Adquirida (AIDS), tem apresenta- do aumento significativo nesta faixa etária. Por estas razões, nas mulheres climatéricas, com queixas urogenitais, deve-se sempre considerar na etapa do diagnóstico, o uso ou não de TRH, pesquisando-se o meio vaginal pelo estudo bacterioscópico, em geral do tipo III nos casos de atrofia acentuada, II na hipotrofia, que são os achados mais comuns em não usuárias de TRH ou quando a mesma Referências 1. Bagnoli VR, Fonseca AM, Ariê WMY. Vulvovaginites recorrentes. Revisão dos as- pectos clínicos relevantes. Rev Ginecol Obstet 2001; 12(1):40-4. 2. Sobel, JD. Vaginitis. N Engl J Med 1997; 337(26):1896-901. Referências Referências 1. Bagnoli VR, Fonseca AM, Ariê WMY. Vulvovaginites recorrentes. Revisão dos as- pectos clínicos relevantes. Rev Ginecol Obstet 2001; 12(1):40-4. 2. Sobel, JD. Vaginitis. N Engl J Med 1997; 337(26):1896-901. O tratamento é etiológico, com os pro- dutos farmacológicos empregados de acor- do com o agente infeccioso. Contudo, al- guns aspectos devem ser ressaltados: quan- do as manifestações decorrerem apenas do hipoestrogenismo, basta a TRH tópica, que oferece excelentes resultados, ou se hou- ver indicação para TRH sistêmica, aplicá-la. Quando o hipoetrogenismo estiver interfe- rindo de forma desfavorável e facilitando as infecções genitais, a TRH como coadjuvan- te do tratamento etiológico, acelera o pro- cesso de cura e reduz os índices de recidiva. VICENTE RENATO BAGNOLI ANGELA MAGGIO DA FONSECA PAULO AUGUSTO DE ALMEIDA JUNQUEIRA WILSON MAÇA YUKI ARIÊ Entretanto, a TRH pode promover o de- senvolvimento de vulvovaginite fúngica por vezes rebelde ao tratamento. 3. Spinillo A, Bernuzzi AM, Cevini C, Gul- minetti R, Luzi S, De Santolo A, et al. The relationship of bacterial vaginosis, candida and trichomonas infection to symptomatic vaginitis in postmenopausal women attending a vaginitis clinic. Maturitas 1997; 27:253-60. Medicina Baseada em Evidências Àbeira do leito não está sendo efetiva, e tipo I quando a TRH for efetiva. Torna-se imperioso nesta avalia- ção a pesquisa dos agentes mais comuns de infecções genitais, pois ao contrário do que se acreditava, também nesta faixa etária estas patologias como as vulvovaginites existem em freqüência significativa, e embora menos freqüente também infecções pelo Papiloma Vírus Humano(HPV), cervicites e doença in- flamatória pélvica, quadros estes nem sem- pre cogitados. O diagnóstico inclui sempre bacterioscopia, colposcopia, vulvoscopia, colpocitologia oncótica e quando necessário cultura e antibiograma de urina e secreção vaginal, à semelhança do roteiro empregado para mulheres no menacme. despercebidas, e utilizar de forma adequada os hormônios sexuais, que conferem resul- tados excelentes no controle das queixas urogenitais que atrapalham significativa- mente o bem-estar da mulher. Rev Assoc Med Bras 2002; 48(2): 93-117 Àbeira do leito Àbeira do leito Referências 1. Nienaber CA, Fattori R, Lund G, Dieckmann C, Wolf W, Von Kodolitsch Y, et al. Nonsurgical reconstruction of thoracic aortic dissection by stent-graft placement. N Engl J Med 1999; 340:1539-45. 1. Nienaber CA, Fattori R, Lund G, Dieckmann C, Wolf W, Von Kodolitsch Y, et al. Nonsurgical reconstruction of thoracic aortic dissection by stent-graft placement. N Engl J Med 1999; 340:1539-45. O desenvolvimento das endopróteses revestidas tem mudado os critérios de indi- cação do tratamento da dissecção tipo B. A experiência recém adquirida com esta nova modalidade terapêutica, endovascular, mostra ser este procedimento seguro, efi- caz e de execução muito mais simples do que o tratamento convencional. Como conseqüência, o espectro de sua indicação para os pacientes com dissecção aórtica tipo B tem sido ampliado. Esta indicação deve ser fundamentada nos seguintes critérios clínicos e anatômicos principais. 2. Dake MD, Kato N, Mitchell RS, Semba CP, Razavi MK, Shimono T, et al. Endo- vascular stent-graft placement for the treat- ment of acute aortic dissection. N Engl J Med 1999; 340:1546-52. 3. Palma JH, Souza JAM, Alves CMR, Car- valho AC, Buffolo E. Self-expandable aortic stent-grafts for treatment of descending aortic dissections Ann Thorac Surg 2002; 73:1138-41. 3. Palma JH, Souza JAM, Alves CMR, Car- valho AC, Buffolo E. Self-expandable aortic stent-grafts for treatment of descending aortic dissections Ann Thorac Surg 2002; 73:1138-41. Critérios Clínicos: dor recorrente; si- nais ou sintomas sugestivos de rotura emi- nente; presença de fluxo pela luz falsa e/ou diâmetro aórtico maior que 40 mm, especi- almente se houver fluxo pela luz falsa. Estes dois últimos, quando presentes, são predi- tores de desenvolvimento de aneurismas toracoabdominais, de difícil correção cirúr- gica no seguimento tardio. 4. Alves CMR, Palma JH, Souza JAM, Carva- lho ACC, Buffolo E. Endovascular treat- ment of thoracic disease: patient selection and a proposal of a risk score. Ann Thorac Surg 2002; 73:1143-8. 4. Alves CMR, Palma JH, Souza JAM, Carva- lho ACC, Buffolo E. Endovascular treat- ment of thoracic disease: patient selection and a proposal of a risk score. Ann Thorac Surg 2002; 73:1143-8. Àbeira do leito Àbeira do leito dor persistente ou sinais de rotura. O trata- mento cirúrgico convencional da dissecção tipo B cursa com altas taxas de mortalidade e morbidade decorrentes da toracotomia, co- lapso do pulmão, perdas sangüíneas substan- ciais durante a abertura da aorta, dificuldades técnicas relacionadas à sutura de uma parede aórtica friável e, principalmente, das altera- ções hemodinâmicas e teciduais induzidas pela isquemia e reperfusão de todos os órgãos intra-abdominais, medula espinhal e extremidades inferiores. Deve ser ressaltado que, em condi- ções especiais, pode-se ocluir a origem da artéria subclávia esquerda. Ao contrário do tratamento cirúrgico clássico, a ocor- rência de paraplegia é rara com o trata- mento endovascular. para tornar-se um parâmetro de avaliação importante durante a ultra-sonografia obs- tétrica de rotina ou morfológica no pré- natal. Vários foram os motivos que leva- ram a um incremento na importância do método, entre eles: a evolução tecno- lógica dos aparelhos de ultra-sonografia com desenvolvimento de sondas trans- vaginais e a maior familiaridade dos médi- cos e pacientes com o exame, diminuindo o preconceito e os tabus em relação ao uso dessas sondas durante a gestação em relação a abortamentos e sangramentos. O surgimento de diversos estudos relacio- nando o comprimento do colo uterino na ultra-sonografia transvaginal durante a ges- tação com a predição do parto prematuro, também contribuiu para universalização do método. O parto prematuro é a princi- pal causa de morbimortalidade pós-natal. A taxa de partos prematuros espontâneos tem se mantido estável ao longo das três últimas décadas, variando de 5,9% a 10,7%. A dificuldade na identificação de gestantes com risco elevado para o parto prematuro e a falta de um método tera- pêutico realmente eficaz para sua inibição são os dois principais motivos para as altas taxas de prematuridade. A ultra-sonografia transvaginal surgiu, então, como o melhor e mais fácil método para rastreamento do parto prematuro grave (aquele com idade gestacional inferior a 33 semanas). O mé- todo pode ser aplicado na população de alto ou baixo - risco, sintomáticas ou assintomáticas. A melhor época para reali- zação do exame é de 22 a 24 semanas de gestação, que também hoje é considerada a melhor época para realização da ultra- sonografia morfológica fetal, uma vez que tem uma melhor sensibilidade na detecção de malformações fetais de aparecimento mais tardio e maior facilidade na visibi- lização de várias estruturas fetais tal como QQQQQUANDO UANDO UANDO UANDO UANDO INDICAR INDICAR INDICAR INDICAR INDICAR OOOOO TRA TRA TRA TRA TRATTTTTAMENT AMENT AMENT AMENT AMENTOOOOO ENDOV ENDOV ENDOV ENDOV ENDOVASCULAR ASCULAR ASCULAR ASCULAR ASCULAR NA NA NA NA NA DISSECÇÃO DISSECÇÃO DISSECÇÃO DISSECÇÃO DISSECÇÃO AÓRTICA AÓRTICA AÓRTICA AÓRTICA AÓRTICA TIPO TIPO TIPO TIPO TIPO B ? B ? B ? B ? B ? Pode-se concluir que a menopausa é um período com características especiais e que, portanto, merece atenção especial, para permitir que a mulher que atualmente vive grande parte de sua vida neste período, sinta-se bem e protegida, bastando que valorizemos entre as queixas também as urogenitais, e as tratemos corretamente. Deve-se enfatizar a necessidade de excluir a presença de neoplasias dos órgãos ge- nitais e urinários, que muitas vezes passam A dissecção aórtica é a patologia de maior mortalidade entre as que se manifestam por dor torácica. Classicamente, a dissecção tipo A (quando há o envolvimento da aorta ascen- dente) é tratada cirurgicamente, enquanto que na dissecção tipo B (quando a aorta ascendente não está comprometida) o trata- mento cirúrgico só está indicado quando há Rev Assoc Med Bras 2002; 48(2): 93-117 101 Obstetrícia Obstetrícia Bioética Bioética O O O O O QUE QUE QUE QUE QUE FFFFFAZER AZER AZER AZER AZER NA NA NA NA NA ÚLCERA ÚLCERA ÚLCERA ÚLCERA ÚLCERA IDIOPÁTICA IDIOPÁTICA IDIOPÁTICA IDIOPÁTICA IDIOPÁTICA ANAL ANAL ANAL ANAL ANAL NO NO NO NO NO DOENTE DOENTE DOENTE DOENTE DOENTE HIV HIV HIV HIV HIV POSITIVO POSITIVO POSITIVO POSITIVO POSITIVO????? A literatura preconiza (TT) até os 5 anos de idade, não sendo necessário esperar o surgi- mento de indicador clínico, radiológico ou laboratorial de doença ativa. O exame anato- mopatológico da peça revela apenas hiper- plasia de células C, ou mesmo não encontra doença. Também pode revelar o carcinoma ainda microscópico e passível de cura. Referência Homem com 35 anos, portador de car- cinoma medular de tireóide (CMT) tem uma filha que apresenta o RET protoon- cogene em uma amostra de sangue do cordão umbilical. Posso indicar a tireoidec- tomia total (TT) na criança? Freyer G, Dazord A, Schlumberger M, Conte-Devolx B, Ligneau B, Trillet-Lenoir V, et al. Psychosocial impact of genetic testing in familial medullary-thyroid carcino- ma: a multicentric pilot-evaluation. Ann Oncol 1999; 10:87-95. O CMT ocorre nas formas esporádica e familiar. Na forma familiar é descrito o RET protooncogene, que está relacionado ao desenvolvimento de doença clínica até os 13 anos de idade. Após instalação da doen- ça, a cura é mais difícil. A pesquisa do RET é indicada em todo recém-nascido filho de portador deste gene. Clínica Cirúrgica MÁRIO HENRIQUE BURLACCHINI DE CARVALHO A úlcera anal idiopática está associada aos estádios avançados da infecção pelo HIV, embora possa ser a primeira manifes- tação da AIDS. A anamnese revela dor anal incapacitante de forte intensidade, contí- nua, de longa duração e acompanhada com secreção purulenta avermelhada. O exame proctológico só é possível sob analgesia e revela úlcera profunda do canal anal, cober- ta com secreção purulenta vermelho-acas- tanhada. Diferente da fissura anal, é inva- siva, mais cranial no canal anal e ocorre mesmo com hipotonia esfincteriana. Loca- liza-se preferivelmente na parede posterior do canal anal, tem contorno irregular, com fundo necrótico e exsudativo, sendo sua base formada pelo esfincter anal interno e às vezes associada a abscesso interesfincte- riano crônico. Àbeira do leito Àbeira do leito o coração. Desta forma, é preconizada a realização da avaliação do comprimento do colo uterino no momento da ultra- sonografia morfológica. O parâmetro mais importante no exame é o comprimento do colo uterino, medido linearmente do seu orifício interno ao externo. Quanto menor o comprimento, maior o risco para o parto prematuro. O risco torna-se impor- tante quando o colo uterino está medindo menos que 2,0 cm de comprimento, sendo que este risco é muito elevado quando o colo uterino mede 1,5 cm ou menos. As gestantes com colo uterino curto devem ser orientadas quanto às medidas gerais para prevenção do parto prematuro (repouso, abstinência sexual, pesquisa e tratamento de infecção urinária). A circlagem do colo uterino também tem mostrado ser útil com redução de 50% na incidência de partos prematuros graves no grupo de gestantes com colo < 1,5cm. pesquisa bem conduzida, fundamentos da boa prática médica. O benefício trazi- do reforça e incentiva a pesquisa e sua aplicação prática. CCCCCOMO OMO OMO OMO OMO AAAAAVVVVVALIAR ALIAR ALIAR ALIAR ALIAR OOOOO COL COL COL COL COLOOOOO UTERINO UTERINO UTERINO UTERINO UTERINO NUMA NUMA NUMA NUMA NUMA UL UL UL UL ULTRA TRA TRA TRA TRA-----SONOGRAFIA SONOGRAFIA SONOGRAFIA SONOGRAFIA SONOGRAFIA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA????? Critérios anatômicos: colos de ater- risagem, proximal e distal ao orifício de en- trada, menor que 30-32 mm; o implante da endoprótese não pode comprometer a ori- gem de vasos importantes, incluindo os tron- cos supra-aórticos e os vasos esplâncnicos; o acesso arterial, especialmente do sistema ilía- co-femoral, deve ser compatível com o cali- bre do sistema de liberação da endoprótese de 20 French (6,3mm de diâmetro). CCCCCOMO OMO OMO OMO OMO AAAAAVVVVVALIAR ALIAR ALIAR ALIAR ALIAR OOOOO COL COL COL COL COLOOOOO UTERINO UTERINO UTERINO UTERINO UTERINO NUMA NUMA NUMA NUMA NUMA UL UL UL UL ULTRA TRA TRA TRA TRA-----SONOGRAFIA SONOGRAFIA SONOGRAFIA SONOGRAFIA SONOGRAFIA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA OBSTÉTRICA????? A avaliação do colo uterino, nos últi- mos anos, deixou de ser uma curiosidade 102 Rev Assoc Med Bras 2002; 48(2): 93-117 IIIIINDICARIA NDICARIA NDICARIA NDICARIA NDICARIA OPERAÇÃO OPERAÇÃO OPERAÇÃO OPERAÇÃO OPERAÇÃO COM COM COM COM COM BASE BASE BASE BASE BASE APENAS APENAS APENAS APENAS APENAS EM EM EM EM EM EXAME EXAME EXAME EXAME EXAME GENÉTICO GENÉTICO GENÉTICO GENÉTICO GENÉTICO????? MARCOS ROBERTO TAVARES SIDNEY ROBERTO NADAL CARMEN RUTH MANZIONE SIDNEY ROBERTO NADAL CARMEN RUTH MANZIONE Referências 1. Nadal SR, Manzione CR, Horta SHC, Galvão VM. Management of idiopathis ulcer of the anal canal by excision in HIV-positive patients. Dis Colon Rectum 1999; 42:1598-601. Referências 1. Nadal SR, Manzione CR, Horta SHC, Galvão VM. Management of idiopathis ulcer of the anal canal by excision in HIV-positive patients. Dis Colon Rectum 1999; 42:1598-601. 2. Schmidt SL, Wexner SD, Nogueras JJ, Jagel- man DG. Is aggresive management of perianal ulcers in homosexual HIV-positive men jus- tified? Dis Colon Rectum 1993; 36:240-6. 3. Wilcox CM, Schwartz DA. Idiopathis anorectal ulceration in patients with human immunodeficiency virus inection. Am J Gas- troenterol 1994; 89:599-604. Assim, a indicação da biópsia pulmonar a céu aberto deve ser feita para as pneu- mopatias difusas evoluindo com insuficiência respiratória, não-responsiva à terapêutica empírica inicial. Esta indicação não pode ser retardada na sua indicação, pois o desenvol- vimento de fibrose pulmonar é progressivo e além de determinada situação, inviabiliza uma melhor evolução da criança, mesmo com tratamento correto da etiologia primária. 2. Schmidt SL, Wexner SD, Nogueras JJ, Jagel- man DG. Is aggresive management of perianal ulcers in homosexual HIV-positive men jus- tified? Dis Colon Rectum 1993; 36:240-6. 3. Wilcox CM, Schwartz DA. Idiopathis anorectal ulceration in patients with human immunodeficiency virus inection. Am J Gas- troenterol 1994; 89:599-604. Essa estratégia elucida o diagnóstico e indica a mudança da terapêutica inicial em até 80% dos casos. É um procedimento seguro, com taxas de complicações baixas. A par do esclarecimento etiológico, os dados anatomopatológicos são importantes na formulação prognóstica do caso. Essa estratégia elucida o diagnóstico e indica a mudança da terapêutica inicial em até 80% dos casos. É um procedimento seguro, com taxas de complicações baixas. O uso de antimicrobianos e analgé- sicos, preconizado por alguns, não trata a doença. A aplicação de corticoide intra-lesional alivia os sintomas durante curto espaço de tempo. A opção cirúr- gica é defendida nos casos de falência da terapia clínica. Nossa proposta inicial tem sido o tratamento operatório, com o qual temos obtido sucesso. QQQQQUANDO UANDO UANDO UANDO UANDO INDICAR INDICAR INDICAR INDICAR INDICAR EEEEE QUAL QUAL QUAL QUAL QUAL OOOOO RESUL RESUL RESUL RESUL RESULTTTTTADO ADO ADO ADO ADO DDDDDAAAAA BIÓPSIA BIÓPSIA BIÓPSIA BIÓPSIA BIÓPSIA PULMONAR PULMONAR PULMONAR PULMONAR PULMONAR AAAAA CÉU CÉU CÉU CÉU CÉU ABERT ABERT ABERT ABERT ABERTOOOOO????? A par do esclarecimento etiológico, os dados anatomopatológicos são importantes na formulação prognóstica do caso. Portanto, a biópsia pulmonar a céu aberto é um procedimento que deve ser sempre con- siderado nas condições expostas de indicação. Realizamos ressecção mucocutânea em raquete, incluindo a úlcera, sob raquianestesia. Após este procedimento, notamos a presença de abscesso laminar que descola o plano intersfincteriano. Abrimos a parede retal longi- tudinalmente sobre o abscesso e fazemos a curagem do leito. Indicamos sutura contínua com catgut 000 cromado na borda da ferida operatória com finalidade hemostática. O do- ente é medicado com antimicrobiano, anal- ségico e banhos de assento. Há alívio dos sintomas no pós-operatório imediato. O exa- me anatomopatológico revela processo infla- matório inespecífico e na cultura crescem ger- Referências 1. Iams JD. Cervical ultrasonography. Ultra- sound Obstet Gynecol 1997; 10:156-60. 2. Heath VCF, Southall TR, Elisseou A, Nicolaides KH. Cervical length at 23 weeks of gestation: prediction of spontaneous preterm delivery. Ultrasound Obstet Gyne- col 1998; 12:312-17. Cabe aos pais autorizar a retirada da tireóide, obrigando seu filho a reposição hormonal por toda a vida, ou recusar e esperar o desenvolvimento da doença, que pode levar à morte por volta dos 30 anos de idade. 3. Iams JD, Goldenberg RL, Meis PJ, Mercer B, Moawad A, Das A et al. The length of the cervix and the risk of spontaneous premature delivery. N Engl J Med 1996; 334:567-72. 3. Iams JD, Goldenberg RL, Meis PJ, Mercer B, Moawad A, Das A et al. The length of the cervix and the risk of spontaneous premature delivery. N Engl J Med 1996; 334:567-72. 4. Heath VCF, Souka AP, Erasmus I, Gibb DMF, Nicolaides KH. Cervical length at 23 weeks of gestation: the value of Shirodkar suture for the short cervix. Ultrasound Obstet Gynecol 1998; 12:318-22. Trata-se de diagnóstico precoce basea- do no conhecimento e tecnologia, que per- mite ação prévia à doença. A consequente ablação de um órgão é procedimento radi- cal para tratamento preventivo. Todo procedimento pode ser aplica- do se validado pela prática clínica ou por 103 Rev Assoc Med Bras 2002; 48(2): 93-117 EVANDRO R. BALDACCI O diagnóstico etiológico nas pneumopatias agudas, difusas e graves é um desafio muito grande. O dilema do pediatra na busca da defi- nição etiológica passa por duas estratégias. A primeira diz respeito à utilização de técnicas não- invasivas (estudos de imagem, sorologias, cultu- ras de sangue e secreção pulmonar, PCR no sangue e secreções), que poderão dar no máxi- mo 50% de resposta e quase sempre muito demoradas, retardando uma terapêutica mais objetiva o que pode ser crítico para a sobrevida do paciente. Os casos graves exigem a par da introdução de uma terapêutica empírica inicial, Àbeira do leito Tanto sua etiologia, como o tratamento apropriado, necessitam ser determinados. As culturas e biópsias são habitualmente conclusi- vas. É possível que os vírus presentes em grande número nos homossexuais portadores de HIV não sejam responsáveis pela doença, que talvez seja causada por outros microor- ganismos ou combinação de agentes ainda não identificados e resistentes aos antimicrobianos usados. Sua origem poderia ser o trauma repetido, pelo coito anal receptivo e outras práticas homossexuais, já que a sepse anor- retal e os abscessos intersfincterianos crôni- cos são comuns nestes doentes. Sugerimos a origem criptogenética. Parece-nos um abs- cesso com evolução diferente da esperada, provavelmente pelo imunodepressão destes pacientes, pois temos observado em nossos casos que a úlcera localiza-se no canal anal, envolve a linha pectínea e comunica-se com abscesso laminar intersfincteriano. baseada na freqüência etiológica local, técnicas com respostas rápidas em relação à etio- logia para re-alinhamento da terapêutica. mes da flora intestinal. A cicatrização completa ocorre em até seis meses. A remissão dos sintomas, o controle das doenças oportunistas associadas e a melhora da qualidade de vida, após a operação, demonstram a necessidade de tratamento em caráter de urgência. Essa segunda estratégia é útil na elucidação das infecções primárias ou hospitalares, e fun- damental nas situações de insuficiência respira- tória grave, quando a mortalidade sem uma intervenção mais agressiva e objetiva no diag- nóstico e terapêutica ultrapassa os 60%. SIDNEY ROBERTO NADAL CARMEN RUTH MANZIONE Referências Referências 1. Bousso A, Baldacci ER, Fernades JC, Fernandes ICO, Cordeiro AMG, Otoch JP, et al. Contribuição da biópsia pulmonar a céu aberto na avaliação de pneumopatias difusas e agudas em unidade de terapia intensiva pediátrica. J Pneumol 2002; 28:30-40. 2. Sakane PT, Katayama DM, Freddi NA, Carvalho MF, Saldiva PHN, Pillegi FL, et al. Biopsia pulmonar a ceú aberto em crianças para diagnóstico histopatológico de doença pulonar difusa de evolução grave. AMB Rev Assoc Med Bras 1987; 33:7-10 1. Bousso A, Baldacci ER, Fernades JC, Fernandes ICO, Cordeiro AMG, Otoch JP, et al. Contribuição da biópsia pulmonar a céu aberto na avaliação de pneumopatias difusas e agudas em unidade de terapia intensiva pediátrica. J Pneumol 2002; 28:30-40. p 2. Sakane PT, Katayama DM, Freddi NA, Carvalho MF, Saldiva PHN, Pillegi FL, et al. Biopsia pulmonar a ceú aberto em crianças para diagnóstico histopatológico de doença pulonar difusa de evolução grave. AMB Rev Assoc Med Bras 1987; 33:7-10 104 Rev Assoc Med Bras 2002; 48(2): 93-117
https://openalex.org/W4242811871	https://jneuroinflammation.biomedcentral.com/counter/pdf/10.1186/1742-2094-3-9	English	null	Correction: Microglial inflammation in the parkinsonian substantia nigra: relationship to alpha-synuclein deposition	Journal of neuroinflammation	2,006	cc-by	438	BioMed Central BioMed Central BioMed Central nigra: relationship to alpha synuclein deposition Emilie Croisier1, Linda B Moran1, David T Dexter2, Ronald KB Pearce1 and Manuel B Graeber1 Address: 1Department of Neuropathology, Division of Neuroscience and Mental Health, Imperial College London, and Hammersmith Hospitals Trust, London, UK and 2Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Imperial College L d L d UK Address: 1Department of Neuropathology, Division of Neuroscience and Mental Health, Imperial College London, and Hammersmith Hospitals Trust, London, UK and 2Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Imperial College London, London, UK Email: Emilie Croisier - e.croisier@imperial.ac.uk; Linda B Moran - l.moran@imperial.ac.uk; David T Dexter - d.dexter@imperial.ac.uk; Ronald KB Pearce - ronald.pearce@imperial.ac.uk; Manuel B Graeber - m.graeber@imperial.ac.uk * C di th mail: Emilie Croisier* - e.croisier@imperial.ac.uk; Linda B Moran - l.moran@imperial.ac.uk; David T Dexter - d.dex onald KB Pearce - ronald.pearce@imperial.ac.uk; Manuel B Graeber - m.graeber@imperial.ac.uk * Corresponding author Published: 06 April 2006 Published: 06 April 2006 Received: 06 April 2006 Accepted: 06 April 2006 Journal of Neuroinflammation 2006, 3:9 doi:10.1186/1742-2094-3-9 This article is available from: http://www.jneuroinflammation.com/content/3/1/9 s article is available from: http://www.jneuroinflammation.com © 2006 Croisier 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. Upon re-examination of all dates of birth and death of cases described in this paper [1], the ages at death of the following cases presented in Table 1 need to be corrected as follows: All other clinical information is accurate, and this does not affect the findings of the paper. Journal of Neuroinflammation Open Access Page 1 of 1 (page number not for citation purposes) References References 1. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB: Micro- glial inflammation in the parkinsonian substantia nigra: rela- tionship to alpha-synuclein deposition. J Neuroinflammation 2005, 2:14. 1. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB: Micro- glial inflammation in the parkinsonian substantia nigra: rela- tionship to alpha-synuclein deposition. J Neuroinflammation 2005, 2:14. 1. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB: Micro- glial inflammation in the parkinsonian substantia nigra: rela- tionship to alpha-synuclein deposition. J Neuroinflammation 2005, 2:14. Table 1: Case Reported age at death Actual age at death 3 71 70 6 83 82 7 73 72 8 77 76 9 75 76 12 75 74 13 83 82 14 77 76 16 83 84 Table 1: Page 1 of 1 (page number not for citation purposes)
https://openalex.org/W4289171396	https://zenodo.org/record/6947526/files/02-01-2022.pdf	English	null	High-quality Development Effectiveness of Provincial Regions in China: Data-driven Evaluation and Spatio-temporal Characteristics	International Journal of Current Science Research and Review	2,022	cc-by	6,692	2806 Corresponding Author: Diping Zhang I. INTRODUCTION "High-quality development" is the main theme of China's economic and social development in the future. It is an epoch- making and forward-looking judgment based on a new historical starting point and conditions of the times when the quantitative economy of China's high-speed development has reached a certain level. Different from high-speed growth, high quality development has a rich connotation, which includes various segments of development and is still evolving in the current development practice. High-quality development is no longer confined to the goal of "seeking efficient development in the pure economic field", but extends this development model to the national governance system and governance capacity, the ecological civilization system, and even the fields that can have a profound impact on increasing people's sense of access and happiness. Therefore, considering the multi-dimensional and dynamic characteristics of high-quality development, and the fact that most of the current research remains at the theoretical level, whether at the national level, it can help to grasp the "time window" of accelerating economic transformation and upgrading, or at the social level, it can further meet the people's needs for a better and quality life, build a set of a scientific, reasonable and comprehensive index system of high-quality development, dig into the current situation of high quality development of China's economy, and respond to high quality development with high quality assessment, this should be the research direction we need to stick to and work hard for. KEYWORDS: High-quality Development, Entropy method, Temporal Differentiation, Spatial Agglomeration KEYWORDS: High-quality Development, Entropy method, Temporal Differentiation, Spatial Agglomeration Yingying Chu1, Diping Zhang2 1,2 School of Science, Zhejiang University of Science and Technology, Hangzhou, China Yingying Chu1, Diping Zhang2 1,2 School of Science, Zhejiang University of Science and Technology, Hangzhou, China Yingying Chu1, Diping Zhang2 ABSTRACT: Healthy and sustainable high-quality development needs to rely on scientific planning and design of assessment system, enabling high-quality assessment to become an important tool to lead and drive high-quality development in China. A high-quality development evaluation index system is proposed, consisting of six dimensions: economic operation, innovation- driven, coordinated development, green ecology, open development, and sharing and harmony, and the temporal and spatial Differentiation in China's inter-provincial high-quality development levels are discussed after being measured using the entropy weight method. The results show that from the time dimension, China's overall high quality development level shows a fluctuating upward trend with an obvious growth rate, and Jiangsu, Zhejiang and Shanghai maintained a high level of high-quality development during this period. From the spatial dimension, it is found that China's high-quality development level has a significant positive spatial correlation between regions from 2014 to 2020, with a stable "high-high" agglomeration in the eastern coastal areas and the middle and lower reaches of the Yangtze River, the "low-high" agglomeration is mainly distributed in the central plains, and the "low-low" agglomeration effect in the western provinces and regions has spread on a larger scale after 2015, with significant performance in the north-western provinces and regions. International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org www.ijcsrr.org Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 International Journal of Current Science Research and Review ISSN: 2581-8341 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 and opening up, urban and rural development and ecological environment. LIU (2018) believed that the quality of economic development should be analyzed from a diversified perspective, which requires more balance in economic structure, more attention to the improvement of service quality and more emphasis on the market as well as the role of demand. Based on the meaning of economic development quality, LI, SHI and XU (2019) summarize the connotation of high-quality development and divide it into three categories: (1) from the perspective of "new development concept" and the main social contradiction, they point out that high- quality development is development with "meeting the people’s growing needs for a better life" as the fundamental purpose and "the five development concepts of innovation, coordination, green, openness and sharing" as the fundamental concept (Zhan and Pei (2016), JIN (2018)). (2) from the perspective of high-quality economic development, it is pointed out that "high quality" is the fundamental requirement in high quality development, and "sustainable" is the fundamental way. (3) from the perspective of distinguishing the different requirements of micro and macro, it suggests that high-quality development needs to meet both products and services at the micro level and structures and efficiency at the macro level (WEI and LI (2018), AN (2018)). Many researchers have studied regional econimic development measurement and evaluation in recent decades, which has shown a dynamic development process. The methods of evaluation are different at different stages of development and are divided into two main types. The first one advocates the use of a single indicator to evaluate the quality of economic development, such as Total Factor Productivity (TFP) (Zhang, Liu, and Huang (2020), Zeng, Shu and Ye (2022).) or Value-added rate, etc. Mlachila, Tapsoba and Tapsoba (2017) established an indicator for developing countries to measure the quality of their economic development that reflects the intrinsic nature of economic growth and the effectiveness of economic growth at the social level. Yu, Zhang, and Zhang (2019) added environmental factors to total factor productivity to measure the quality development of China’s economy. International Journal of Current Science Research and Review ISSN: 2581-8341 The second advocates building evaluation index systems to evaluate economic development, the results of research have been reflected (Kristensen and Mosgaard (2020), State, Bulin, Oehler-Sincai and et al (2019), SzopikDepczyńska, Cheba, Bąk and et al (2018), et al (2012)). Among the evaluations on China’s high-quality development, some scholars consider that the conclusions obtained by a single indicator method may be one-sided and limited. So they also set up evaluation indicator systems, but with slightly different focuses. Song, Zhang, and Yi (2015) defined the quality of economic development as "the extent to which economic development meets the requirements of social and sustainabledevelopment" at the macro level and constructed an evaluation index system from three dimensions: competitive quality, livelihood quality and ecological quality. SHI and Ren (2018) proposed the evaluation index system from two dimensions: growth fundamentals and social outcomes. CHEN and SHI (2019) established the index system from six dimensions: innovation, coordination, green, openness, sharing, and efficiency, guided by the "Five Development Concepts". However, there are few studies on the spatial and temporal differentiation of high-quality development. Lu, Xing, and Yang (2019) concluded that there is a high spatial dependence effect of high- quality economic development in different regions of China by using a spatial correlation model, forming a distribution pattern of "high in the east", "flat in the middle" and “low in the west”. CHEN and SHI (2019) pointed out that the high-quality level of China’s economy in the eastern coastal region is much higher than that in the central and western regions, and the effectiveness of high-quality development has accelerated in recent years, with the largest gap in the eastern region and the smallest gap within the northeastern region. Fang and Ma (2019) explored the regional clustering of China’s high-quality development with the combination of GIS and exploratory spatial data analysis (ESDA) methods, pointing out that there is an obvious and still continuously enhancing agglomeration effect of inter-provincial high quality development level in China, and it spreads from the eastern coast to the central and western inland regions. According to the above discussion, the consensus has been basically reached that high-quality development is a dynamic and multidimensional concept, and it is relatively mature to measure the level of high-quality development by constructing an indicator system, but there remain the problems of incomplete indicator system and redundancy of indicator settings. II. LITERATURE REVIEW High-quality development is a new concept for the quality of China's economic development. To accurately understand the connotation of high-quality development, the first lies in clarifying the meaning of the quality of economic development. The current relevant literature can be referred to: Barro (2002) explained the quality of economic growth from six perspectives: life expectancy, fertility rate, environmental conditions, income equity, political system, and religious beliefs. HUI and CHAO (2010) have pointed out that quality focuses on the qualitative improvement in the process of economic growth, reflecting its quality in terms of process, outcome, and prospect. REN and WEN (2018) emphasized that the quality of economic development is a multidimensional concept with rich connotations, which is not only expressed as increasing the quantity of total economic volume and material wealth, but also a value judgment of the level, specifically including four aspects of economic development, reform Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 2807 Corresponding Author: Diping Zhang III. EVALUATION INDEX SYSTEM High-quality development is a description of the quality of economic growth, which is different from the speed of economic growth, and focuses on solving the problem of "how to develop in different spheres of economic process". Secondly, high-quality development is a concrete manifestation of the new development concept, representing a comprehensive economic development model of higher quality, more efficient, more stable, and more open in the new era of China. And it requires development on multiple aspects: dynamic and sustainable healthy development of the national economy without blindly pursuing GDP growth; high-efficiency development driven by innovation; green development with continuous improvement of the ecological environment; and the development with continuous improvement of people's quality of life. Therefore, the evaluation of high quality development has puts forward new and higher requirements for the reform and innovation of the statistical index system: we should pay more attention to the performance of development in terms of effectiveness, coordination, innovation, openness and sharing when judging the level of high-quality development, and the evaluation index should also reflect the changes in the main contradictions of Chinese society and the characteristics of economic development in the new era. Based on the above, we construct an evaluation index system of China's inter-provincial high-quality development from three levels: target layer, criterion layer and indicator layer. The target layer is about the research purpose, which is China's high-quality development, the criterion layer is constructed around the "economic growth" and the five contents of the new development concept, including six indices of economic operation, innovation-driven, coordinated development, green ecology, open development, sharing and harmony, according to the core connotation of each index and the established target tasks, we further determine quantitative indicators to measure the degree of their completion, i.e. the indicator layer. Table 1 specifically displays the constructed evaluation index system of high-quality development. 2808 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Table 1. International Journal of Current Science Research and Review International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 Volume 05 Issue 08 August 2022 International Journal of Current Science Research and Review ISSN: 2581-8341 And there is a lack of quantitative research on the spatial and temporal characteristics of high-quality development. Therefore, with the guidance of economic growth and "new development concept", the index system is constructed from six dimensions: economic operation, innovation-driven, coordinated development, green ecology, open development, sharing and harmony. We measure the level of high-quality development in China’s inter-provincial economy from 2014 to 2020 and analyze its spatial and temporal differentiation, providing support for exploring ways to achieve quality development. 2807 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org 2808 Corresponding Author: Diping Zhang III. EVALUATION INDEX SYSTEM Comprehensive evaluation index system of High‐quality development Target layer Guideline layer Index layer Index Company Index direction Economic operation Economic development intensity GDP growth rate % + Fiscal revenue million yuan + Social Labor Productivity % + Rationalization of economic development Service industry value added as a percentage % + Contribution rate of residential consumption % + Innovation drive Innovation inputs R&D investment intensity % + R&D staff full-time equivalent person per year + Innovation output Patent effective volume piece + Technical market turnover million yuan + Coordinate development Rural and urban development Urban-rural income disparity yuan - Urbanization rate % + Industry structure development Rationalization of industrial structure % - Material and spiritual civilization development The proportion of cultural expenditure to fiscal expenditure % + Per capita cultural consumption expenditure of residents yuan + Green Ecology Ecological Environment Energy consumption per unit of GDP ton of standard coal / million yuan - Percentage of days with good air quality % + Greening coverage of built-up areas % + Table 1. Comprehensive evaluation index system of High‐quality development International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Sustainability Sewage treatment rate % + Harmless disposal rate of domestic waste % + General industrial solid waste comprehensive utilization rate % + Opening up development Trade opening External trade dependency % Investment opening Actual Foreign Direct Investment(FDI) million yuan + Tourism opening 100 10000 people + Sharing Harmony People's lives Per capita disposable income of residents yuan + Urban registered unemployment rate % - Number of urban workers' pension insurance participants person + Development achievements Number of beds in health facilities per 1,000 people + Public library holdings per capita + General university student-teacher ratio % + The weight of each index is determined by the entropy weight method. The steps are as follows: (1)For m evaluation units and n evaluation indicators the original data matrix is formed: The weight of each index is determined by the entropy weight method. The steps are as follows: The weight of each index is determined by the entropy weight method. The steps are as follows: (1)For m evaluation units and n evaluation indicators the original data matrix is formed: The weight of each index is determined by the entropy weight method. 2809 Corresponding Author: Diping Zhang 2810 Corresponding Author: Diping Zhang IV. ANALYSIS OF EMPIRICAL RESULTS A. Temporal Differentiation of High-Quality Development in China After comparing the average value of China's high-quality development level, it is found that there is a fluctuating upward trend from 2014 to 2020, rising from 0.3556 in 2014 to 0.3782 in 2020. From the magnitude of change in the mean value, there is a most significant growth in the overall national level of quality development from 2016 to 2017, and since 2017 the growth has been stable year by year. And Jiangsu, Zhejiang and Shanghai have the highest level of high-quality development and will continue to play the role of high-quality development "leader" in the future, they ensure a more leading position but also maintain the most stable, lasting, and excellent development trend. However, Tibet, Qinghai, Xinjiang, Ningxia, and Gansu are lagging in high-quality development, which fully confirms the characteristics of the long-standing development gap between the east and the west in China. B. Spatial Differentiation of High-Quality Development in China wij is the spatial weight matrix, and here a 0-1 weight distance matrix is used, 1 if the province is spatially adjacent to the province and 0 if it is not, while taking into account the special case: Hainan Province is an island, so Guangdong and Hainan are considered as each other's neighboring regions. The Global Moran's I index takes values in the range of (-1, 1). When Moran's I index > 0, it indicates that there is a spatially positively correlated agglomeration effect of China's high-quality development, while <0 is the opposite, and the closer the index value is to -1 or +1 indicates a higher degree of agglomeration, the index value of 0 means that there is no spatial correlation. Table 2 shows the Moran's I index of China's high quality development level for the period 2014- 2020 Table 2: global Moran's I Index for China's high-quality development during 2014-2020 Year 2014 2015 2016 2017 2018 2019 2020 Moran’s I 0.21 0.20 0.19 0.22 0.24 0.26 0.26 Z-value 3.20 3.05 2.96 3.24 3.55 3.68 3.85 P-value 0.0016 0.0023 0.0030 0.0012 0.0004 0.0001 0.0001 Table 2: global Moran's I Index for China's high-quality development during 2014-2020 Year 2014 2015 2016 2017 2018 2019 During this period, the Global Moran's I index showed positive values, and the p-values all passed the 1% significance level test, which indicated that the spatial distribution of China's high quality economic development level was not completely random, but there was a significant positive spatial correlation, that is, provinces (cities and autonomous regions) with higher development level were adjacent to each other, and provinces (cities and autonomous regions) with lower level were adjacent to each other. It can also be found that 2016 was the turning point, with Moran's I index declining continuously during 2014-2016 and starting to show an upward trend of a larger magnitude after 2016, which reflects the spatial dependence of high-quality development level in China's provinces has been strengthening in recent years, and the forward pace of high-quality development across the region has accelerated significantly, with inter-regional collaboration further deepening. 2) Spatial local autocorrelation analysis: Local autocorrelation analysis describes the similarity between a spatial region and its neighboring regions, which is generally measured by the Local Moran's I index. B. Spatial Differentiation of High-Quality Development in China Considering the variability in the level of China's provincial economic quality development, we analyze its spatial characteristics further. Global and local spatial autocorrelation analysis are used to explain the spatial clustering and differences of China's economic high-quality development. g q y p 1) Spatial global autocorrelation analysis: The Global Moran's I index is used to determine the spatial correlation of economic quality development differences among provinces. The global Moran's I index is defined as follows: correlation analysis: The Global Moran's I index is used to determine the spatial correlation of economic erences among provinces. The global Moran's I index is defined as follows: 1) Spatial global autocorrelation analysis: The Global Moran's I index is used to determine the spatial quality development differences among provinces. The global Moran's I index is defined as follows:      1 1 1 1 1 n n ij i j i j n n n i ij i i j w x x x x n I x x w              (6) (6) where, n is the number of provinces in China, xi denotes the observed value of the ith region, and denotes the regional average. wij is the spatial weight matrix, and here a 0-1 weight distance matrix is used, 1 if the province is spatially adjacent to the province and 0 if it is not, while taking into account the special case: Hainan Province is an island, so Guangdong and Hainan are considered as each other's neighboring regions. The Global Moran's I index takes values in the range of (-1, 1). When Moran's I index > 0, it indicates that there is a spatially positively correlated agglomeration effect of China's high-quality development, while <0 is the opposite, and the closer the index value is to -1 or +1 indicates a higher degree of agglomeration, the index value of 0 means that there is no spatial correlation. Table 2 shows the Moran's I index of China's high quality development level for the period 2014- 2020 where, n is the number of provinces in China, xi denotes the observed value of the ith region, and denotes the regional average. III. EVALUATION INDEX SYSTEM The steps are as follows: n units and n evaluation indicators, the original data matrix is formed: (1)For m evaluation units and n evaluation indicators, the original data matrix is formed:  ij m n R r   (1) (1) (2)The individual evaluation indicators are homogenized, that is, the indicator values are transformed into weights P of the indicator values of the ith program under the jth indicator: 1 ij ij m ij i x P x   1 ij ij m ij i x P x   (2) (2) (3)Calculate the entropy coefficient of the jth indicator: 1 ln m j ij ij i e k p p     (3) 1 ln m j ij ij i e k p p     (3) where    1 0 0 j k ,e lnm (4) Calculate the information entropy redundancy of the jth indicator: 1 i i g e  (4) Calculate the information entropy redundancy of the jth indicator: 1 i i g e  (5) Calculate the weights of each indicator: (4) Calculate the information entropy redundancy of the jth indicator: 1 i i g e  (4) Calculate the information entropy redundancy of the jth indicator: 1 i i g e  (5) Calculate the weights of each indicator: 1 i j n i i g a g   (4) (6) Calculate the composite score: 1 n i i ij j v a p   (5) 1 i j n i i g a g   (4) 1 n i i ij j v a p   (5) 1 i j n i i g a g   (6) Calculate the composite score: 1 n i i ij j v a p   1 i j n i i g a g   (6) Calculate the composite score: (4) 1 n i i ij j v a p   (5) 2809 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 2809 Corresponding Author: Diping Zhang 2809 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Available at: ijcsrr.org Page No.-2806-2815 2811 Corresponding Author: Diping Zhang B. Spatial Differentiation of High-Quality Development in China The Local Moran's I index is defined as follows: 2) Spatial local autocorrelation analysis: Local autocorrelation analysis describes the similarity between a spatial region and its neighboring regions, which is generally measured by the Local Moran's I index. The Local Moran's I index is defined as follows: Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Volume 05 Issue 08 August 2022 g Available at: ijcsrr.org Page No.-2806-2815 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org    International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org ( ) ( ) n n x x w x x    Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org g DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org 1 1 ( ) ( ) ( ) ( ) n i ij i j i i ij j n j i i n x x w x x I d z w z x x                   (7) (7) where zi, zj are the normalized values of the i-th and j-th observations. When Ii (d) is greater than 0, it means that region i and its neighboring regions have similar spatial distribution, in which case there will be the same high level or the same low level. On the contrary, when Ii (d) is less than 0, it means that region i and its neighboring regions have opposite spatial distribution (high-low aggregation distribution or low-high aggregation distribution). Thus four patterns of spatial distribution will exist: high-high pattern; high-low pattern; low-low pattern and low-high pattern. We visualize the spatial distribution of China's provincial economic quality development by plotting the Lisa aggregation map (in Figure1). Figure 1. China’s provincial high-quality development aggregation in 2014-2020 Figure 1. China’s provincial high-quality development aggregation in 2014-2020 2811 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 JCSRR @ 2022 www.ijcsrr.o 812 Corresponding Author: Diping Zhang Volume 05 Issue 08 August Available at: ijcsrr Page No.-2806- Figure 1. China’s provincial high-quality development aggregation in 2014-2020(Continued from previous figure) www.ijcsrr.org Figure 1. China’s provincial high-quality development aggregation in 2014-2020(Continued from previous figure) ina’s provincial high-quality development aggregation in 2014-2020(Continued from previous figure) Figure 1. International Journal of Current Science Research and Review ISSN 2581 8341 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijc www.ijcsrr.org DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 In general, the regions with "high - high pattern" are the most, basically 5-7 provinces can be stabilized every year, which reflects that China's economy has shown good results in achieving high quality development. And there is a relatively stable high- high aggregation in China's eastern coastal region and some regions in the middle and lower reaches of Yangtze River, especially Jiangsu, Shanghai, Zhejiang, Hubei, etc., which have shown a high level of high-quality economic development and obvious spillover effects in the past five years. They are mostly adjacent to each other and have formed a spatially clustered and mutually reinforcing whole, which is a crucial part of China's goal of achieving high-quality economic development. The second is the region with "low-high pattern", which Hebei, Henan as well as Jiangxi provinces belonged for four of the last six years (2015-2018), with their low high-quality development level and a significant gap between them and their neighboring provinces. Hebei locates in the Beijing-Tianjin-Hebei city cluster, close to the Bohai Sea. Despite its geographical location, but in the early stage of development, it lagged far behind compared with the neighboring Beijing and Tianjin. On the one hand, it may be related to the fact that the cooperation between Beijing and Tianjin had not been deepened, the Beijing-Tianjin-Hebei cooperative development strategy was first proposed in 2014, and the effectiveness was not revealed by some factors such as inappropriate cooperation methods and unstable cooperation models, on the other hand, it may also lie in the fact that the two regions, Beijing and Tianjin, attract a large amount of human, financial and other resources from the surrounding area, resulting in a greatly restricted development space. In 2019 a turnaround was ushered in, with further synergistic development and closer and more effective inter-regional cooperation, the positive radiation effect of the Beijing-Tianjin region has been released and the low - high agglomeration phenomenon in Hebei province disappears. 2813 Corresponding Author: Diping Zhang International Journal of Current Science Research and Review ISSN 2581 8341 Similarly, although Henan and Jiangxi have obvious location advantages, close to the Yangtze River Delta region with active economic development, high degree of openness and strong innovation capacity, they did not bring it into play, their high-quality development derailed from surrounding areas, which may be related to the lack of close cooperation with the surrounding areas weak initiative to absorb the successful experience of high-quality development. Now Henan has gotten rid of the dilemma of high-quality development and entered a high aggregate of high area. It is reported that regions with "low-low pattern" are mainly distributed in western China, with Xinjiang and Gansu added to the original Tibet and Qinghai after 2015, and the low- low agglomeration of the four provinces has been basically stable, indicating that the reverse driving role of the low region has exceeded the original positive driving role played. Although China's overall high quality economic development level has improved significantly, the situation in the western region is not optimistic. The western region should further accelerate the breaking of geopolitical restrictions and seek high quality cross-regional cooperation in the future. V. CONCLUSIONS AND POLICY RECOMMENDATIONS After using the entropy method to measure the level of high-quality economic development in China's provinces from 2014 to 2020, we explore the characteristics of their spatial and temporal differences, and the results are as follows: p p p (1) Since 2015, China's economic quality development trend has been generally positive, with most provinces improving their quality development level year by year. (1) Since 2015, China's economic quality development trend has been generally positive, with most provinces improving their quality development level year by year. (2) China's provincial economic quality development shows a significant positive spatial correlation: the eastern coastal region and the middle and lower reaches of the Yangtze River belong to the "high-high agglomeration" pattern, and this phenomenon has basically stabilized in the region. The low-low agglomeration phenomenon in the western region intensifies after 2015. (3) The spatial and temporal differences fully illustrate that there is also a problem of unbalanced and insufficient development between regions in China's high-quality development. (3) The spatial and temporal differences fully illustrate that there is also a problem of unbalanced and insufficient development between regions in China's high-quality development. Therefore, against the background of good quality development of China's economy, but with development differences between regions, further achieving the goal of high-quality development still requires the joint efforts of all regions: Therefore, against the background of good quality development of China's economy, but with development differences between regions, further achieving the goal of high-quality development still requires the joint efforts of all regions: (1) As the main driver of China's high-quality economic development, the eastern coastal regions should continue to develop new models of high-quality development in various fields and promote the implementation of various policies and strategies. At the same time, it should further play an active radiating role to drive the development of its neighboring regions and take the initiative to strengthen cooperation with the central and western regions, including sending local high-quality counterpart technologies, professional talents and investment projects with high return rates and high feasibility, helping them break through bottlenecks in economic development. (2) Geographically, the central regions act as a bridge between the eastern and western regions and has both opportunities and challenges in promoting high-quality economic development. International Journal of Current Science Research and Review China’s provincial high-quality development aggregation in 2014-2020(Continued from 2812 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 2812 Corresponding Author: Diping Zhang 2812 Corresponding Author: Diping Zhang 2812 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 2814 Corresponding Author: Diping Zhang International Journal of Current Science Research and Review ISSN: 2581-8341 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 05 Issue 08 August 2022 DOI: 10.47191/ijcsrr/V5-i8-02, Impact Factor: 5.995 IJCSRR @ 2022 www.ijcsrr.org development experience of the eastern region to build a suitable development system for itself, and also bear the challenge of transferring resources and driving the high-quality development of the western region under the premise of ensuring its own benign development goals. So the central regions need to clarify own orientation, actively seek interaction with neighboring regions, and use geographical advantages to grasp, or even create opportunities to achieve high-quality economic development. (3) The western region is the focus of China's high-quality economic development, the introduction and implementation of various national support policies can certainly bring strong development momentum to them, coupled with their own unique characteristics in nature, culture, environment and other resources, they can fully break through the geographical restrictions, while changing the mode of economic development, optimizing the economic structure with the introduction of talent, technology, capital and other active policies, with the aim of achieving the leap to high-quality development. ACKNOWLEDGEMENTS This study was funded by General Research Project of Zhejiang Provincial Education Department (Postgraduate Special) Y202146820) REFERENCES 1. AN, S.X., 2018. 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Interpretation of sustainable development using a set of macroeconomic and social indicators for a group of nine emerging economies. Sustainability 11. 10.3390/su11113095 Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Cite this Article: Yingying Chu, Diping Zhang (2022). High-quality Development Effectiveness of Provincial Regions in China: Data-driven Evaluation and Spatio-temporal Characteristics. International Journal of Current Science Research and Review, 5(8), 2806-2815 18. Yu, Y.Z., Zhang, Y.X., Zhang, S.H., 2019. Research on the characteristics of time and space conversion of china’s economy from high-speed growth to high-quality development. The Journal of Quantitative & Technical Economics 36, 3–21. https://doi.org/10.13653/j.cnki.jqte.2019. 06.001 International Journal of Current Science Research and Review www.ijcsrr.org www.ijcsrr.org 16. Szopik-Depczyńska, K., Cheba, K., Bąk, I., et al, 2018. 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Zeng, S.L., Shu, X.f., Ye, W.x., 2022. Total factor productivity and high-quality economic development: A theoretical and empirical analysis of the yangtze river economic belt, china. International Journal of Environmental Research Public Health 19, 2783. 10.3390/ijerph19052783. 20. Zhan, X.Y., Pei, P.C., 2016. Estimation and evaluation of china’s provincial quality of economic growth −− empirical analysis based on "five development concepts". Public Finance Research , 40–53. https: //doi.org/10.19477/j.cnki.11-1077/f.2016.08.004. Cite this Article: Yingying Chu, Diping Zhang (2022). High-quality Development Effectiveness of Provincial Regions in China: Data-driven Evaluation and Spatio-temporal Characteristics. International Journal of Current Science Research and Review, 5(8), 2806-2815 2815 Corresponding Author: Diping Zhang Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815 Volume 05 Issue 08 August 2022 Available at: ijcsrr.org Page No.-2806-2815
https://openalex.org/W1975738789	https://europepmc.org/articles/pmc3966373?pdf=render	English	null	Pandemic<i>Vibrio parahaemolyticus</i>, Maryland, USA, 2012	Emerging infectious diseases	2,014	cc-by	2,611	LETTERS LETTERS should be initiated immediately be- cause β-lactam antimicrobial drugs are inefficient for the treatment of rickettsioses (9). strains are endemic to the West Coast (2) and have been historically linked to outbreak-associated V. parahaemo- lyticus infections caused by consump- tion of raw oysters harvested from the region (5). Spain, 2007–2011. Emerg Infect Dis. 2013;19:267–9. http://dx.doi.org/10.3201/ eid1902.111706 7. Edouard S, Parola P, Socolovschi C, Davoust B, La Scola B, Raoult D. Clus- tered cases of Rickettsia sibirica mon- golitimonae infection, France. Emerg Infect Dis. 2013;19:337–8. http://dx.doi. org/10.3201/eid1902.120863 This research was funded by the Uni- té de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), Marseille, France. In August 2012, a V. parahaemo- lyticus outbreak involving 6 persons occurred in Maryland, USA. The pa- tients (members of 2 dining parties) had eaten in the same restaurant on the same day; raw and cooked seafood was served at the restaurant. Party A comprised 4 diners, of whom 2 had laboratory-confirmed illness and 2 were probable case-patients. Party B comprised 2 diners, of whom 1 had laboratory-confirmed illness and 1 was a probable case-patient. Probable case-patients were epidemiologically linked to confirmed case-patients, but V. parahaemolyticus was not detected in their stool samples. The epidemio- logic investigation did not conclusive- ly identify the specific food respon- sible for the outbreak. The affected diners had not eaten oysters, lobster, or mussels, but they had eaten cooked clams, fish, crab, and shrimp. Because the patients had not eaten oysters, a traceback investigation was not con- ducted. The outbreak possibly was caused by cross-contamination during food preparation. No other cases were reported from this restaurant or the surrounding area. g 8. Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier PE, Sotto A, et al. Warmer weather linked to tick attack and emergence of severe rickettsioses. PLoS Negl Trop Dis. 2008;2:e338. http://dx.doi. org/10.1371/journal.pntd.0000338 D.R. is a cofounder of the biotechnol- ogy company Inodiag (www.inodiag.com) and is the inventor of a serologic diagnos- tic method for which the patent is held by Aix-Marseille University; this method was not used in this study. All other authors have no conflicts of interest. 9. Botelho-Nevers E, Socolovschi C, Raoult D, Parola P. Treatment of Rickettsia spp. infections: a review. Expert Rev Anti Infect Ther. 2012;10:1425–37. References 1. Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev. 2013;26:657–702. http://dx.doi.org/10.1128/CMR.00032-13 DOI: http://dx.doi.org/10.3201/eid2002.130575 DOI: http://dx.doi.org/10.3201/eid2002.130575 Julie Solary, Cristina Socolovschi, Camille Aubry, Philippe Brouqui, Didier Raoult, and Philippe Parola Author affiliations: Aix-Marseille Université, Marseille, France LETTERS http:// dx.doi.org/10.1586/eri.12.139 Address for correspondence: Philippe Parola, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), WHO Collaborative Center for Rickettsioses and Other Arthropod-borne Bacterial Diseases, Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France; email: philippe. parola@univ-amu.fr Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012 A similar graph was obtained by using chromosome II of the same strain as reference (data not shown). In brief, the BIGSdb genome comparator tool performs wgMLST, which produces a color-coded wgMLST output (online Technical Appendix 2, wwwnc.cdc.gov/EID/article/20/4/13-0818-Techapp2.xlsx) that facilitates comparison among isolates. This loci output is further categorized into loci that are 1) variable among all isolates, 2) identical among all isolates, 3) missing in all isolates, and 4) incomplete because of being located at the ends of contigs. The variable loci among all isolates are the loci used for assessing relationships and producing a distance matrix based on the number of variable alleles; the strains are resolved into a network by using the NeighborNet algorithm (9). MLST, multilocus sequence typing; CC, clonal complex; ST, sequence type. The whole genomes of the 3 Maryland strains were sequenced by using the Ion Torrent personal ge- nome machine (Life Technologies, Grand Island, NY, USA); in silico multilocus sequence typing (MLST) (2) showed that the isolates were all ST3, the most common ST belong- ing to CC3. Bioinformatic analysis of the whole genomes was conducted with the Bacterial Isolate Genome Sequence Database (6) genome com- parator tool available within the V. parahaemolyticus MLST database (http://pubmlst.org/vparahaemolyti- cus) (7,8). Results confirmed that these outbreak isolates were linked to the O3:K6 pandemic clone of V. parahaemolyticus (Figure). We iden- tified 2,613 variable loci in this anal- ysis by using as reference genome the prototype pandemic V. parahae- molyticus clonal strain RIMD221633 (available from GenBank, www.ncbi. nlm.nih.gov/genome/?term=vibrio parahaemolyticus) (10). Differences in variable loci and the absence of certain genes indicated that, although indistinguishable by MLST and PFGE, these strains are easily differ- entiated from RIMD2210633 (online Technical Appendix 1). The draft ge- nome sequences for the 3 strains are available at the V. parahaemolyticus MLST database (identification nos. 1187 [Vp16MD], 1188 [Vp17MD], and 1189 [Vp18MD]). V. parahaemolyticus strains be- longing to the pandemic CC have caused thousands of infections and a V. parahaemolyticus pandemic (3). Foodborne illnesses caused by pan- demic V. parahaemolyticus are un- commonly reported in the United States. In Maryland, 12 and 21 cases of V. parahaemolyticus–associated gastroenteritis were reported in 2012 and 2013, respectively. We report that the pandemic CC was still causing US outbreaks as recently as August 2012. Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012 2. Wang JM, Hudson BJ, Watts MR, Karagiannis T, Fisher NJ, Anderson C et al. Diagnosis of Queensland tick typhus and African tick bite fever by PCR of lesion swabs. Emerg Infect Dis. 2009;15:963–5. http://dx.doi.org/10.3201/eid1506.080855 V. parahaemolyticus was isolated from stool samples of 3 of the pa- tients. The isolates were characterized by real-time PCR for virulence-relat- ed genes (tdh and trh). All 3 isolates were tdh positive and lacked the trh gene. Pulsed-field gel electrophoresis (PFGE) was run, using Sfil and Notl; the resulting K16S12.0138 (Sfil) and K16N11.0143 (Notl) patterns were indistinguishable. The PFGE pat- tern combination was queried against combination entries made in PulseNet (www.cdc.gov/pulsenet/) during Feb- ruary 4, 2010–April 16, 2013, and found to be indistinguishable from other clinical entries (online Technical 3. Mouffok N, Socolovschi C, Benabdellah A, Renvoise A, Parola P, Raoult D. PCR diag- nosis of rickettsiosis on eschar swabs, Al- geria. Emerg Infect Dis. 2011;17:1968–9. http://dx.doi.org/10.3201/eid1710.110332 3. Mouffok N, Socolovschi C, Benabdellah A, Renvoise A, Parola P, Raoult D. PCR diag- nosis of rickettsiosis on eschar swabs, Al- geria. Emerg Infect Dis. 2011;17:1968–9. http://dx.doi.org/10.3201/eid1710.110332 To the Editor: Since 1996, an in- creasing number of infections caused by Vibrio parahaemolyticus strains belonging to a pandemic clonal com- plex (CC), CC3, typically O3:K6, have been observed worldwide (1–3); most of these strains are sequence type (ST) 3. In the summer of 1998, out- breaks linked to O3:K6 occurred in Galveston Bay, Texas, and Oyster Bay, New York, USA; the illnesses were associated with oyster consumption (4). Strains belonging to CC36 are the leading cause of V. parahaemolyticus infections in the United States. These 4. Socolovschi C, Renzulli A, Brouqui B, Parola P, Raoult D. The use of eschar swabs for the diagnosis of African tick-bite fever. Ticks Tick-borne Dis. 2012;3:361–3. 5. Fournier PE, Gouriet F, Brouqui P, Lucht F, Raoult D. Lymphangitis-associat- ed rickettsiosis, a new rickettsiosis caused by Rickettsia sibirica mongolotimonae: seven new cases and review of the litera- ture. Clin Infect Dis. 2005;40:1435–44. http://dx.doi.org/10.1086/429625 6. Ramos JM, Jado I, Padilla S, Masia M, Anda P, Gutierrez F. Human infection with Rickettsia sibirica mongolitimonae, 718 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014 LETTERS LETTERS LETTERS Figure. Neighbor-Net graph generated with the Bacterial Isolate Genome Sequence Database genome (BIGSdb) comparator tool implemented within the Vibrio parahaemolyticus MLST database (http://pubmlst.org/vparahaemolyticus) (7,8) using 2,613 variable loci. Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012 These loci were identified by using as a reference (ref) the V. parahaemolyticus strain RIMD2210633 chromosome I (3,080 genes) and conducting a whole-genome MLST (wgMLST) for V. parahaemolyticus genomes available through GenBank (AN-5034 O4:K68 ST3, Peru-466 ST3, K5030 ST3, 16_VP16T, AQ3810 ST87, AQ4037 ST96, PCV08–7 ST390, BB220P ST88, and SNUVpS-1) and 3 Maryland outbreak strains (Vp16MD, Vp17MD, and Vp18MD). This typing showed that these 3 strains belonged to the pandemic CC3. A similar graph was obtained by using chromosome II of the same strain as reference (data not shown). In brief, the BIGSdb genome comparator tool performs wgMLST, which produces a color-coded wgMLST output (online Technical Appendix 2, wwwnc.cdc.gov/EID/article/20/4/13-0818-Techapp2.xlsx) that facilitates comparison among isolates. This loci output is further categorized into loci that are 1) variable among all isolates, 2) identical among all isolates, 3) missing in all isolates, and 4) incomplete because of being located at the ends of contigs. The variable loci among all isolates are the loci used for assessing relationships and producing a distance matrix based on the number of variable alleles; the strains are resolved into a network by using the NeighborNet algorithm (9). MLST, multilocus sequence typing; CC, clonal complex; ST, sequence type. Appendix 1 Table, wwwnc.cdc.gov/ EID/article/20/4/13-0818-Techapp1. pdf). This PFGE pattern combina- tion has been seen 25 times; all pat- terns were for strains from humans (N. Facundo, pers. comm.). In 2012, this PFGE pattern combination was observed in 3 US states—California (6 cases), Arizona (6 cases), and Tex- as (5 cases)—but those isolates were not further tested (S.G. Stroika, pers. comm.), suggesting that other cases of pandemic V. parahaemolyticus in- fections have occurred in the United States but were not identified as being caused by pandemic clones. Figure. Neighbor-Net graph generated with the Bacterial Isolate Genome Sequence Database genome (BIGSdb) comparator tool implemented within the Vibrio parahaemolyticus MLST database (http://pubmlst.org/vparahaemolyticus) (7,8) using 2,613 variable loci. These loci were identified by using as a reference (ref) the V. parahaemolyticus strain RIMD2210633 chromosome I (3,080 genes) and conducting a whole-genome MLST (wgMLST) for V. parahaemolyticus genomes available through GenBank (AN-5034 O4:K68 ST3, Peru-466 ST3, K5030 ST3, 16_VP16T, AQ3810 ST87, AQ4037 ST96, PCV08–7 ST390, BB220P ST88, and SNUVpS-1) and 3 Maryland outbreak strains (Vp16MD, Vp17MD, and Vp18MD). This typing showed that these 3 strains belonged to the pandemic CC3. References 1. González-Escalona N, Cachicas V, Acevedo C, Rioseco ML, Vergara JA, Cabello F, et al. Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004. Emerg Infect Dis. 2005;11:129–31. http://dx.doi. org/10.3201/eid1101.040762 Address for correspondence: Narjol Gonzalez- Escalona, Food and Drug Administration, Center for Food and Applied Nutrition, 5100 Paint Branch Pkwy, College Park, MD 20740, USA; email: narjol.gonzalez-escalona@fda.hhs.gov 2. González-Escalona N, Martinez-Urtaza J, Romero J, Espejo RT, Jaykus LA, DePaola A. Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing. J Bacteriol. 2008;190:2831–40. http:// dx.doi.org/10.1128/JB.01808-07 3. Nair GB, Ramamurthy T, Bhattacharya SK, Dutta B, Takeda Y, Sack DA. Global dissemination of Vibrio parahaemolyticus serotype O3:K6 and its serovariants. Clin Microbiol Rev. 2007;20:39–48. http:// dx.doi.org/10.1128/CMR.00025-06 For feasibility reasons, we used excess serum samples. Seventy-six samples were from healthy volun- teers who gave informed consent; 318 clinical blood samples from patients had been obtained by private labora- tories and by the surveillance labora- tory of the National Malaria Control Programme (PNLP) during August 1–October 8, 2011. The Ministère de la Santé, de la Solidarité et de la Pro- motion du Genre of the Union of the Comoros, authorized the serologic investigation (authorization no. 1175/ MSSPG/DNS). Julie Haendiges, Marvin Rock, Robert A. Myers, Eric W. Brown, Peter Evans, and Narjol Gonzalez-Escalona Author affiliations: Department of Health and Mental Hygiene, Baltimore, Maryland, USA (J. Haendiges, M. Rock, R.A. Myers); and Food and Drug Administration, Col- lege Park, Maryland, USA (E.W. Brown, P. Evans, N. Gonzalez-Escalona) of bacteria, resulting in increased ex- posure of humans to leptospirosis- causing pathogens (1). Among islands in the southwestern Indian Ocean, hu- man leptospirosis is endemic to Mayo- tte, France, and La Réunion (2–4) and to the Seychelles, where the incidence of leptospirosis is one of the highest worldwide (5). Leptospirosis is poorly documented in other islands in the re- gion, including Mauritius, Madagas- car, and the Union of the Comoros (2,6–8). Whether the scant documen- tation indicates underdiagnosis or re- flects local epidemiologic specificities is unknown. To improve knowledge of Leptospira infection in the region, we conducted a study in the Union of the Comoros to serologically assess the presence or absence of leptospirosis in humans. The Union of the Comoros consists of 3 islands: Grande-Comore, Mohéli, and Anjouan. Together with a fourth, southern island, Mayotte, these islands form the Comoros Archipelago. 8. Jolley KA, Maiden MC. Automated extraction of typing information for bac- terial pathogens from whole genome se- quence data: Neisseria meningitidis as an exemplar. Euro Surveill. 2013;18:20379. 9. Bryant D, Moulton V. Neighbor-net: an agglomerative method for the construc- tion of phylogenetic networks. Mol Biol Evol. 2004;21:255–65. http://dx.doi. org/10.1093/molbev/msh018 10. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, et al. Genome sequence of Vibrio para- haemolyticus: a pathogenic mechanism distinct from that of V. cholerae. Lancet. 2003;361:743–9. http://dx.doi.org/10.1016/ S0140-6736(03)12659-1 DOI: http://dx.doi.org/10.3201/eid2004.130818 Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012 It is possible that complete availability of PFGE patterns during the outbreaks (online Technical Appendix 1) could have provided additional insight into the scope of the outbreak and impli- cated food sources. The application of rapid, whole-genome sequencing technology aided our discovery that the Maryland outbreak strains were part of the pandemic CC and likely related to V. parahaemolyticus strains that shared common PFGE patterns and that were reported as the cause of illnesses in several states around the same time as the Maryland outbreak. The presence of this virulent V. parahaemolyticus strain in Maryland is an ongoing public health concern, requiring continued microbiological surveillance. This pandemic strain also indicates the need for establish- ing a V. parahaemolyticus genome database that is accessible worldwide. Such a database would enable im- proved tracking and faster responses to emergent and dangerous pandemic clonal strains. 719 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014 LETTERS Serologic Evidence of Leptospirosis in Humans, Union of the Comoros, 2011 4. DePaola A, Kaysner CA, Bowers J, Cook DW. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl Environ Microbiol. 2000;66:4649–54. http://dx.doi.org/10.1128/AEM.66.11. 4649-4654.2000 To the Editor: Leptospirosis is a worldwide bacterial zoonosis caused by infection with pathogenic Leptospira spp. (Spirochaetales, Lep- tospiraceae). Most mammals can be infected, but rats are considered the main reservoir, maintaining Lepto- spira spirochetes in the lumen of renal tubules and contaminating the envi- ronment with bacteria-infected urine. Transmission to humans is accidental, occurring through contact with animal secretions or with contaminated envi- ronmental materials. 5. Abbott SL, Powers C, Kaysner CA, Takeda Y, Ishibashi M, Joseph SW, et al. Emergence of a restricted bioserovar of Vibrio parahaemolyticus as the predomi- nant cause of vibrio-associated gastro- enteritis on the West Coast of the United States and Mexico. J Clin Microbiol. 1989;27:2891–3. We used the microscopic aggluti- nation test (MAT) to test serum sam- ples; the MAT was based on a panel of 15 Leptospira strains, enabling the screening of all recently reported sero- groups for human and animal cases on neighboring Mayotte (2,4,9). A list of the tested strains follows, shown as Ge- nus species Serogroup/Serovar (type strain): L. borgpetersenii Ballum/Cas- tellonis (Castellon 3), L. borgpeterse- nii Sejroe/Hardjobovis (Sponselee), L. borgpetersenii Sejroe/Sejroe (M 84), 6. Jolley KA, Maiden MC. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioin- formatics. 2010;11:595. http://dx.doi. org/10.1186/1471-2105-11-595 7. Jolley KA, Hill DM, Bratcher HB, Harrison OB, Feavers IM, Parkhill J, et al. Resolution of a meningococ- cal disease outbreak from whole- genome sequence data with rapid Web-based analysis methods. J Clin Mi- crobiol. 2012;50:3046–53. http://dx.doi. org/10.1128/JCM.01312-12 In temperate countries, human leptospirosis is a sporadic disease; inci- dence is much higher in the tropics be- cause climate and environmental con- ditions are conducive to the survival 720 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014
https://openalex.org/W4200247885	http://pure-oai.bham.ac.uk/ws/files/180926131/RickardF2022Clinical.pdf	English	null	Clinical Handover Communication at Maternity Shift Changes and Women's Safety in Banjul, The Gambia: A Mixed-Methods Study	Research Square (Research Square)	2,021	cc-by	12,504	Clinical handover communication at maternity shift changes and women's safety in Banjul, The Gambia Rickard, Faith ; Lu , Fides ; Gustafsson , Lotta ; MacArthur, Christine; Cummins, Carole; Coker, Ivan ; Wilson, Amie; Mane, Kebba ; Manneh, Kebba ; Manaseki-Holland, Semira DOI: 10 1186/s12884 022 05052 9 Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Rickard, F, Lu , F, Gustafsson , L, MacArthur, C, Cummins, C, Coker, I, Wilson, A, Mane, K, Manneh, K & Manaseki-Holland, S 2022, 'Clinical handover communication at maternity shift changes and women's safety in Banjul, The Gambia: a mixed-methods study', BMC pregnancy and childbirth, vol. 22, 784. https://doi.org/10.1186/s12884-022-05052-9 Citation for published version (Harvard): Rickard, F, Lu , F, Gustafsson , L, MacArthur, C, Cummins, C, Coker, I, Wilson, A, Mane, K, Manneh, K & Manaseki-Holland, S 2022, 'Clinical handover communication at maternity shift changes and women's safety in Banjul, The Gambia: a mixed-methods study', BMC pregnancy and childbirth, vol. 22, 784. https://doi.org/10.1186/s12884-022-05052-9 Link to publication on Research at Birmingham portal General rights 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. 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Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of th When citing, please reference the published version. University of Birmingham University of Birmingham Abstract Background: Clinical handover is a vital communication process for patient safety; transferring patient responsibility between healthcare professionals (HCPs). Exploring handover processes in maternity care is fundamental for service quality, addressing continuity of care and maternal mortality. Methods: This mixed-methods study was conducted in all three maternity hospitals in Banjul, The Gambia. Shift- to-shift maternity handovers were observed and compared against a standard investigating content and environ‑ ment. Semi-structured interviews and focus group discussions with doctors, midwives and nurses explored handover experience. Results: One hundred ten nurse/midwife shift-to-shift handovers were observed across all shift times and maternity wards; only 666 of 845 women (79%) were handed over. Doctors had no scheduled handover. Shift-leads alone gave/ received handover, delayed [median 35 min, IQR 24–45] 82% of the time; 96% of handovers were not confidential and 29% were disrupted. Standardised guidelines and training were lacking. 6 of 28 topics [IQR 5–9] were communicated per woman. Information varied significantly by time, ifi i d l i F i l b 10 [IQR 8 14] i h d d 8 [IQR 5 11] A median 6 of 28 topics [IQR 5–9] were communicated per woman. Information varied significantly by time, high-risk classification and location. For women in labour, 10 [IQR 8–14] items were handed-over, 8 [IQR 5–11] for women classed ‘high-risk’, 5 [IQR 4–7] for ante/postnatal women (p < 0.001); > 50% had no care management plan communicated. Twenty-one interviews and two focus groups were conducted. Facilitators and barriers to effective handover sur‑ rounding three health service factors emerged; health systems (e.g. absence of formalised handover training), organi‑ sation culture (e.g. absence of multidisciplinary team handover) and individual clinician factors (e.g. practical barriers such as transportation difficulties in getting to work). Conclusion: Maternity handover was inconsistent, hindered by contextual barriers including lack of team com‑ munication and guidelines, delays, with some women omitted entirely. Findings alongside HCPs views demonstrate feasible opportunities for enhancing handover, thereby improving women’s safety. Keywords: Handover, maternity, communication, safety, quality improvement © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Clinical handover communication at maternity shift changes and women’s safety in Banjul, the Gambia: a mixed‑methods study Faith Rickard1†, Fides Lu1†, Lotta Gustafsson1, Christine MacArthur2, Carole Cummins2, Ivan Coker3, Amie Wilson2, Kebba Mane3, Kebba Manneh4 and Semira Manaseki‑Holland2* Take down policy down policy the University of Birmingham exercises care and attention in making items available there are rare occasions when an it ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 https://doi.org/10.1186/s12884-022-05052-9 Open Access Backgroundh In 2015, The Gambia in West Africa, had an estimated maternal mortality rate (MMR) of 706 maternal deaths per 100,000[1], one of the ten highest worldwide. Since 1990, no significant improvement had been made despite 2 Institute of Applied Health Research, College of Medical and Dental Sciences, University of Brimingham, Edgbaston, Birmingham, UK Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 2 of 13 approaches to measure quality of handover for numerous comparable LMIC’s [30]. global attention to maternity provisions [1, 2]. The major- ity of maternal deaths in low-middle income countries (LMICs) are preventable [3, 4]. To combat this, the World Health Organisation (WHO) has increased focus on the quality of maternity care [5], and has highlighted com- munication failures during patient handovers as one of the top five patient safety problems[6] and a critical com- ponent of WHO standards of quality maternity services and women’s safety [5]. Hence, optimal communication has been identified as a key strategy to reduce adverse events and improve outcomes [5, 7]. The aim of this study was to investigate handover prac- tices between HCPs at shift changes on maternity wards in The Gambia; specifically to assess the quality of hando- ver including barriers and facilitators, as defined by envi- ronment of handover, staff participation and content of clinical information handed over. Findings will be devel- oped into a conceptual model with specific recommenda- tions which could enhance maternity care in The Gambia and parallel LMICs. Shift-to-shift clinical handover is a vital communica- tion point for patient information transfer, fundamental to patient safety [6, 8, 9]. It is defined as the transfer of patient responsibility and accountability between col- laborating healthcare professionals (HCPs). Study design A mixed-methods cross-sectional study was conducted from January to March 2018 in maternity units in all three government hospitals in the urban Greater Banjul region of The Gambia; Hospital 1 (tertiary), 2 (secondary) and 3 (secondary). The mixed-methods design included both quantitative and qualitative methodology. (See Additional File 1 details of health systems and hospitals). Shift-to-shift handover is particularly important within maternity [8, 16, 17]. Complications during pregnancy are frequent and changes in observations and clinical condition occur rapidly affecting both mother and baby [18–20]. In The Gambia and many LMICs, this is exacer- bated by the fact that many women in hospital are high- risk pregnancy referrals. Furthermore, the high rate of grand-multiparity (fertility rate of 5.8 births per woman [21]) increases the risk of complications. Many high- income country (HIC) settings still struggle with opti- mal shift-to-shift maternity handover [22], with studies reporting inconsistent and even inaccurate handovers [23, 24] leading to the implementation of guidelines to standardise handover in several HICs [25, 26]. whilst our systematic review indicates that no research has taken place in LMICs. [27] High-quality handover enables continuity of care between medical providers, facilitating shared awareness of patients’ con- ditions and planned management [10]. Inadequate hand- overs can lead to delays in diagnosis, treatment errors and life-threatening adverse events [9–15]. Statistical analysis It was calculated that a conservative sample size of 385 individual women’s handovers would detect a 50% prev- alence of the handover items communicated with 95% confidence intervals with a 5% margin of error. Informa- tion topics (handover contents) recommended for good handover were categorised by Situation (10 items), Back- ground (11 items), Assessment (3 items), Recommenda- tions (4 items) (SBAR) [25, 26, 31, 32]. Non-parametric tests, Mann–Whitney U or Kruskal–Wallis, were used where appropriate for univariate analysis. Multiple linear regression with the number of items handed over as the dependent variable and the independent variables hos- pital, ward location, time of shift change, day observed, handover lead, whether in active labour, high risk woman, reason for admission, whether questions were asked at handover, whether notes were used, interrup- tions, handover delay, handover duration and number of women handed over was conducted to identify potential confounding factors from amongst those decided a priori using evidence from the literature. [9, 25, 26] Data were analysed using SPSS version 24.0. ff A median of 3 (IQR 2–4) staff changed at each ward shift but only the lead nurse/midwife for the shift received handover as a one-to-one, face-to-face com- munication from the previous lead. There was no fur- ther handover from the shift-lead to the rest of the team except for delegation of specific tasks. Staff read the patient notes or asked the shift-lead if they had questions regarding the women they were caring for. Table 1 Characteristics of observed nurse and midwife handover sessions (no planned doctor handover sessions took place in the period of the study) * non-urgent interruptions; questions from other staff, power cuts, removing relatives, phones ringing, new admissions or finding patient notes Handover characteristics Total no. nurses n Total no. midwives n Total no. doctors n 61 58 42 No. of shift changes observed total n (%) 110 (12) No. of individual women handed over total n (%) 666 (79) No. of shift changes observed Weekdays n (%) Weekends/Bank holidays n (%) 83 (75) 27 (25) No. of shift changes observed n (%) 8am 40 (36) 2 pm 39 (35) 8 pm 31 (28) Duration of handover session in minutes median (IQR) 4 (3–7) No. of handovers delayed > 5 min after shift change n (%) 90 (82) Handover delay in minutes median (IQR) 35 (24–45) No. of shifts where handover not occurred n (%) 5 (4.8) No. Quantitative data collection I f d i Informed written consent was gained to observe staffs (including nurses, midwives, medical officers and doc- tors) who conduct handover on the maternity ward at shift changes for the timeframe of the study. An observation tool covering handover content and environment was completed on the wards by three trained UK medical student researchers, each observing one shift at a time (Additional File 2). Environmental fac- tors included: distractions, maintaining confidentiality, location, use of non-technical language, environment supportive of questions, communication method and documentation, time set aside and HCP involvement. As no prior handover standards existed in The Gam- bia, the observation checklist was adapted from a study in the USA [24] to also include the British Royal Col- lege of Obstetricians and Gynaecologists [25] and the American College of Obstetricians and Gynaecologists [26] guidelines. The researchers classified each woman as “in active labour” or not, and either ’high-risk’ or not (obstetric emergencies or risk factors for complications as defined by the National Institute for Health and Care Excellence and WHO guidelines [18, 19]). Classifica- tion was recorded from information communicated during handover and by asking the staff afterwards. The tool was piloted in The Gambia before use. All hando- vers were conducted in English. To reduce bias, a simi- lar proportion of morning, afternoon and evening shift changes were observed across the three hospitals, seven days a week, including public holidays. As handovers Women globally are encouraged to attend health facili- ties for maternity care from a skilled birth attendant; in The Gambia, 55% of women now attend such facilities to give birth [28]. However, with high workloads, continu- ity and quality of care can be overlooked, likely implicat- ing maternal and infant mortality; anecdotally handover communication plays an important part in this. These problems in care quality can impact the benefits of health facility birth [16, 17]. Clinical handover procedure is dependent on con- text, with current practice and culture creating barri- ers and facilitators to handover unique to specialties, roles and countries [29, 30]. However, important lessons and recommendations for health systems can be learnt by in-depth study of one handover system, developing Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 3 of 13 researcher independently double-coded four of the most data-rich transcripts for analyst triangulation [34]. on different wards occurred simultaneously, researchers moved between wards to attempt to increase observa- tions. Quantitative data collection I f d i Data were recorded onto paper and subsequently digitized using Microsoft Excell. Results A total of 110 nurse and midwife shift changes for 666 individual women (see Table 1 and 2) were observed out of a possible 945 shift changes over the 5 weeks in the three hospitals (Additional File 4). The number of women handed over at each shift ranged from 0 to 24, with 179 (21.2%) women on the wards not being handed over. At 5 observed shift changes (4.8%) there was no verbal hando- ver and the staff left before the next staff arrived.f Statistical analysis of women on each ward median (IQR) 5 (3–13) No. of women handed over on each ward median (IQR) 4 (2–8) No. handover sessions with non-urgent interruptions n (%)* 32 (29) Aids used for verbal handover n (%) Staff wrote own notes 54 (8) Maternity cards/Ward notes 566 (85) Whiteboard Questions asked Standard medical terminology used 52 (8) 168 (25) 646 (97) Qualitative interviewsh The qualitative and quantitative components of the study were conducted in parallel. A purposive sampling frame included all cadres of HCPs (nurses, midwives, medical officers and doctors). Methodology combined both focus group discussions (FGDs) to gain diversified perspec- tives and one-to-one semi-structured interviews (SSIs) to examine personal beliefs and attitudes more compre- hensively. The FGDs were challenging as the HCPs were not available at the same time. No staff took part in both FGDs and SSIs. Participants were recruited until the- matic saturation was achieved. One UK medical student trained in qualitative method- ology conducted all the interviews. FGDs and SSIs were conducted at the hospital sites using a pre-determined piloted topic guide (Additional File 3). All interviews were conducted in English, spoken by all HCPs in The Gambia. The interviews were recorded, transcribed ver- batim and anonymised. Field notes and analytic memos were also made after every interview. * non-urgent interruptions; questions from other staff, power cuts, removing relatives, phones ringing, new admissions or finding patient notes Data analysis Inductive thematic analysis based on Braun and Clarke’s six-step approach [33] was undertaken. A second Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 4 of 13 Table 2 Frequency of inclusion of each information item on data schedule * ’high-risk’ as defined by National Institute for Health and Care Excellence and WHO guidelines. Data analysis For breakdown of conditions observed see Additional File 6 and Methods ** Global comments included, "normal delivery", "post caesarean section", "stable", "you know her", "no problems" and "continue treatment" Item on observation tool n = 28 High-risk* Frequency (%) n = 266 Active labour Frequency (%) n = 94 Not active labour/high- risk Frequency (%) n = 347 Total Frequency (%) n = 666 Situation Lead identified 266 (100) 94 (100) 347 (100) 666 (100) Location 266 (100) 93 (99) 345 (99) 663 (99) Woman’s’ Name 177 (66) 68 (72) 236 (68) 445 (67) Vital signs 79 (30) 56 (60) 39 (11) 152 (23) Specific concerns about woman 51 (19) 20 (21) 27 (8) 84 (13) Gravidity/Parity 26 (10) 41 (44) 12 (3) 65 (10) Key woman’s values 22 (8) 6 (6) 19 (5) 43 (6) Gestation 14 (5) 14 (15) 3 (1) 23 (3) Age 5 (2) 4 (4) 5 (1) 12 (2) Resuscitation status 9 (3) 5 (5) 1 (0) 10 (2) Median (IQR) 3 (3–4) 4 (3–5) 3 (2–3) 3 (3–4) Background Current medications 135 (51) 20 (21) 112 (32) 253 (38) Main complaint 151 (57) 48 (51) 66 (19) 236 (35) Brief history 117 (44) 47 (50) 91 (26) 229 (34) Admission date 109 (41) 56 (60) 78 (22) 222 (33) Diagnosis/Active problems 111 (42) 38 (40) 61 (18) 182 (27) Other chart information 39 (15) 45 (48) 35 (10) 105 (16) Physical examination results 47 (18) 68 (72) 8 (2) 98 (15) Progress from admission 101 (38) 55 (58) 140 (40) 81 (12) Treatment response 38 (14) 4 (4) 17 (5) 56 (8) Laboratory results 29 (11) 17 (18) 13 (4) 48 (7) Allergies 0 (0) 0 (0) 0 (0) 0 (0) Median (IQR) 3 (2–4) 4 (3–6) 1 (1–2) 2 (1–4) Assessment Clinical impression of woman 137 (52) 54 (57) 177 (51) 344 (52) Critical assessment of situation 54 (20) 26 (28) 23 (7) 85 (13) Concerns/problems 49 (18) 24 (25) 23 (7) 81 (12) Median (IQR) 1 (0–1) 1 (0–2) 1 (0–1) 1 (1–1) Recommendations Management plan 139 (52) 54 (57) 142 (41) 302 (45) Time scale 60 (23) 20 (21) 40 (12) 106 (16) Requests/Tests 50 (19) 22 (23) 34 (10) 92 (14) Critical features of management 44 (16) 14 (15) 12 (3) 59 (9) Median (IQR) 1 (0–2) 1 (0–2) 0 (0–1) 0 (0–1) Summary data Median no. Global comments included, "normal delivery", "post caesarean section", "stable", "you know her", "no problems" and "continue treatment" Data analysis items handed over (IQR) 8 (5–11) 10 (8–14) 5 (4–7) 6 (5–9) Global comments n (%) 5 (2) 5 (2) 98 (28) 206 (31) Table 2 Frequency of inclusion of each information item on data schedule Methods Global comments included, "normal delivery", "post caesarean section", "stable", "you know her", "no problems" and "continue treatment" Doctors were often not based on the maternity wards and did not conduct routine shift-to-shift handovers. Instead, they attended to review women when called, or only communicated specific concerns to the next shift doctor on the phone. Occasionally, ward meet- ings discussed new admissions. In one hospital, doctors had 24-h on-call rotas which meant they knew about the women admitted. Page 5 of 13 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Health systems factors fi h d Specific handover practices to facilitate effective hando- ver for high-risk patients were described, such as, the use of a whiteboard to inform the next shift about patients who require extra care. A commonly reported barrier to clinical handover was the extreme staff shortages on the maternity wards. Consequently, handovers were often carried out in pressurised work environments with important clinical information being omitted. Many par- ticipants spoke of how the absence of formal protocols and training resulted in a wide variation in the quality and content of handovers, thus were highly supportive of implementing standardised protocols. Individual handover information content During verbal handovers, out of a possible 28 topics, a median (IQR) of 6 (5–9) SBAR items were communi- cated for each woman (Table 2). Handovers commonly discussed more "Situation" and "Background" topics com- pared to "Assessment" and "Recommendation’’ topics, where in around half of handovers, no information was exchanged (see Additional File 5). No SBAR items were consistently communicated for all women (except "lead identified" and "patient location" which were implied with the bedside handover style). Qualitative results Characteristics of the handovers are given in Table 1. Of the 90 (81.8%) delayed handovers, only 4.5% were due to emergencies or situations requiring the shift-lead’s atten- tion. Emergencies included complex births and post- partum haemorrhage. Handovers in all hospitals were conducted at the end of each woman’s bed except in the labour rooms in Hospital 2 where handover took place in front of a completed whiteboard located in the adjoining staff station (12% of handovers) in view of HCPs, patients and visitors. This displayed women’s details including name, gravidity, parity, vital signs and physical exami- nations. All hospital wards had rooms available where handover could potentially have taken place confiden- tially, however many were already used for other pur- poses. No specific written protocols or pro-forma were available to assist with verbal handover. A report book, located at the nursing station on all wards, contained a brief handwritten summary of certain high-risk women on the ward and was updated at the end of each shift. SSIs were conducted with eight doctors, eight mid- wives and five nurses. Two FGDs were conducted with five midwives and four nurses in Hospital 1. Additional File 7 summarises the demographic details of all 30 participants. Three major themes identifying barriers and facilita- tors to optimal handover emerged from thematic analysis these included healthcare systems factors, organisational cultural factors and individual HCP factors. These themes along with their supporting quotations are displayed in Table 4. A summary of the salient points is given below. Organisational cultural factors Many barriers to handover were identified. The major- ity of HCPs expressed a need for a whole team hando- ver. The high patient-to-staff ratio and a large amount of information being passed on from one shift to the next meant that the lead-nurse was often overloaded with information that was difficult to retain. Furthermore, many participants spoke of the inadequacies of the docu- mentation culture. They reported that “incomplete notes” coupled with “illegible handwriting” could lead to infor- mation lapses regarding patient care. A few respondents discussed how these communication failures had resulted in the omission or duplication of treatments, potentially causing patient harm. A small number of respondents expressed concern about how seemingly stable patients could often be missed during handovers as HCPs focused their attention on patients categorised as more critically ill. The majority reported a lack of punctuality of staff for their shift handovers. Participants discussed how this can jeopardise patient safety, for example, “wards can be left unattended when one set of staff have to leave before the next shift arrive”. Women in active labour versus not in active labour had a median of 4 more information items discussed. Those deemed high-risk also had significantly more items com- municated (Tables 2 and 3). The fewest items were dis- cussed in ante and postnatal wards, where 196 (29%) women were handed over with only a global comment. Comparison of handover content On multivariate linear regression analysis (Table 3), the independent variables that significantly influenced the number of items handed over included the following; with fewer items discussed at the ante/postnatal ward, at the early shift change, tertiary hospital and more dis- cussed where were women were in active labour or high- risk, admission for observation, when questions were asked and when handovers lasted longer. The regression predicted 46% of the variation in information communi- cated (R2 = 0.46, p < 0.001). Rickard et al. Comparison of handover content B would all be speaking at the same level… s school, it’s going to be easy for everybody t system and then they have to train you all o is very small… so most of the staffs here, th they work in the government here on morn close, they go to the private for afternoon… double shift, which is affecting the handing “Main problem of the handing over here… is thing that can improve our handing over is w the ground. So when this one is doing the ha listening so if this one forgot some informati remember this one said this and this one said “We need enough staff, that is now a proble like… 10 or 20 patients… so sometimes wh in the morning, you have a full ward and it i senior midwife that is taking over. You have to… to remember, to put in your head and miss one or two information” (M2) [B] “I was working in a clinic where it’s all electr struggling to read the handwriting [B], if I h there will not be any delay in the informatio worry about the folders getting torn or you lot of time will be saved.” (D6) [F] Organisational cultural factors Facilitator • Monitoring of handover practice Barrier • Absence of multidisciplinary team handover • Incomplete documentation for handover • HCPs not performing a detailed handover to the next shift • Healthcare staff tardiness “There are days that the handing over may ately but we, from time to time, we pop in a been doing so as the one who supervises th their toes from time to time to see whether being done or not.” (D4) [F] “Nurse will hand over to the nurse, mostly d to the doctors. Organisational cultural factors Supporting Quotations You have a lot of information to… to remember, to put in your head and sometimes you tend to miss one or two information” (M2) [B] “I was working in a clinic where it’s all electronic… So rather me struggling to read the handwriting [B], if I have that it’s easy… there will not be any delay in the information… you don’t have to worry about the folders getting torn or you losing the papers… a lot of time will be saved.” (D6) [F] Facilitator • Monitoring of handover practice Barrier • Absence of multidisciplinary team handover • Incomplete documentation for handover • HCPs not performing a detailed handover to the next shift • Healthcare staff tardiness “There are days that the handing over may not be done appropri‑ ately but we, from time to time, we pop in and see what they have been doing so as the one who supervises them, I have to be on their toes from time to time to see whether the handing over is being done or not.” (D4) [F] “Nurse will hand over to the nurse, mostly doctors will hand over to the doctors. It’s all separate.” (N5) [B]“…and not all the patients are even written in the book….” (M13) [B] • Healthcare staff tardiness “there is no proper documenting… you may have done some‑ thing for a patient, you did not record it… the one who hand over may come and… give the same medications to the same person.” (N7) [B]“Sometimes even the handing over is not done. You only write report and leave the book there. When they come, they read from the report book… … we only report special cases and you leave the rest because we are rushing to leave… [B] “If I am in a shift and a nurse didn’t come until after one hour, the nurse is late, I leave the patients.” (M13) [B] “when you have to hand over and then the doctor that is sup‑ posed to come is a bit late. Comparison of handover content It’s all separate.” (N5) [B]“…an are even written in the book….” (M13) [B] “there is no proper documenting… you ma thing for a patient, you did not record it… t may come and give the same medication Table 4 Themes with supporting quotations showing barriers and facilitators to effective sh Theme Concept Supporting Quota Health systems / hospital systems factors Facilitator • Some tools and procedures for effective handover Barrier • Absence of confidentiality of notes • Absence of protocols and standards • Absence of formalised handover training • Poor salary • Extreme staff shortages on the ward • Absence of electronic based record system which could help legibility and rapid information exchange “we have a board w staff that this patie as a reminder, the b have to do these th “[For] high-risk pati a red pen to outlin they [the HCPs] see immediately.” (D3) “the manual book come in from anot database you can Comparison of handover content BMC Pregnancy and Childbirth (2022) 22:784 Page 6 of 13 Table 3 Handover characteristics and associated predicted number of items handed over in multiple regression analysis Mann–Whitney U test Handover characteristics Frequency (%) median (IQR) Median (IQR) Items p value Regression coefficient (95% CI): change in n of handover items discussed p value Hospital Hospital 1 190 (28) 5 (4–8) -0.82 (-1.54 to -0.09) 0.028 Hospital 2 172 (26) 8 (5–11) < 0.001 1 Hospital 3 304 (46) 6 (5–9) 0.11 (-0.48 to 0.70) 0.720 Ward Location Labour ward 104 (16) 11 (8.-14) 1 High Dependency Unit 99 (15) 8 (6–11) < 0.001* -0.83 (-1.91 to 0.25) 0.130 Ante/postnatal ward 463 (69) 5 (4–7) -1.77 (-2.85 to -0.68) 0.001 Time of shift change 8am 311 (47) 6 (5–8) -0.84 (-1.41 to -0.27) 0.004 2 pm 196 (29) 7 (5–12) < 0.001* 1 8 pm 159 (24) 7 (5–10) 0.15 (-0.50 to 0.80) 0.964 Day observed Weekday 498 (75) 6 (5–9) 0.130 1 Weekend/Bank holiday 168 (25) 7 (4–9) -0.27 (-0.76 to 0.22) 0.277 Handover lead Midwife 253 (38) 6 (5–9) 1 Nurse 412 (62) 6 (5–9) 0.230 0.43 (-0.10 to 0.96) 0.115 Active labour No 572 (86) 6 (5–9) 1 Yes 94 (14) 10 (8–14) < 0.001 1.06 (0.12 to 1.99) 0.027 High-risk woman No 400 (60) 6 (5–9) 1 Yes 266 (40) 8 (5–11) < 0.001 1.30 (0.79 to 1.82) < 0.001 Reason for admission Labour 615 (92) 6 (5–9) 1 Observation 51 (8) 7 (5–12) 0.411 -1.01 (-1.83 to -0.20) 0.015 Questions asked at handover No 168 (25) 6 (4–9) 1 Yes 498 (75) 7 (5–10) < 0.001 1.11 (0.58 to 1.64) < 0.001 Notes used at handover No 612 (92) 6 (5–9) 1 Yes 54 (8) 6 (5–9) 0.118 0.12 (-0.65 to 0.89) 0.761 Interruptions No 609 (91) 7 (5–9) 1 Yes 57 (9) 5 (8–4) < 0.001** 0.23 (-0.56 to 1.02) 0.573 Handover delay Time after official shift change (mins) 35 (24–45)* - - 0.02 (0.01 to 0.03) 0.002 Handover duration Length (mins) 4 (3–7)* - - 1.99 (1.46 to 2.53) < 0.001 Number of women handed-over Number of women 4 (2–8)* - - 0.08 (0.03 to 0.14) 0.003 *** Kruskal–Wallis test Page 7 of 13 Rickard et al. Supporting Quotations “we have a board where we write the precaution to the incoming staff that this patient is high-risk or critical or needs attention so as a reminder, the board is there to remind the staff that look you have to do these things.” (M6) [F] g “[For] high-risk patients, we do make a special handing over… I use a red pen to outline that information [in patient’s notes] … when they [the HCPs] see that red column, they call their attention more immediately.” (D3) [F] “the manual book that we write the data in… someone can just come in from another ward and open the book [B]… but if it is a database, you can have a code or like a password so it is only us that can get access to the patient data.” (M2) [F] g p “we don’t have a written guideline…having a protocol also gives you a more clear view of what you actually need for the patient need to do for that patient and what emphasis to put on patients’ care.” (D5) [B] “if we have that [formal training of handover] from medical school or nursing school that would be fantastic. Because then at least we would all be speaking at the same level… so that once we finish school, it’s going to be easy for everybody than coming into the system and then they have to train you all over.” (D6) [B]“the salary is very small… so most of the staffs here, they work in two places, they work in the government here on morning shift. When they close, they go to the private for afternoon… you have to work d bl hif hi h i ff i h h di ”(M13) [B] “if we have that [formal training of handover] from medical school or nursing school that would be fantastic. Because then at least we would all be speaking at the same level… so that once we finish school, it’s going to be easy for everybody than coming into the system and then they have to train you all over.” (D6) [B]“the salary is very small… so most of the staffs here, they work in two places, they work in the government here on morning shift. Supporting Quotations When they close, they go to the private for afternoon… you have to work double shift, which is affecting the handing over.” (M13) [B] “Main problem of the handing over here… is short of staff... the only thing that can improve our handing over is when we have staffs on the ground. So when this one is doing the handing over, others are listening so if this one forgot some information, then the others will remember this one said this and this one said this.” (M1) [B] “We need enough staff, that is now a problem… I have more than like… 10 or 20 patients… so sometimes when we are taking over in the morning, you have a full ward and it is only one midwife- senior midwife that is taking over. You have a lot of information to… to remember, to put in your head and sometimes you tend to miss one or two information” (M2) [B] “I was working in a clinic where it’s all electronic So rather me double shift, which is affecting the handing over.” (M13) [B] “Main problem of the handing over here… is short of staff... the only thing that can improve our handing over is when we have staffs on the ground. So when this one is doing the handing over, others are listening so if this one forgot some information, then the others will b thi id thi d thi id thi ”(M1) [B] remember this one said this and this one said this. (M1) [B] “We need enough staff, that is now a problem… I have more than like… 10 or 20 patients… so sometimes when we are taking over in the morning, you have a full ward and it is only one midwife- senior midwife that is taking over. Comparison of handover content BMC Pregnancy and Childbirth (2022) 22:784 Table 4 Themes with supporting quotations showing barriers and facilitat Theme Concept Health systems / hospital systems factors Facilitator • Some tools and procedures for effective handover Barrier • Absence of confidentiality of notes • Absence of protocols and standards • Absence of formalised handover training • Poor salary • Extreme staff shortages on the ward • Absence of electronic based record system which could help legibility and rapid information exchange Organisational cultural factors Facilitator • Monitoring of handover practice Barrier • Absence of multidisciplinary team handover • Incomplete documentation for handover • HCPs not performing a detailed handover to the next shift • Healthcare staff tardiness Table 4 Themes with supporting quotations showing barriers and facilitators to effective shift-to-shift handover Theme Concept Supporting Quotations Health systems / hospital systems factors Facilitator • Some tools and procedures for effective handover Barrier • Absence of confidentiality of notes • Absence of protocols and standards • Absence of formalised handover training • Poor salary • Extreme staff shortages on the ward • Absence of electronic based record system which could help legibility and rapid information exchange “we have a board where we write the preca staff that this patient is high-risk or critical o as a reminder, the board is there to remind have to do these things.” (M6) [F] “[For] high-risk patients, we do make a spec a red pen to outline that information [in pat they [the HCPs] see that red column, they c immediately.” (D3) [F] “the manual book that we write the data in come in from another ward and open the b database, you can have a code or like a pass that can get access to the patient data.” (M2 “we don’t have a written guideline…having you a more clear view of what you actually need to do for that patient and what emph care.” (D5) [B] “if we have that [formal training of handove or nursing school that would be fantastic. Individual HCP factors the information content and environment of handover in all labour, high-dependency, antenatal and postnatal wards in all public maternity hospital units in The Gam- bia’s capital city to establish the facilitators and barriers to optimal handover. Qualitative and quantitative data supported and expanded on findings; despite no formal handover protocol, training or monitoring the concept of handover, including patient safety aspects and need for confidentiality, was widely understood and valued by HCPs. However, numerous aspects of shift-to-shift hand- over need attention in order to reduce errors experienced by HCPs and improve continuity of care. These involved the lack of full staff participation, missed handover ses- sions or missed women, handover environment, variable and scanty clinical content communicated. Important facilitators were that most of the staff were aware of the importance of handover. Transportation difficulties, frequently resulting in staff tardiness, were repeatedly mentioned as a barrier to effective handover. Participants also mentioned social factors, including the shortage of childcare provisions in close proximity to the hospitals, as an additional barrier. Supporting Quotations “I’ve experienced, if you don’t do proper handing over, it leads to some problems. Lapses are made about a patient.” (M13) [B] “there is no proper documenting… you may have done some‑ thing for a patient, you did not record it… the one who hand over may come and… give the same medications to the same person.” (N7) [B]“the only way to solve it [of HCP tardiness] is the hospital to provide transport for the staff… everyone struggle on their own… you will stand one hour, two hours just to have a car to go home, so it’s a problem and the salary is not that much for you to hire a taxi every day.” (M12) [B] “here, we don’t have a centre where you put your kids, a day care… So I think also it could be part of solving the problem [of HCP tardiness], if we had a centre for mothers here where you can put your child.” (M13) [B] “… also the other thing is that their handwriting is bad… you can’t read… Handwriting is important because you’re writing for someone to read so if you’re writing it and someone else can’t read it so it’s useless, it’s like don’t write.” (D7) [B] “Not everyone has basic skills in this computer…it [an electronic system] would be advantageous for us, the ones that have skills on it, but the ones that don’t have skills on it, they will not even look at it, they will not even bother themselves.” (N5) [B] “And sometimes you either work on afternoon here then go to private at night, so like for example, those on morning shift here, they are always in a haste… to go early to that other hospital they are working… Because you that feel here, the salary is not much for you, you have to work double shift, which is affecting the hand‑ ing over.” (M13) [B] Abbreviations: The code letter after each quotation refers to the cadre of HCP; doctor (D), midwife (M) and nurse (N). [F] illustrates a facilitator; [B] shows a barrier Mixed method triangulationh The quantitative and qualitative results were triangu- lated in order to guide intervention development [35]. The health systems, organisational cultural and indi- vidual HCP factors that interact and influence effective handover as barriers and facilitators, in our setting were applied to develop a conceptual diagram (Fig. 1). For example: need for standardised protocols is recognised by motivated individuals, health systems are required to drive development, with organisational support essential to address standardisation, use of tools and promoting inclusion of all women in shift-to-shift handover. We have translated the research findings into a frame- work of systems culture, organisational culture and indi- vidual HCP factors. These themes identified aligned with those in the Australian Commission on Safety and Qual- ity in Health Care [36] and Humphries et al. in India [37]; indicating that the framework embraces factors applica- ble to maternity units in other LMICs alongside other specialities. The following discussion utilises this foun- dation to develop interventions and recommendations supporting wider maternity service assessment and care improvement. Supporting Quotations difficulty with transport getting to work • Absence of childcare facilities in close proximity to the hospitals • Illegible handwritten notes • Many HCPs are computer illiterate • HCPs working in multiple places and working double shifts “I’ve experienced, if you don’t do proper handing over, it leads to some problems. Lapses are made about a patient.” (M13) [B] “there is no proper documenting… you may have done some‑ thing for a patient, you did not record it… the one who hand over may come and… give the same medications to the same person.” (N7) [B]“the only way to solve it [of HCP tardiness] is the hospital to provide transport for the staff… everyone struggle on their own… you will stand one hour, two hours just to have a car to go home, so it’s a problem and the salary is not that much for you to hire a taxi every day.” (M12) [B] “here, we don’t have a centre where you put your kids, a day care… So I think also it could be part of solving the problem [of HCP tardiness], if we had a centre for mothers here where you can put your child.” (M13) [B] “… also the other thing is that their handwriting is bad… you can’t read… Handwriting is important because you’re writing for someone to read so if you’re writing it and someone else can’t read it so it’s useless, it’s like don’t write.” (D7) [B] “Not everyone has basic skills in this computer…it [an electronic system] would be advantageous for us, the ones that have skills on it, but the ones that don’t have skills on it, they will not even look at it, they will not even bother themselves.” (N5) [B] “And sometimes you either work on afternoon here then go to private at night, so like for example, those on morning shift here, they are always in a haste… to go early to that other hospital they are working… Because you that feel here, the salary is not much for you, you have to work double shift, which is affecting the hand‑ ing over.” (M13) [B] Abbreviations: The code letter after each quotation refers to the cadre of HCP; doctor (D), midwife (M) and nurse (N). [F] illustrates a facilitator; [B] shows a barrier Supporting Quotations You have to sit and wait until she comes or he comes… that is affecting the handing over because you have to go home, you have another activity to do.” (D2) [B] “most of the time when they come in the morning, either the night staff are rushing to go home or the morning staff are late, so they don’t usually hand over properly.” (D6) [B] “there is no proper documenting… you may have done some‑ thing for a patient, you did not record it… the one who hand over may come and… give the same medications to the same person.” (N7) [B]“Sometimes even the handing over is not done. You only write report and leave the book there. When they come, they read from the report book… … we only report special cases and you leave the rest because we are rushing to leave… [B] “If I am in a shift and a nurse didn’t come until after one hour, the nurse is late, I leave the patients.” (M13) [B] “when you have to hand over and then the doctor that is sup‑ posed to come is a bit late. You have to sit and wait until she comes or he comes… that is affecting the handing over because you have to go home, you have another activity to do.” (D2) [B] “most of the time when they come in the morning, either the night staff are rushing to go home or the morning staff are late, so they don’t usually hand over properly.” (D6) [B] Page 8 of 13 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Table 4 (continued) Theme Concept Individual healthcare professional factors Facilitator • HCP knowledge and experience of importance of handover in patient safety Barrier • Practical barriers e.g. difficulty with transport getting to work • Absence of childcare facilities in close proximity to the hospitals • Illegible handwritten notes • Many HCPs are computer illiterate • HCPs working in multiple places and working double shifts Table 4 (continued) Theme Concept Supporting Quotations Individual healthcare professional factors Facilitator • HCP knowledge and experience of importance of handover in patient safety Barrier • Practical barriers e.g. Discussion Information communication at handover presents a major patient safety challenge globally [6]. Based on set criteria, we assessed the rate of women handed over, Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 9 of 13 Fig. 1 Conceptual diagram for Influential Factors—Model showing triangulation of interaction between health systems, organisational culture and individual HCP factors, and their contributing influence on maternity handover m for Influential Factors—Model showing triangulation of interaction between health systems, organisational culture and d their contributing influence on maternity handover Fig. 1 Conceptual diagram for Influential Factors—Model showing triangulation of interaction between health systems, o individual HCP factors, and their contributing influence on maternity handover Standardisation protocols and trainingh and sustainable solutions [5, 30, 40, 41]. Staff awareness and experience, verbalising the potential dangers of inade- quate or missed handovers, is a vital individual factor facil- itating these recommendations and to improve a patient safety culture prioritising handover communication. The important obstacles to most aspects of optimal handover were health systems factors, primarily lack of standards, protocols and training which were strongly supported by our HCPs. Studies in HICs alongside WHO standards for HICs and LICs, currently advocate guide- lines and pro-forma for maternity handovers; apply- ing tools such as SBAR for efficient communication and reducing adverse events. A simple checklist would further support information retention in staff who felt overloaded with information [5, 25, 26, 38]. A continuum of culturally- sensitive training, formal and informal, running alongside behaviour change methods (such as non-monitory incen- tives, handover champions, team supervision and moni- toring) focussed on increasing the impact of handover, is needed to embed handover processes with the aim of improving patient outcomes [39]. These highlight low-cost Verbal versus written handover practices Handovers were commonly verbal only. Alongside a lack of standardised handover documentation and vari- able legibility of notes instead left staff reportedly rely- ing upon memory or judgement for patient care. This was viewed as an organisational culture issue, where the importance of complete and legible notes was not a pri- ority, even though many staff reported the likely result of errors in care [14, 15, 25, 49]. Practices including writing notes on scraps of paper and the report book (located at all nursing stations containing a brief handwritten sum- mary of some high-risk women, updated each shift) was not unfamiliar and could be built upon to facilitate handover. Whiteboards were one method employed successfully in one ward to provide handover structure and clarity. For the larger wards (30 beds), creative use of boards needs to be considered [50, 51]. Adoption of electronic databases has been shown to increase the efficiency of information exchange and was supported by our HCPs [52]. However, unreliable electricity supply, funds for establishing such a system, alongside varying HCP com- puter literacy, are barriers to this solution currently in The Gambia’s health system. Location‑specific handover with inclusion of all women Location specific handover with inclusion of all women Critically over 20% of women were missed from hando- ver, and most others had little clinical information com- municated. The WHO recommends in the interest of patient safety [43], quality improvement and optimal pregnancy outcomes, a standardised handover with clear, accurate information transfer should be performed and promoted for all pregnant women [40]. Co-ordinated care for every woman is arguably a priority safety con- cern in sub-Saharan Africa (SSA) in order to reduce the persistently high maternity death rates [1, 5, 44]. In The Gambia and SSA, many women admitted in mater- nity wards are high-risk (40% in our study with many referred from local health centres). In active labour, but also ante-natal and post-partum stages, these women by definition need close monitoring for unexpected changes in condition [5, 8]; with over half of maternal and new- born deaths occurring in the first few days post-partum [20, 44]. Health systems based intervention, addressing specific communication requirements in different stages, utilising targeted tools such as the WHO Safe Childbirth Checklist, could reduce handover variability and missed women, guiding continuity of care. Delays and interruptions Lack of protocols led to a number of downstream barri- ers to comprehensive handovers. With no set times for shift-to-shift handover, timing varied largely leading to absence of either coming or leaving staff or shortened handovers, with some handovers skipped completely. Exacerbated by staff shortages, this was a major barrier blurring the transition of patient responsibility [41]. At the HCP level, prompt handover was not prioritised and significant individual barriers such as finding childcare and transport to and from work were identified. Provi- sion of transport services for staff alongside availability of childcare centres around the hospitals were suggested and could be paid for by the HCPs (as they currently bear the cost) are potential facilitators to punctuality. These organisational interventions, could improve staff motiva- tion and encourage HCPs’ full and equal engagement in work [45, 46]. Clinician team participation Other health systems barriers to adequate handover existed in that doctors have no set handover routine, obtaining information through asking on-ward staff, patients’ notes or calling doctors on the last shift. Fur- thermore, for nurses/midwives, only the shift-leads gave and received handover which was not formally passed on to the rest of the team. This limits proactive monitoring/treatment of women, amplifying the risk of adverse events with potentially critical gaps in clinical information transferred to the majority of HCPs [7]. As Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 10 of 13 Page 10 of 13 Compared to other times, 8am handover had fewer information items communicated. Fatigue from 12-h shifts alongside handover delays may have played an important role in this [46]. Interruptions during hando- ver were frequent and acceptable from organisational culture perspective. This is a recognised cause of short- ened, inaccurate or incomplete handovers, exacerbated by lack of protected time and staff shortage [47, 48]. Addressing these systems based standards, monitoring a protected handover time and prioritising attendance over other tasks (excluding emergencies) could avoid delays, improving information transfer [29]. suggested by our participants, promoting engagement of all junior and senior staff in handover could improve teamwork, quantity of information communicated[8] and provide valuable learning opportunities for junior staff to confidently support optimal handover culture [39, 42]. However, practically implementing a multidisci- plinary team handover in these busy maternity hospitals would require a change in organisational culture with support and staffing [8]. Promoting service utilisation in LMICsl g Reflecting on handover beyond simply provision of care, handover is intricately linked with patient satisfac- tion and patient-centred care [5, 47]. Holistic concepts (patient values and concerns) were scarcely discussed and all but 4% of handovers were not confidential [48]. This was an organisational culture issue with little pri- oritisation given to patient-centred care, likely given the clinical pressure of work and lack of human resources. For example, all hospital wards had private/staff rooms available where handover could potentially have taken place confidentially, however these were often used for other purposes. In a country where only 55% of women attend health facilities for childbirth, improving women’s Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 11 of 13 Page 11 of 13 further triangulating quantitative and qualitative data, the richness of data was maximised to create a versatile framework. experiences has the potential to encourage women to seek healthcare, improving national maternity outcomes [4, 28]. Although the majority of the SSIs were conducted in private, some were carried out on the ward (HCPs unable to leave the ward unattended) which could have com- promised participants speaking freely. Quantitative data analysis was limited by the observation tool placing equal weighting on each SBAR information item. Some items may have greater impact on patient care, particularly across different wards. Additionally, researcher presence could have influenced handover, with some HCPs con- ducting a more detailed handover and others omitting information. However, we consider the large sample size would have diluted these effects. Furthermore, classifica- tion of high-risk relied on information given at handover and clarifying with HCPs afterwards. This may have over- estimated the quality of information discussed for this group since some high-risk women may not have been identified. The checklist used was adopted from a study in USA as we could find nothing similar from a LMIC setting. The qualitative findings however supported use of the checklist as HCPs demonstrated and volunteered the importance of similar principles, facilitating the development of specific and targeted recommendations. Challenges to adequate handover as demonstrated in this LMIC are ongoing globally across maternity services [8, 22–24, 47]. Handover interventions proposed utilise a low-cost, culturally-sensitive, multi-faceted approach (Table 5). This involves health system managers along- side staff participation with activities that prioritise patient-safety and patient-centred culture [5, 7, 8, 41, 43]. Strengths and limitationshi This study reports the first research on handover prac- tices in maternity care in Africa. Given the high MMR in SSA, this is a significant contribution to maternity care improvement. Although handover is location, con- text and culture specific, important lessons and rec- ommendations for comparable LMIC’s can be learnt by in-depth study of one handover system such as this. Further research should investigate rural regions of The Gambia and other LMICs to increase generalisability and transferability.h Promoting service utilisation in LMICsl Importantly, our qualitative data demonstrated HCPs’ willingness and drive to provide high-quality care and improve skills. However, clear evidence and consensus on specific information and environment required at mater- nity handovers requires further development [8, 23, 24, 38, 47, 53]. Our framework (Fig. 1) could begin to pro- vide a conceptual guide for this process. Conclusion Cl l h f Clinical shift-to-shift handover in maternity care is a critical communication moment with major impli- cations for maternity care safety globally. This study has identified culturally-sensitive recommendations to address the complex barriers to effective commu- nication in one sub-Saharan African capital city, rec- ommending that it should become routine to discuss all women at handover (even if briefly) and reducing This study minimised selection bias by provid- ing observations across all three-government mater- nity hospitals in the capital city; researchers observed all maternity wards across a random selection of days and times, including weekends and public holidays. By Table 5 Shift-to-shift handover recommendations Factors Proposed improvements Healthcare systems Developing targeted national handover guidelines + re-audit to refine situational appropriateness Implement handover training within educational curricula + ongoing professional development Consider solutions to staff shortages and salary driving HCPs to work at multiple jobs Further assess the role of handover in quality of patient care with further research into the links proposed Individual clinicians Facilitate work attendance and punctuality by considering transport and childcare options Encourage and support HCPs existing awareness and recognition of the importance of handover Address note taking and written handover including the consideration of handwriting legibility Organisational Encourage full team participation in handover for HCPs training, teamwork and accurate handover Support handover of all women to minimum criteria as supported by guideline development Consider the impact of incomplete documentation and the availability of resources for written handover Address the organisational tardiness culture alongside improvements linked to developing handover protocol and approaching the individual factors as above Table 5 Shift-to-shift handover recommendations Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Rickard et al. BMC Pregnancy and Childbirth (2022) 22:784 Page 12 of 13 Page 12 of 13 information omission, particularly for antenatal and postnatal women. Alongside this, investigating innova- tive methods to improve punctuality and to implement a protected handover time/location is an important next step. Identification of standardised priority top- ics to be communicated in different locations will aid the development of specific guidelines and reduce com- munication lapses. We advocate a multi-level approach prioritising systems approaches such as hospital policy alongside formalising handover guidelines, monitoring and training in The Gambia. Importantly, much of the factors affecting handover visualised in our framework are also relevant to other acute specialities and wards in LMICs and add to the generalisability of our findings in hospital settings. Funding This study was financed by an Intercalated Award funded by the Arthur Thompson Trust and by BMedSc Population and Health Sciences student funding. 10. Guthrie B, Saultz JW, Freeman GK, et al. Continuity of care matters. BMJ. 2008;337: a867. 10. Guthrie B, Saultz JW, Freeman GK, et al. Continuity of care matters. BMJ. 2008;337: a867. Author details 1 The online version contains supplementary material available at https://doi. org/10.1186/s12884-022-05052-9. 1 University of Birmingham Medical School, Edgbaston, Birmingham, UK. 2 Institute of Applied Health Research, College of Medical and Dental Sciences, University of Brimingham, Edgbaston, Birmingham, UK. 3 Bundung Maternal and Child Health Hospital, Banjul, The Gambia. 4 Kanifing General Hospital, Banjul, The Gambia. Additional file 1. Hospital settings and background information. Additional file 2. Quantitative Data observation tool. Additional file 3. Interview Topic Guide. Supplementary file4. Graph showing spread of observations across days of week, hospitals and shift changes. Additional file 5. Table of total SBAR items discussed at handover. Additional file 6. Table of high-risk and obstetric emergency conditions observed. Additional file 7. Table of characteristics of participants in SSIs and FDGs. Additional file 1. Hospital settings and background information. Additional file 2. Quantitative Data observation tool. Additional file 3. Interview Topic Guide. Supplementary file4. Graph showing spread of observations across days of week, hospitals and shift changes. Additional file 5. Table of total SBAR items discussed at handover. Additional file 6. Table of high-risk and obstetric emergency conditions observed. Additional file 7. Table of characteristics of participants in SSIs and FDGs. Received: 9 November 2021 Accepted: 28 July 2022 Received: 9 November 2021 Accepted: 28 July 2022 Supplementary file4. Graph showing spread of observations across days of week, hospitals and shift changes. Additional file 6. Table of high-risk and obstetric emergency conditions observed. Acknowledgements We would like to thank Dr Buba Manjang for his support in The Gambia. Thank you to Dr Sayeed Haque and Dr Gilles de Wildt for their expertise and guid‑ ance. We would also like to thank the hospital administrators in The Gambia, Ahmadou Samateh, Malang Ndong, Kebba Mane, Baboucarr Saine, Kebba Manneh and all the staff, nurses, doctors and medical officers in all three hospitals for their cooperation and time. 3. Paxton A, Maine D, Freedman L, et al. The evidence for emergency obstetric care. Int J Gynecol Obstet. 2005;88(2):181–93. 4. Thaddeus S, Maine D. Too far to walk: maternal mortality in context. Soc Sci Med. 1994;38(8):1091–110. 5. WHO. Sexual and reproductive health. Standards for improving quality of maternal and newborn care in health facilities. 2016. Available: http:// www.who.int/reproductivehealth/publications/maternal_perinatal_ health/improving-mnh-health-facilities/en/. Accessed Nov 2018. Additional file 7. Table of characteristics of participants in SSIs and FDGs. Additional file 7. Table of characteristics of participants in SSIs and FDGs. 2. UN General Assembly. Transforming our world: The 2030 Agenda for Sus‑ tainable Development, 2015. Available: http://www.refworld.org/docid/ 57b6e3e44.html [Accessed Oct 2018]. Competing interests All authors declare no competing financial, personal or professional interests that may have influenced the research. Authors’ contributions The authors FR and FL recognise each other as contributing jointly to this study in conception, collation, analysis and writing. They share joint first authorship. LG was involved in study design and data collection. SMH computerised the study, CM and CC were involved in the design of this study and interpretation of the study findings. IC was involved in setting up the study and responsible for presenting the projects to the ethics commit‑ tee in The Gambia. AW, KM, KM were involved in the write up of the study and expertise in maternal health. The author(s) read and approved the final manuscript. 6. WHO. High 5s project - Action on patient safety. World Health Organisa‑ tion; 2013. Available: https://www.high5s.org/bin/view/Main/WebHome. Accessed Nov 2018. 6. WHO. High 5s project - Action on patient safety. World Health Organisa‑ tion; 2013. Available: https://www.high5s.org/bin/view/Main/WebHome. Accessed Nov 2018. 7. Wilson RM, Michel P, Olsen S, et al. Patient safety in developing countries: retrospective estimation of scale and nature of harm to patients in hospi‑ tal. BMJ. 2012;344: e832. 7. Wilson RM, Michel P, Olsen S, et al. Patient safety in developing countries: retrospective estimation of scale and nature of harm to patients in hospi‑ tal. BMJ. 2012;344: e832. 8. Lyndon A, Zlatnik MG, Watcher RM. Effective physician-nurse commu‑ nication: a patient safety essential for labor and delivery. Am J Obstet Gynecol. 2011;205(2):91–6. 8. Lyndon A, Zlatnik MG, Watcher RM. Effective physician-nurse commu‑ nication: a patient safety essential for labor and delivery. Am J Obstet Gynecol. 2011;205(2):91–6. 9. British Medical Association, NHS Modernising Agency, NHS National Patient Safety Agency. Safe Handover, Safe Patients: Guidance on Clinical Handover for Clinicians and Managers. London: British Medical Associa‑ tion, 2004. Ethical Approval and consent to participate Local ethical approval was granted by the Scientific Coordinating Committee and Gambia Government/MRC Joint Ethics Committee on behalf of all data collection sites on the 26th January 2018 (SCC 1582v1.1 and SCC 1581V1.1). Ethical approval was granted by the University of Birmingham BMedSc Popu‑ lation Sciences and Humanities Internal Ethics Review Committee on 12th January 2018 (IREC2017/1414091and IREC2017/1410620). Informed written consent was gained from all HCPs delivering and receiving handover, prior to observations and participation in interviews. The data observation tool did not request names or other identifying variables to ensure anonymity of data for research purpose only. All methods were carried out in accordance with relevant guidelines and regulations. Local ethical approval was granted by the Scientific Coordinating Committee and Gambia Government/MRC Joint Ethics Committee on behalf of all data collection sites on the 26th January 2018 (SCC 1582v1.1 and SCC 1581V1.1). Ethical approval was granted by the University of Birmingham BMedSc Popu‑ lation Sciences and Humanities Internal Ethics Review Committee on 12th January 2018 (IREC2017/1414091and IREC2017/1410620). Informed written consent was gained from all HCPs delivering and receiving handover, prior to observations and participation in interviews. 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 31. Availability of data and materials 19. WHO. Pregnancy, childbirth, postpartum and newborn care: A guide for essential practice (3rd edition): World Health Organisation, 2015. Avail‑ able: http://www.who.int/maternal_child_adolescent/documents/imca- essential-practice-guide/en/ [Accessed Oct 2018]. 44. Li XF, Fortney JA, Kotelchuck M, et al. The postpartum period: the key to maternal mortality. Int J of Gyne & Obs. 1996;54(1):1–10. 45. International Finance Corporation. Tackling Childcare: The Business Case for Employer-Supported Childcare. 2017. 20. Lawn JE, Cousens S, Zupan J. 4 million neonatal deaths: when? Where? Why? The Lancet. 2005;365(9462):891–900. 46. Fealy G, Munroe D, Riordan F, et al. Clinical handover practices in maternity services in Ireland: A qualitative descriptive study. Midwifery. 2016;39:20–6. 21. The World Bank. Fertility rate, total (births per woman). 2015. Available: https://data.worldbank.org/indicator/SP.DYN.TFRT.IN?locations=GM [Accessed Oct 2018]. 47. Riesenberg LA, Leisch J, Cunningham JM. Nursing handoffs: a systematic review of the literature. Am J Nurs. 2010;110(4):24–34. 22. MBRRACE-UK. Saving babies lives, improving mothers’ care. Maternal, neonatal and infant clinical outcome review programme. 2016. 48. Farhan M, Brown R, Woloshynowych M, et al. The ABC of handover: a qualitative study to develop a new tool for handover in the emergency department. Emerg Med J. 2012;29(12):941–6. 23. Holly C, Poletick EB. A systematic review on the transfer of information during nurse transitions in care. J Clin Nurs. 2014;23(17–18):2387–96. 49. Bhabra G, Mackeith S, Monteiro P, et al. An experimental comparison of handover methods. The Annals of The Royal College of Surgeons of England. 2007;89(3):298–300. 24. Goff SL, Knee A, Morello M, et al. Handoff quality for obstetrical inpatients varies depending on time of day and provider type. J Reprod Med. 2014;59(3–4):95. 25. Royal College of Obstetricians and Gynaecologists. Improving Patient Handover (Good Practice No.12). 2010. Available: https://www.rcog. org.uk/en/guidelines-research-services/guidelines/good-practice-12/ [Accessed Nov 2018]. 50. Wong HJ, Caesar M, Bandali S, et al. Electronic inpatient whiteboards: improving multidisciplinary communication and coordination of care. Int J Med Informatics. 2009;78(4):239–47. 51. Xiao Y, Schenkel S, Faraj S, et al. What whiteboards in a trauma center operating suite can teach us about emergency department communica‑ tion. Ann Emerg Med. 2007;50(4):387–95. 26. American College of Obstetricians and Gynecologists. Communica‑ tion Strategies for Patient Handoffs (Committee Opinion Number 517). 2012. Available from: http://www.acog.org/Resources-And-Publications/ Committee-Opinions/Committee-on-Patient-Safety-and-Quality-Impro vement/Communication-Strategies-for-Patient-Handoffs [Accessed Nov 2018]. 52. Bakti NI, Williamson M, Sehjal R, et al. The use of Microsoft Excel as an electronic database for handover and coordination of patients with trauma in a District General Hospital. BMJ Innovations. 2017;3(3):130–6. 52. Publisher’s Note The Institute for Healthcare Improvement and NHS Institute for Innova‑ tion and Improvement. Situation, Background, Assessment and Recom‑ mendation (SBAR). London: IHI and NHS Institute for Innovation and Improvement. 2006. 31. The Institute for Healthcare Improvement and NHS Institute for Innova‑ tion and Improvement. Situation, Background, Assessment and Recom‑ mendation (SBAR). London: IHI and NHS Institute for Innovation and Improvement. 2006. 32. Thomas V, Dixon A. Improving safety in maternity services. A toolkit for teams. 2012: The King’s Fund. 32. Thomas V, Dixon A. Improving safety in maternity services. A toolkit for teams. 2012: The King’s Fund. 33. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006;3(2):77–101. 33. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006;3(2):77–101. 34. Carter N, Bryant-Lukosius D, DiCenso, A et al. The use of triangulation in qualitative research. Oncology nursing forum. 2014;41(5):545–7. 34. Carter N, Bryant-Lukosius D, DiCenso, A et al. The use of triangulation in qualitative research. Oncology nursing forum. 2014;41(5):545–7. 35. O’Cathain A, Murphy E, Nicholl J. Three techniques for integrating data in mixed methods studies. BMJ. 2010;341: c4587. 35. O’Cathain A, Murphy E, Nicholl J. Three techniques for integrating data in mixed methods studies. BMJ. 2010;341: c4587. 36. Australian Comission on Safety and Quality in Health Care. Clinical Handover and Patient Safety—Literature Review Report. 2005: 10–28. 36. Australian Comission on Safety and Quality in Health Care. Clinical Handover and Patient Safety—Literature Review Report. 2005: 10–28.
https://openalex.org/W2134422543	https://orca.cardiff.ac.uk/id/eprint/130482/1/c4ce01411a.pdf	English	null	Energetics, thermal isomerisation and photochemistry of the linkage-isomer system [Ni(Et<sub>4</sub>dien)(η<sup>2</sup>-O,ON)(η<sup>1</sup>-NO<sub>2</sub>)]	CrystEngComm	2,015	cc-by	11,245	Introduction photoexcited metastable state(s) back to the stable ground state is blocked, allowing them to be characterised through single-crystal X-ray diffraction. Above a certain critical temperature, the so-called metastable limit,1 the onset of decay back to the ground-state structure occurs. Around the metastable limit, additional short-lived species may be observed in experiments where the crystal is continuously pumped with light during the data collection and thus reaches a “pseudo steady state” population of isomers.1,10 Linkage isomerisation is an interesting phenomenon whereby the binding mode of a ligand to the transition-metal centre in a coordination or organometallic complex changes in response to an external stimulus, typically thermal or photoactivation.1 Particularly in the solid state, where linkage isomerisation represents a single-crystal-to-single-crystal transition, these systems have attracted much interest. The canonical example, and one of the earliest systems studied, is perhaps sodium nitroprusside (Na[Fe(CN)5(NO)]), in which two long-lived metastable species identified spectroscopically following photoactivation2–4 were found to correspond to different binding modes of the NO ligand.5 Since this pioneering work, many more solid-state linkage-isomerisation systems have been found, with two prototypical families of compounds, viz. Ni–NO2 (ref. 6–11) and Ru–SO2,12–15 being widely studied. A particular challenge from a materials-design standpoint is to engineer the molecular solid so as to obtain a large photoconversion yield while maintaining the reversibility of the transition. In our recent work with linkage isomeric sys- tems, we have chosen to employ a simple crystal-engineering approach, aiming to produce a large “reaction cavity” within which the isomerisation can take place. The reaction cavity serves both to reduce the steric barriers to the transition, and also the stress it places on the crystal.6,15 Using this design principle, several Ni–NO2 systems with reversible, 100% photoconversion yields have been synthesised.6,9,10 Solid-state linkage isomerism can be studied experimen- tally through single-crystal photocrystallography measure- ments, in which the crystal is irradiated with light in situ on the diffractometer.1 At low temperatures, the decay of the [Ni(Et4dien)(η2-O,ON)(η1-NO2)] represents a particularly interesting linkage-isomer system because the isomerisation has been shown to be thermally as well as photochemically activated.7,10 Skeletal structures of the complex and of the three binding modes of the isomerisable NO2 group are shown in Fig. 1. The octahedral Ni centre is coordinated at three sites by the tridentate Et4dien ligand, with another two sites being taken up by an η2-bound NO2 group. Department of Chemistry, University of Bath, Claverton Down, Bath, BA1 7AY, UK. E-mail: a.walsh@bath.ac.uk † Electronic supplementary information (ESI) available: Includes additional calculation results, including the determination of the magnetic ground state of the GS and MS1 crystals, the GS-MS1 enthalpy differences obtained in solid-state and molecular calculations with different parameters, and complete TD-DFT datasets from calculations with several different functionals. See DOI: 10.1039/c4ce01411a ‡ JMS and RCO contributed equally to this work. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: CrystEngComm, 2015, 17, 383 Jonathan M. Skelton,‡ Rachel Crespo-Otero,‡ Lauren E. Hatcher, Stephen C. Parker, Paul R. Raithby and Aron Walsh* We present the results of a detailed theoretical study of the linkage isomerisation in [Ni(Et4dien)(η2-O,ON) (η1-NO2)] (Et4dien = N,N,N′,N′-tetraethyldiethylenetriamine). We probe the structure and bonding of the three experimentally-identified isomers in this system through electronic-structure calculations, and we establish possible transition pathways between them using transition-state modelling and periodic solid-state molecular-dynamics simulations. We also explore the photochemical isomerisation reaction using time-dependent density-functional theory. These results provide a thorough account of the linkage isomerisation in this compound, and add insight to ongoing experimental work on this and related systems. Received 8th July 2014, Accepted 21st August 2014 Received 8th July 2014, Accepted 21st August 2014 DOI: 10.1039/c4ce01411a www.rsc.org/crystengcomm CrystEngComm, 2015, 17, 383–394 \| 383 This journal is © The Royal Society of Chemistry 2015 CrystEngComm PAPER View Article Online View Journal \| View Issue CrystEngComm PAPER View Article Online View Journal \| View Issue CrystEngComm Periodic solid-state calculations DFT calculations with periodic boundary conditions were carried out using the VASP code.17 The semi-local PBEsol exchange-correlation functional18 was used for the majority of the calculations, although we also tested several other functionals, viz. PBE,19 the semi-empirical dispersion-corrected PBE-D220 and PBE-D321 functionals, and the non-local vdw-DF22 and vdw-DF223 functionals. Projector-augmented wave pseudopotentials24 were used, and the Brillouin zone was sampled at the Γ point. A plane-wave cut-off of 944.5 eV was used during geometry optimisations and single-point calculations, and molecular-dynamics (MD) simulations were performed with a smaller cut-off of 755.6 eV. As described in the results section, a Hubbard U correction of 5.32 eV was applied to the Ni d bands during the MD simulations, using the scheme of Dudarev et al.25 For these calculations, non-spherical contributions to the gradient corrections within the PAW spheres were accounted for. state (GS), and is formed by cooling in the absence of illumi- nation. The metastable O-bound endo-nitrito isomer (MS1) can be generated from the GS complex by photoactivation at 100 K and, additionally, can be generated thermally in signif- icant population at higher temperatures.7 More recently,10 a second O-bound exo-nitrito isomer (MS2) was observed in pseudo-steady-state photocrystallographic experiments at temperatures close to the metastable limit of ~150–160 K, which indicates that this species has a lifetime that is shorter than the duration of the X-ray data collection. While the steady-state structures of the various isomers in this system have been well characterised, little is presently known of the transition pathways which connect them. In this respect, the thermal isomerisation exhibited by this sys- tem makes it an ideal candidate for theoretical investigation. Whereas exploring excited-state potential-energy surfaces can pose a significant challenge for quantum chemistry, charac- terisation of ground-state properties is much less difficult. An understanding of the thermal isomerisation pathways could then, in principle, provide a basis for modelling the photo- chemical reaction. Geometry optimisation was performed by allowing the atomic positions to relax until the magnitude of the forces on the ions was less than 10−2 eV Å−1. After convergence, we found that with PBEsol the stress on the simulation cell was less than 0.2 GPa in both models, so we opted to fix the volume and cell shape at the experimentally-determined parameters. Periodic solid-state calculations MD simulations were carried out with the constraint that only the isomerisable NO2 ligands be allowed to move; by fixing the lighter atoms, we were then able to use a relatively long integration timestep of 5 fs. A Berendsen thermostat26 was used to fix the temperature during the dynamics simulations. In this article, we present the results of a computational study of the linkage isomerisation in [Ni(Et4dien)(η2-O,ON) (η1-NO2)], using a combination of solid-state and molecular quantum-chemistry calculations. We perform detailed electronic-structure calculations on the three species, and identify the isomerisation pathways connecting them using transition-state modelling and solid-state molecular dynamics. We also show that the crystal environment significantly influences the energetics of the isomerisation. Finally, we present some preliminary data from ongoing photochemical modelling, which provides some insight into possible excited-state isomerisation pathways. Our modelling provides important theoretical insight into the dynamics of linkage isomerisation in this system, and will support ongoing exper- imental work on this and related materials. Introduction The sixth site is occupied by a second η1-coordinated NO2 group, the binding mode of which can be switched between three known forms. The N-bound nitro isomer is the stable ground CrystEngComm, 2015, 17, 383–394 \| 383 This journal is © The Royal Society of Chemistry 2015 383 CrystEngComm View Article Online CrystEngComm View Article Online View Article Online Paper Paper Fig. 1 Schematic structure of the [Ni(Et4dien)(η2-O,ON)(η1-NO2)] system. The four skeletal drawings show the structure of the molecule (left) and the three different binding modes of the isomerisable NO2 group (right), viz. the ground-state (GS) nitro and metastable endo- and exo-nitrito forms (MS1/MS2, respectively). Paper Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Schematic structure of the [Ni(Et4dien)(η2-O,ON)(η1-NO2)] system. The four skeletal drawings show the structure of the molecule (left) and the three different binding modes of the isomerisable NO2 group (right), viz. the ground-state (GS) nitro and metastable endo- and exo-nitrito forms (MS1/MS2, respectively). 384 \| CrystEngComm, 2015, 17, 383–394 Computational methods All computational modelling was carried out within the Kohn–Sham density-functional theory (DFT) formalism.16 Models of the ground- and metastable-state crystal structures were created from the crystallographic data published with ref. 7. For the molecular quantum-chemistry calculations, single complexes were extracted from these published struc- tures, and an initial model of the MS2 complex was made from the MS1 structure by rotating the O-bound NO2 ligand. Separate software packages were used for the solid-state and molecular calculations, as outlined below. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Transition-state modelling calculations were performed in the gas phase with the 6-31G(d) and 6-311++G(d,p) basis sets and the M06 and PBE0 functionals. Calculations with water as a polarisable continuum were performed using the 6-31G(d) basis set for geometry optimisation, and the 6-311++G(d,p) basis set for single-point energy calculations. Harmonic vibrational-frequency calculations were performed to confirm the nature of all stationary points found, and Intrinsic Reaction Coordinate (IRC) calculations35 were carried out to check the connection between the reactants, transition states and products. Given that the Ni centre is a central component in the iso- merisation process, it is possible that the discrepancy arises from common issues which (semi-)local DFT functionals can encounter when treating strongly-correlated 3d-electrons.39 We therefore investigated the effect of applying a Hubbard U correction to the Ni d states on the energy difference between the two structures. Strikingly, we found that the difference reduced systematically with the magnitude of the U term (see ESI†), such that for a value of 5.32 eV the experimental enthalpy difference was reproduced to within 0.2 kJ mol−1. The size of this correction is reasonable, and is not too dis- similar to, for example, the typical values used in DFT + U calculations on NiO in the literature (e.g. ref. 25 and 40). We also note in passing that extrapolating the trend suggests that a larger on-site potential of around 10 eV would zero the energy difference between the isomers. Finally, it is worth mentioning that while, in the present case, the U correction was chosen to match experimental data, as is fairly common practice, an alternative, a less empirical linear-response method exists to obtain the value from first-principles;41 however, this technique is not presently implemented in VASP. To study the photochemistry of the complexes, time- dependent DFT (TD-DFT) calculations on both the triplet and singlet excited states were carried out, using the corre- sponding triplet and singlet ground-state wavefunctions as references. Different functionals, viz. M06,31 B3LYP,29 PBE0,28 M06HF36,37 and wB97xd38 were considered in combi- nation with the LANL2DZ pseudopotential and basis set for Ni, with a 6-311++G(d,p) basis set being used for the light atoms (this mixed basis set is referred to as LANL2DZ/ 6-311++G(d,p) in the text). The TD-DFT calculations were performed with water as a polarisable continuum. Results and discussion A second factor which may play an important role in the energetics is dispersion, another effect which many DFT functionals struggle to describe accurately, and which is likely to be significant in molecular crystals such as this. The PBEsol functional does not include any explicit dispersion corrections, and so to quantify the differences such corrections might make we performed additional geometry optimisations on the GS and MS1 crystals using a selection of other functionals, viz. PBE,19 PBE-D220 and PBE-D3,21 and vdw-DF22 and vdw-DF2.23 The D2/D3 func- tionals apply a semi-empirical correction to the PBE energies to approximately account for dispersion interactions, while the vdw-DF functionals are non-empirical and attempt to treat dispersion more accurately through a non-local electron correlation. After geometry optimization, we compared the calculated GS–MS1 energy differences, and also various bond lengths around the Ni centre, between the six functionals to experimental data (see ESI†). We found that the energy differ- ences computed with PBE and the two vdw-DF functionals came closest to the experimental values, whereas the disper- sion corrections applied by the PBE-D2/D3 methods yielded values similar to PBEsol; however, in all cases the difference Energetics and ground-state magnetism This journal is © The Royal Society of Chemistry 2015 Molecular calculations All isolated molecular calculations were performed using Gaussian09 program.27 A range of different functionals were employed for studying energetics, viz. PBE,19 the PBE0,28 B3LYP,29and TPSSH30 hybrid functionals, the M06 meta- hybrid,31 and the CAMB3LYP long-range-corrected hybrid.32 The M06 and PBE0 functionals were used for the transition- state and photochemistry modelling, due to their good general-purpose performance when describing a broad range of properties.33 Triplet and singlet states were computed 384 \| CrystEngComm, 2015, 17, 383–394 This journal is © The Royal Society of Chemistry 2015 View Article Online CrystEngComm Paper using the unrestricted and restricted approaches, respec- tively. The LANL2DZ basis set and corresponding pseudo- potential were used to treat the Ni atom, with various combinations of the split-valence 6-31G(p), 6-311G(d) and 6-311++G(d,p) basis sets being used for the lighter atoms. Polarisable continuum model (PCM) calculations, carried out to study the possible effects of the crystal environment on properties,34 were performed to mimic three different solvents with increasing dielectric constants, viz. toluene (ε = 2.4), ethanol (ε = 24.9) and water (ε = 78.4). states with the moments on different pairs of Ni atoms oriented in opposite directions. We found no significant difference in energy between these configurations; the maximum 0.08 kJ mol−1 per molecule is well within the error of the calculations. To compare the calculated GS–MS1 energy difference against experiment, we fitted the temperature dependence of the isomer populations reported in ref. 7 to a Boltzmann expres- sion, which yielded an enthalpy difference of 9.69 kJ mol−1 per molecule (see ESI†). This value is less than half the difference computed in these calculations, and so we therefore opted to investigate briefly the effect of some computational parameters on the computed ΔE. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, bearing this caveat in mind, these two sets of tests suggest that to accurately capture the energetics of this system requires both a good description of the Ni d electrons, and also accounting for dispersion forces. An important question about the isomerisation process is whether or not it proceeds in a concerted manner, i.e. whether one complex isomerising influences subsequent events at neighbouring sites. To investigate this, we created variants of the GS and MS1 crystal structures in which 1–4 of the η1-bound NO2 ligands were swapped with their isomers “by hand”, thereby converting 1–4 of the symmetry- equivalent molecular complexes to the other isomer. The geometries of these models were then optimized with PBEsol, and, after verifying that the isomerisation was not reversed, we compared the energy change from flipping 1–4 of the ligands in tandem against the sum of the energy required to flip the same ligands in isolation (Fig. 2a). p g ( g ) For the GS-to-MS1 isomerisation, up to three flips (75% conversion) leads to a change in energy which is more or less equal to the sum of those for the individual iso- merisations, whereas the fourth leads to a disproportion- ately large relative increase in energy. We observed a linear increase in the stress on the unit cell with successive isomerisations (Fig. 2b), which is consistent with the fact that the MS1 structure has a larger unit-cell volume than the GS one. If this build-up of stress makes the final isomerisation energetically more difficult, one might infer that, during very fast (e.g. laser-induced) photo- isomerisation processes, the final 25% conversion may be constrained by lattice relaxation (phonon coupling). In keeping with this picture, we found that for the reverse MS1-to-GS transition, where isomerisation leads to a negative stress, the energy required to flip all four ligands was practically identical to the sum of the individual isomerisation energies. These results thus suggest that, at equilibrium, the isomerisation is not a concerted process, in either direction, and is likely to happen randomly throughout the crystal. This is consistent with kinetic data from photocrystallographic measurements.10 Fig. 2 Effect of the GS →MS1 (blue line) and reverse MS1 →GS (red line) isomerisation on the energy of (a) and external pressure on ( pext; b) the GS/MS1 crystal structures. This journal is © The Royal Society of Chemistry 2015 Energetics and ground-state magnetism The energies and stresses are computed from single-point PBEsol + U calculations performed on the PBEsol-optimised structures. These calculations suggest that the linear increase in the stress on the unit cell with conversion during the GS →MS1 transition makes the final isomerisation energetically more difficult, in the absence of lattice relaxation. CrystEngComm View Article Online CrystEngComm View Article Online CrystEngComm View Article Online View Article Online View Article Online Paper was consistently overestimated. The better match of the computed energy difference with experimental data obtained with the vdw-DF functionals might be taken as an indication that dispersion forces make a significant contribution to the energetics, although this should be a tentative conclusion, since it is quite possible that the dispersion corrections in these functionals may be compensating for other issues, e.g. the aforementioned inability of standard DFT functionals to treat the correlated Ni 3d electrons. This journal is © The Royal Society of Chemistry 2015 ive energy differences between the three species, using various functionals and basis sets, are Table 1. Corresponding energy differences between S2 may be found in Table 2. Interestingly, MS1 is lower in energy than the GS in the gas phase for all ations considered, save for PBE/LANL2DZ/6-311G(d) −13 kJ mol−1), which suggests that PBE may exhibit urable error compensation. To investigate this created periodic molecular models of the GS and exes, by placing the molecules in a simulation arge vacuum gap to separate them from adjacent ages. After converging the energy as a function size and optimizing the geometry with PBEsol, an energy difference of ΔEGS–MS1 = −14.6 kJ mol−1 le (see ESI†). Single-point calculations with a correction of 5.32 eV and with the bare PBE func- ed energy differences of −1.4 and −9.3 kJ mol−1, The reasonably good correspondence between the t of the GS →MS1 (blue line) and reverse MS1 →GS merisation on the energy of (a) and external pressure on GS/MS1 crystal structures. In plot (a), the sum of the ges which occur when 1–4 ligands are isomerised in hed lines) is compared against the energy change on same number of ligands in tandem. The energies and computed from single-point PBEsol + U calculations n the PBEsol-optimised structures. These calculations the linear increase in the stress on the unit cell with ring the GS →MS1 transition makes the final isomerisation more difficult, in the absence of lattice relaxation. Energetics and ground-state magnetism Solid-state calculations. To explore the energetics and magnetic properties of the system, we first performed a series of single-point calculations on the optimised GS and MS1 crystal structures. The Ni atom in both complexes is for- mally Ni(II) in an octahedral environment, and thus a simple crystal-field model of the d-orbital splitting would predict two unpaired electrons in the eg orbitals. We found that this open-shell magnetic state was the lowest-energy in both structures, with energy differences of ΔET–S = −35.4 and −39.0 kJ mol−1 per molecule in the GS and MS1 isomers, respectively. For comparison, these are both considerably larger than the difference in energy between the isomers themselves, which for the triplet states was calculated to be ΔEGS–MS1 = −23.9 kJ mol−1 per molecule. Given that the crystal structures are formed of essentially isolated complexes, one would not expect long-range magnetic interactions to occur between the Ni centres. To confirm this, we compared the relative energies of a ferro- magnetic state, with all the Ni magnetic moments aligned in the same direction, and three different antiferromagnetic This journal is © The Royal Society of Chemistry 2015 CrystEngComm, 2015, 17, 383–394 \| 385 385 CrystEngComm View Article Online The relative energy differences between the three species, calculated using various functionals and basis sets, are collected in Table 1. Corresponding energy differences between MS1 and MS2 may be found in Table 2. Interestingly, MS1 is found to be lower in energy than the GS in the gas phase for all the combinations considered, save for PBE/LANL2DZ/6-311G(d) (ΔEGS–MS1 = −13 kJ mol−1), which suggests that PBE may exhibit some favourable error compensation. To investigate this further, we created periodic molecular models of the GS and MS1 complexes, by placing the molecules in a simulation cell with a large vacuum gap to separate them from adjacent periodic images. After converging the energy as a function of the gap size and optimizing the geometry with PBEsol, Fig. 2 Effect of the GS →MS1 (blue line) and reverse MS1 →GS (red line) isomerisation on the energy of (a) and external pressure on ( pext; b) the GS/MS1 crystal structures. In plot (a), the sum of the energy changes which occur when 1–4 ligands are isomerised in isolation (dashed lines) is compared against the energy change on flipping the same number of ligands in tandem. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The main difference between the GS and MS1 isomers lies in the relative stabilities of the Ni–N and Ni–O bonds. A Natural Bond Orbital (NBO) second-order perturbation analysis42 shows that the most important stabilisation of both bonds is through electron transfer from the ligand to the metal. The Ni–N bond is more energetically favourable by around 9 kJ mol−1 due to the better donor capacity of N (Δ2ELP(N)→Ni = 160 kJ mol−1, Δ2ELP(O)→Ni = 151 kJ mol−1 at the M06/6-311++G(d,p) level with a continuum of water). latter value and the molecular calculations suggests that the 6-311G(d) basis set used in the latter is approaching the conver- gence limit with respect to this property. For PBE, PBE0 and M06, ΔEGS–MS1 becomes more negative as the basis set size is increased, although even with the larg- est basis sets the latter two still predict an incorrect relative ordering. Comparing the PBE0, TPSSH and PBE functionals, which notionally have decreasing exchange-energy contribu- tions, the energy difference is seen to decrease; this is similar to the effect of including the U correction in the periodic PBEsol calculations, and illustrates that the exchange poten- tial makes an important contribution to the energy difference between the isomers. An additional source of stabilisation is the weak hydrogen bond between the ligand O atom and the NH group on the Et4dien molecule (classified as such based on intermolecular distance criteria43). The effect of this interaction is most pronounced in the gas phase; the presence of a solvent increases the O–H–N distances, leading to a consequent weakening of the interaction due to dielectric screening. Comparing the hydrogen-bond distances between the iso- mers, we obtain 2.01–2.21 and 1.93–2.05 Å in the GS and MS1 complexes, respectively, depending on the level of theory. To obtain the correct size of the energy difference between the isomers, we found that the basis set for the lighter elements needed to be of at least triple-zeta quality, while for the metal the use of the LANL2DZ basis set and pseudo- potential did not affect significantly the results, leading to a change in ΔEMS1–GS of only 0.6 kJ mol−1 with the M06 func- tional. To obtain the correct sign of ΔEGS–MS1, a combination of a triple-zeta basis set for the light elements and the contin- uum solvent model was needed. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In plot (a), the sum of the energy changes which occur when 1–4 ligands are isomerised in isolation (dashed lines) is compared against the energy change on flipping the same number of ligands in tandem. The energies and stresses are computed from single-point PBEsol + U calculations performed on the PBEsol-optimised structures. These calculations suggest that the linear increase in the stress on the unit cell with conversion during the GS →MS1 transition makes the final isomerisation energetically more difficult, in the absence of lattice relaxation. The relative energy differences between the three species, calculated using various functionals and basis sets, are collected in Table 1. Corresponding energy differences between MS1 and MS2 may be found in Table 2. Interestingly, MS1 is found to be lower in energy than the GS in the gas phase for all the combinations considered, save for PBE/LANL2DZ/6-311G(d) (ΔEGS–MS1 = −13 kJ mol−1), which suggests that PBE may exhibit some favourable error compensation. To investigate this further, we created periodic molecular models of the GS and MS1 complexes, by placing the molecules in a simulation cell with a large vacuum gap to separate them from adjacent periodic images. After converging the energy as a function of the gap size and optimizing the geometry with PBEsol, we obtained an energy difference of ΔEGS–MS1 = −14.6 kJ mol−1 per molecule (see ESI†). Single-point calculations with a Hubbard U correction of 5.32 eV and with the bare PBE func- tional yielded energy differences of −1.4 and −9.3 kJ mol−1, respectively. The reasonably good correspondence between the Molecular calculations. The solid-state calculations suggest that, to a good approximation, the molecular units behave as isolated complexes, influenced by the dielectric environment of the crystal. We therefore carried out molecu- lar quantum-chemistry calculations on complexes extracted from the GS and MS1 crystal structures, plus a model of the MS2 intermediate obtained by rotating the O-bound NO2 group in MS1. 386 \| CrystEngComm, 2015, 17, 383–394 This journal is © The Royal Society of Chemistry 2015 386 View Article Online View Article Online CrystEngComm CrystEngComm Paper Table 1 Calculated energy differences between the GS and MS1 complexes (ΔEGS–MS1). The entries compare, variously, the effect of different exchange-correlation (XC) functionals, basis sets and polarisable dielectric continuums on the energies of the two isomers. CrystEngComm, 2015, 17, 383–394 \| 387 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In tests where a pseudopotential was used to describe the Ni core electrons, the first entry in the basis-set column gives the basis used for the Ni atom, and the second gives that used for all other atoms We note that differences in vibrational zero-point energy are not expected to affect significantly the relative stabilities of the species; energy corrections estimated within the harmonic approximation at the M06/LANL2DZ/6-31G(d) level of theory change ΔEGS–MS1 by less than 1 kJ mol−1 (ΔE + ZPVE = 8.76 kJ mol−1, compared to ΔE = 9.33 kJ mol−1). XC functional Basis set Continuum ΔEGS–MS1/kJ mol−1 XC functional Basis set Continuum ΔEGS–MS1/kJ mol−1 PBE LANL2DZ/6-31G(d) None 2.45 PBE0 None 11.7 B3LYP None 15.70 TPSSH None 9.71 M06 None 9.33 CAMB3LYP None 19.70 PBE LANL2DZ/6-311G(d) None −13.01 PBE0 None 6.03 M06 None 1.62 M06 6-311G(d) None 2.39 M06 LANL2DZ/6-311+ +G(d,p) None 1.84 M06 PhMe −1.81 M06 EtOH −6.96 M06 H2O −7.54 We now consider the optimised geometries of the molecu- lar complexes. Fig. 3 shows the geometry in the plane defined by the nitro ligands and the Ni centre in the GS, MS1 and MS2 isomers. Comparing with available experimental data (see ESI†), the agreement between the bond lengths in the optimised complexes and the crystal structures is generally very good, with the only exception being a signifi- cant underestimation of the length of the O(η1-NO2)–HN distance. We note that a crystal structure containing the MS2 geometry was not available to compare this set of data against. This journal is © The Royal Society of Chemistry 2015 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. he GS, MS1 and MS2 complexes with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set, with water Fig. 3 Optimised geometries of the GS, MS1 and MS2 complexes with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set, with water as a dielectric continuum. However, this appears to be largely a dielectric effect, as the periodic calculations suggest a minimal interaction, if any, between complexes in the molecular crystal; this observation extends to the isomerisation process itself, as the present cal- culations confirm the experimental finding that the isomeri- zation is likely to be a random, rather than a concerted, process. The pronounced effect that the crystal environment has on the isomerization energetics reinforces the idea that crystal engineering, e.g. tuning the size of the “reaction cavity”, could be used to control the switching process, as has already been discussed in relation to a number of linkage- isomer systems.6,15 While these transition states are superficially similar to TS1 and TS2, the Ni–O bond length remains constant between MS1 and MS2, indicating that this O remains bonded during the rotation; consequently, the MS1-to-MS2 isomerisation requires roughly half the energy of the GS-to-MS1 conversion. We also explored possible direct isomerisation pathways between the GS and MS2 isomers by a relaxed scan of the Ni–O–N angle in the Ni–O2N plane. This yielded a continuous increase in energy, with no further stabilisation. When inter- mediate geometries were optimised as transition states, an additional transition state, TS3, was found (see ESI†), but IRC calculations indicated that this does not connect GS and MS2; rather, this transition state appears to be an in-plane rotation of the O2N ligand connecting two further high- energy metastable structures which have not been observed experimentally. Whether these structures represent transient species (e.g. detectable in ultrafast laser-excitation experi- ments), or are merely artifacts, is presently unclear. Thermal isomerisation: molecular calculations. The main difference between the GS, MS1 and MS2 isomers is the position of the NO2 group. Considering the plane formed by the Ni atom and the isomerisable NO2 group, the three isomers can be connected by simple rotations of the ligand either in or out of this plane. Hypothetical GS-to-MS1/2 isomerisations would also require the breaking or weakening of the bonds between N/O and Ni. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The symmetry of the complex is such that, for each process, two similar transition states may be relevant – indeed, in general both possibilities were found to be saddle points on the potential energy surfaces, albeit with similar energies. Fig. 4 illustrates the transition states connecting the GS, MS1, and MS2 structures, labelled TS1/TS2 and MTS1/MTS2, respectively; the corresponding energies, computed with M06 and PBE0 and the LANL2DZ/6-311++G(d,p) basis set, are listed in Table 3. The kinetic measurements reported in ref. 10 yielded an activation barrier for the decay of MS1 to the GS of 48.6 kJ mol−1, which is closer to the calculated barrier for the MS1-to-MS2 transition than to the MS1-to-GS one. In the absence of a direct GS-to-MS2 isomerisation pathway, this would suggest that the calculated barriers for the GS-to-MS1 transition have been overestimated considerably. However, if a GS-to-MS2 path did exist, and had an activation barrier lower than that for the MS1-to-MS2 transition, then the rate- limiting step in the decay would then be the MS1 →MS2 isomerisation, and the calculated barrier for this is much closer to the experimental value. With regard to the accuracy of the calculated barriers, it is worth noting that the functional, basis set and continuum were optimised to reproduce only the enthalpy differences, and not necessarily the transition barriers. Moreover, since the energetics in this system are evidently quite sensitive to the continuum, it is possible that the use of the high dielectric constant of water in the PCM model may be compensating for deficiencies in either or both of the functional or basis set. TS1 and TS2 correspond to the rotation of the NO2 group up and down with respect to the plane, respectively. The energy barriers for these processes, computed at the M06/LANL2DZ/6-311++G(d,p) level of theory, are both around 70 kJ mol−1, and the presence of water as a continuum increases both barrier heights by roughly 10 kJ mol−1. With the PBE0 functional, both barriers are reduced. The transition states connecting MS1 and MS2, labelled MST1 and MST2, are (anti)clockwise rotations of the ligand about the Ni–O bond. In both the gas phase and continuum calculations, and for both the M06 and PBE0 functionals, the two were found to have very similar energies and geometries. This journal is © The Royal Society of Chemistry 2015 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. With the M06 functional in combination with the 6-311++G(d,p) basis set, PCM simu- lations with the dielectric constant of toluene, ethanol and water gave ΔEGS–MS1 values of −1.81, −6.96 and −7.54 kJ mol−l, respectively. As in MS1, the coordination of the NO2 group to Ni in the MS2 complex is through O. However, due to the geometry of the ligand, the hydrogen-bonding interaction occurs via N, and the length is comparable to the bond in the GS complex, making it weaker than the bond in the MS1 isomer. As a result of the effect of a dielectric medium on the hydrogen-bonding interaction, the energy difference between MS1 and MS2 is reduced significantly when a polarisable continuum is included in the calculations, ranging from ΔEMS2–MS1 = 18.72 kJ mol−1 in the gas phase to 4.83 kJ mol−1 in water using the M06 functional, and from 8.69 to 0.86 kJ mol−1 with PBE0. Table 2 Calculated energy differences between the MS1 and MS2 com- plexes (ΔEMS2–MS1). These entries compare the values obtained for two dif- ferent DFT functionals, viz. M06 and PBE0, in the gas phase and with water as a polarisable continuum This journal is © The Royal Society of Chemistry 2015 Table 2 Calculated energy differences between the MS1 and MS2 com- plexes (ΔEMS2–MS1). These entries compare the values obtained for two dif- ferent DFT functionals, viz. M06 and PBE0, in the gas phase and with water as a polarisable continuum XC functional Basis set Continuum ΔEMS2–MS1/kJ mol−1 M06 LANL2DZ/6-311G+ +(d,p) None 14.31 M06 H2O 4.83 PBE0 None 8.69 PBE0 H2O 0.86 In summary, these calculations suggest that the energy differences between the three linkage isomers is governed by a combination of the influence of the ligand on the Ni 3d states, and hydrogen-bonding interactions between the ligand and the N–H group on the Et4dien ligand. Whether modelled explicitly or implicitly, it is apparent that the crystalline environment significantly influences the relative energies of the three isomers compared to in the gas phase. XC functional Basis set Continuum ΔEMS2–MS1/kJ mol−1 M06 LANL2DZ/6-311G+ +(d,p) None 14.31 M06 H2O 4.83 PBE0 None 8.69 PBE0 H2O 0.86 CrystEngComm, 2015, 17, 383–394 \| 387 387 Paper Paper Fig. 3 Optimised geometries of the GS, MS1 and MS2 complexes with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set, with water as a dielectric continuum. y g p 388 \| CrystEngComm, 2015, 17, 383–394 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To investigate this further requires the calculation or measurement of the macroscopic dielectric constant of the crystal, which is the subject of ongoing work. 388 \| CrystEngComm, 2015, 17, 383–394 This journal is © The Royal Society of Chemistry 2015 View Article Online CrystEngComm Paper Fig. 4 Schematic of the isomerisation pathways in [Ni(Et4dien)(η2-O,ON)(η1-NO2)], modelled using the M06 functional and the LANL2DZ/6-311++G(d,p) basis set, and with water as a continuum. The energy profile illustrates the relative energies of the GS, MS1 and MS2 isomers, and the transition states connecting them, while the four models show the optimised geometries of the four transition states. e. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. rticle is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Schematic of the isomerisation pathways in [Ni(Et4dien)(η2-O,ON)(η1-NO2)], modelled using the M06 functional and the LANL2DZ/6-311++G(d,p) basis set, and with water as a continuum. The energy profile illustrates the relative energies of the GS, MS1 and MS2 isomers, and the transition states connecting them, while the four models show the optimised geometries of the four transition states. Table 3 Energy barriers for the formation of the most important transi- tion states between the GS and MS1 isomers (TS1, TS2) and the MS1 and MS2 isomers (MTS1, MTS2). For each transition state, the barrier height has been computed with the PBE0 and M06 functionals using the LANL2DZ/ 6-311++G(d,p) basis set, and with and without water as a continuum the MS1-to-MS2 isomerisation will be accessible, but slow (on crystallographic data-collection timescales), resulting in a small but detectable steady-state population of the MS2 state. Solid-state molecular dynamics. Given the apparent importance of the crystalline environment, we performed short (50 ps) constrained periodic molecular-dynamics (MD) simulations on the MS1 structure at 370 K, being the highest of the temperatures investigated experimentally in ref. 7, to see whether we could observe the transition pathways found in the molecular calculations. The energy and forces were calculated using the PBEsol functional, with a Hubbard U correction of 5.32 eV, as discussed in the section on energet- ics; we opted for this combination as an inexpensive means of reproducing the experimental enthalpy difference between the GS and MS1 isomers. This journal is © The Royal Society of Chemistry 2015 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The most important excited states, at the TD-M06/LANL2DZ/6-311++G(d,p) level of theory with water as a continuum, are listed in Table 4. We show only the excited states that could be relevant for the photoisomerisation process, but the complete TD-DFT dataset, including the transitions obtained with all the functionals tested, may be found in the ESI.† The overall barrier height for the endo-to-exo transition is ~48 kJ mol−1, and the energy difference between the endo and exo forms is ΔEMS2–MS1 = 2.13 kJ mol−1. The discrepancy between these results and those from the molecular calcula- tions is most likely due in part to the use of the semi-local PBEsol functional, as opposed to the more sophisticated (meta)hybrids available for molecular calculations, plus the fact that a large part of the structure was constrained to the initial MS1 geometry, thus preventing the rest of the molecu- lar backbone relaxing during the transition. The main orbitals involved in the transitions in the GS isomer are shown in Fig. 6. The first six triplet excited states (T1 to T6) in both the GS and MS are transitions between the HOMOβ−5/HOMOβ−6 and LUMOβ/LUMOβ+1 orbitals; the orbital labelling is based on the beta spin channel (that which does not contain the two unpaired electrons) in which we found the most important calculated transitions took place. These electronic excitations all involve a redistribution of the electron density over the NO2 ligands and the metal, and as such can be assigned as delocalized metal-to-ligand (ML) transitions. However, the fact that the same transition states connecting MS1 and MS2 as were observed in the molecular calculations are seem in the MD calculations – in which the system is able to explore its potential energy surface more fully – serves as a pow- erful validation of the mechanism established from the former. The photochemical process: excited states. The [Ni(Et4dien) (η2-O,ON)(η1-NO2)] system studied here is unique in that its isomerisation is both thermally and photochemically activated. Although the former is the main focus of this work, it is of interest to see how photoexcitation may influence the thermal reaction pathway established from the transition- state calculations. HOMOβ−5 contains an important contribution from the Ni dxz and ONO (static ligand) px orbitals, while HOMOβ−6 is made up of the Ni dxy and NO2 (isomerisable ligand) px orbitals. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. While we were not able to observe the MS1-to-GS isomerisation over this timescale, we did observe both the MS1-to-MS2 and the reverse MS2-to-MS1 transitions. To obtain energy profiles for these processes, we extracted the configurations of the isomerisable NO2 group during the transition and performed a series of single-point total-energy calculations while keeping the other three groups fixed at their initial positions. Fig. 5 shows the energy profile for the MS1-to-MS2 transition, together with snapshots of the complex at different points on the curve; a profile for the reverse MS2-to-MS1 isomerisation may be found in the ESI.† Species Continuum ΔEPBE0/kJ mol−1 ΔEM06/kJ mol−1 TS1 None 66.3 71.4 H2O 60.7 79.7 TS2 None 66.6 70.8 H2O 68.1 81.1 MTS1 None 31.2 35.9 H2O 32.2 33.8 MTS2 None 31.1 35.1 H2O 33.8 33.1 In spite of these potential issues, the present calculations establish an intuitive isomerization pathway between the three isomers. The isomerisation between MS1 and MS2 has a (relatively) low activation energy, which would allow equilibration between them over a range of temperatures, and, as found from the energetics calculations, MS1 is the more stable of the two. This low thermal barrier to conver- sion between the endo and exo isomers naturally explains the observation of the latter as a transient intermediate in a narrow temperature range – when the system is heated close to the relatively low-temperature metastable limit, CrystEngComm, 2015, 17, 383–394 \| 389 This journal is © The Royal Society of Chemistry 2015 389 View Article Online View Article Online View Article Online CrystEngComm Paper Paper Fig. 5 Energy profile for the endo-to-exo transition in [Ni(Et4dien)(η2-O,ON)(η1-NO2)], obtained from constrained ab initio molecular-dynamics simulations. The energies are expressed relative to the lowest-energy point on the curve. Snapshots of the geometry of the complex at various local maxima and minima on the profile are shown, with the atoms colour coded as follows: N – blue, O – red, Ni – silver. The largest energy barrier involved in the switching is 48.14 kJ mol−1, and corresponds to a clockwise rotation of the NO2 group by approximately 45° from the endo position. The snapshots were created with the VMD software.44 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 Energy profile for the endo-to-exo transition in [Ni(Et4dien)(η2-O,ON)(η1-NO2)], obtained from constrained ab initio molecular-dynamics simulations. This journal is © The Royal Society of Chemistry 2015 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The energies are expressed relative to the lowest-energy point on the curve. Snapshots of the geometry of the complex at various local maxima and minima on the profile are shown, with the atoms colour coded as follows: N – blue, O – red, Ni – silver. The largest energy barrier involved in the switching is 48.14 kJ mol−1, and corresponds to a clockwise rotation of the NO2 group by approximately 45° from the endo position. The snapshots were created with the VMD software.44 The experimentally-reported UV-visible spectrum of the GS complex in ref. 10 shows an intense absorption band around 400 nm (3.1 eV), and we would hence expect to predict a signif- icant absorption in this spectral region from the molecular calculations, given a suitable choice of polarisable continuum. However, the prediction of accurate absorption energies and oscillator strengths represents a significant challenge for com- putational chemistry,45,46 and for most of the levels of theory considered in this work the first excited state with significant intensity appears at much higher energies; only M06 and B3LYP predict bands with significant oscillator strengths (around 0.02) close to the correct spectral region (3.64 and 3.82 eV, respectively). The most important excited states, at the TD-M06/LANL2DZ/6-311++G(d,p) level of theory with water as a continuum, are listed in Table 4. We show only the excited states that could be relevant for the photoisomerisation process, but the complete TD-DFT dataset, including the transitions obtained with all the functionals tested, may be found in the ESI.† This analysis suggests that the largest energy barrier in the switching process is the point at which the ligand is rotated by ~45° with respect to the plane in which it sits in the endo/exo positions. This configuration is around 12.2 kJ mol−1 higher in energy than the one with the ligand at 90°, although the latter is still a local energy maximum. Indeed, a second posi- tion with the ligand at 45° to the endo/exo plane is also an energy maximum, and in this particular trajectory appears to block the ligand from making a complete rotation when it falls from the transition state to the local exo minimum. considered in this work the first excited state with significant intensity appears at much higher energies; only M06 and B3LYP predict bands with significant oscillator strengths (around 0.02) close to the correct spectral region (3.64 and 3.82 eV, respectively). Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. State E/eV (nm) Oscillator strength Assignment State E/eV (nm) Oscillator strength Assignment GS-T1 0.98 (1260) 0.0002 ML MS1-T1 1.01 (1230) 0.0000 ML GS-T2 1.01 (1230) 0.0001 ML MS1-T2 1.08 (1145) 0.0000 ML GS-T3 1.08 (1145) 0.0000 ML MS1-T3 1.55 (799) 0.0003 ML GS-T4 1.55 (799) 0.0002 ML MS1-T4 1.65 (752) 0.0000 ML GS-T5 1.65 (752) 0.0000 ML MS1-T5 1.82 (682) 0.0002 ML GS-T6 1.82 (682) 0.0001 ML MS1-T6 2.73 (455) 0.0001 ML GS-T7 2.73 (455) 0.0002 MLCT (NO2) MS1-T7 2.88 (431) 0.0000 MLCT (NO2) GS-T8 2.88 (431) 0.0000 MLCT (O2N) MS1-T8 3.41 (363) 0.0000 MLCT (O2N) GS-T9 3.41 (363) 0.0073 ML MS1-T9 3.61 (343) 0.0015 MLCT (NO2) GS-T10 3.61 (343) 0.0005 MLCT (NO2) MS1-T10 3.64 (340) 0.0197 ML GS-T11 3.64 (340) 0.0376 ML MS1-T11 3.67 (338) 0.0218 ML GS-T12 3.67 (338) 0.0205 ML MS1-T12 3.73 (332) 0.0011 ML GS-T17 3.98 (312) 0.0238 ML MS1-T15 3.98 (312) 0.0379 ML GS-T18 4.02 (309) 0.1216 ML MS1-T16 4.00 (310) 0.1044 ML GS-T19 4.05 (306) 0.0272 ML MS1-T17 4.03 (308) 0.0104 ML Fig. 6 Schematic of the molecular orbitals involved in the most important electronic transitions in the GS isomer, computed with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set with water as a continuum. The labelling convention is based on the beta spin channel (that which does not contain the two unpaired electrons), in which the most important calculated electronic transitions take place. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Schematic of the molecular orbitals involved in the most important electronic transitions in the GS isomer, computed with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set with water as a continuum. The labelling convention is based on the beta spin channel (that which does not contain the two unpaired electrons), in which the most important calculated electronic transitions take place. Fig. 6 Schematic of the molecular orbitals involved in the most important electronic transitions in the GS isomer, computed with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set with water as a continuum. The labelling convention is based on the beta spin channel (that which does not contain the two unpaired electrons), in which the most important calculated electronic transitions take place. NO2 and ONO groups, respectively. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The LUMOβ and LUMOβ+1 orbitals have anti- bonding contributions between the Ni d orbitals in the yz 390 \| CrystEngComm, 2015, 17, 383–394 This journal is © The Royal Society of Chemistry 2015 390 View Article Online CrystEngComm CrystEngComm Paper Table 4 Triplet excited states calculated at the TD-DFT/M06 level with the LANL2DZ/6-311++G(d,p) basis set and water as a polarisable continuum; the ground-state reference was the triplet electronic ground state. Each state is listed together with its corresponding calculated oscillator strength, and is assigned as either a metal-to-ligand delocalised (ML) or a metal-to-ligand charge-transfer (MLCT) state. The left portion of the table lists the excited states computed for the GS molecule (prefixed “GS”), while the right portion lists states computed for the MS1 isomer (prefixed “MS1”) plane and the py and pz orbitals on the nitro ligands. Conse- NO2 and ONO groups, respectively. The equivalent excited p (p ), g p p (p ) State E/eV (nm) Oscillator strength Assignment State E/eV (nm) Oscillator strength Assignment GS-T1 0.98 (1260) 0.0002 ML MS1-T1 1.01 (1230) 0.0000 ML GS-T2 1.01 (1230) 0.0001 ML MS1-T2 1.08 (1145) 0.0000 ML GS-T3 1.08 (1145) 0.0000 ML MS1-T3 1.55 (799) 0.0003 ML GS-T4 1.55 (799) 0.0002 ML MS1-T4 1.65 (752) 0.0000 ML GS-T5 1.65 (752) 0.0000 ML MS1-T5 1.82 (682) 0.0002 ML GS-T6 1.82 (682) 0.0001 ML MS1-T6 2.73 (455) 0.0001 ML GS-T7 2.73 (455) 0.0002 MLCT (NO2) MS1-T7 2.88 (431) 0.0000 MLCT (NO2) GS-T8 2.88 (431) 0.0000 MLCT (O2N) MS1-T8 3.41 (363) 0.0000 MLCT (O2N) GS-T9 3.41 (363) 0.0073 ML MS1-T9 3.61 (343) 0.0015 MLCT (NO2) GS-T10 3.61 (343) 0.0005 MLCT (NO2) MS1-T10 3.64 (340) 0.0197 ML GS-T11 3.64 (340) 0.0376 ML MS1-T11 3.67 (338) 0.0218 ML GS-T12 3.67 (338) 0.0205 ML MS1-T12 3.73 (332) 0.0011 ML GS-T17 3.98 (312) 0.0238 ML MS1-T15 3.98 (312) 0.0379 ML GS-T18 4.02 (309) 0.1216 ML MS1-T16 4.00 (310) 0.1044 ML GS-T19 4.05 (306) 0.0272 ML MS1-T17 4.03 (308) 0.0104 ML Fig. 6 Schematic of the molecular orbitals involved in the most important electronic transitions in the GS isomer, computed with the M06 functional and the LANL2DZ/6-311++G(d,p) basis set with water as a continuum. The labelling convention is based on the beta spin channel (that which does not contain the two unpaired electrons), in which the most important calculated electronic transitions take place. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This journal is © The Royal Society of Chemistry 2015 Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The equivalent excited states in the MS1 complex are T7, T8, T9 and T12. plane and the py and pz orbitals on the nitro ligands. Conse- quently, the Ni–N and Ni–O bonds weaken when these excited states are populated. Although these states are not directly populated during the photoabsorption, they most likely do play a role in the deactivation process – if the molecule behaves according Kasha's rule, it is expected to persist longest in the T1 state. Around 3.6 eV, there are two excited states with oscillator strengths larger than 0.02, T11 and T12, which both involve electronic transitions from the HOMOβ to LUMOβ+1 orbitals, yielding a redistribution of electron density from the orbitals in the plane to those perpendicular to it. The brightest com- puted excited states appear around 4 eV, and are T17–T19(GS) and T15–T17(MS1). The main electronic transitions giving rise to these states are from the HOMOβ and HOMOβ−1 to the LUMOβ and LUMOβ+1 orbitals. For the GS molecule, the T7, T8 and T10 excited states are metal-to-ligand charge-transfer (MLCT) states, with the electron density transferred largely to the LUMOβ+2 and LUMOβ+3 orbitals, both of which are mainly localized on the CrystEngComm, 2015, 17, 383–394 \| 391 This journal is © The Royal Society of Chemistry 2015 391 CrystEngComm View Article Online View Article Online View Article Online Paper CrystEngComm CrystEngComm The photocrystallographic experiments in ref. 10 were performed irradiating at the absorption maximum at 400 nm, and also at 500 nm to allow for better penetration of the light into the crystal. In both cases, the photo- isomerisation reaction occurs with good conversion.7,10 These conditions should lead to population of excited states around T11, plus vibrationally-excited states of higher-energy electronic configurations which could contain contributions from the brightest states (e.g. T18). All these states lead to an increased antibonding interaction between the Ni and N atoms, which is consistent with their activating the GS →MS1 isomerisation. the relaxation to T1; 2) isomerisation once the system is in T1; and 3) isomerisation in T0 if the excitation energy is transferred into vibrations, and the isomerisation then happens in the vibrational hot-ground state (i.e. vibronic coupling). Considering that the key experimental observa- tions can be explained based on ground-state calculations, the third mechanism seems to be a likely candidate. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. It is also possible that the photochemical process could occur through a combination of all these mechanisms. To distinguish between them, non-adiabatic dynamics simulations would be required. At present, such simulations are rare for organometallic compounds, due to the complexity of their potential-energy surfaces, which typically contain a high density of excited states and crossings between states of different multiplicities.45,48,49 This is an area of ongoing research that we aim to explore in our future work. To explore this further, we computed the excited states of all the species involved in the thermal isomerisation mecha- nisms. Photochemical processes typically involve multiple states, and crossing between states with different spin multi- plicity can also be important; therefore, singlet as well as triplet excited states were considered. The energy difference between the ground-state triplet and singlet states for the GS species is 1.31 eV, and there are three triplet excited states lower in energy than the singlet ground state. Fig. 7 shows the energy profiles for the lowest-energy GS →MS1 photoisomerisation pathway; data for the other pathways, including for the MS1 →MS2 transition, may be found in the ESI.† The calculated isomerisation barriers in the singlet state are of the same order of magnitude or higher than the corresponding triplet barriers. Since the singlet and triplet states near in energy have different character, and the coupling between singlets and triplets requires this differ- ence in electronic nature,47 plus a small energy difference to increase the spin–orbit coupling, crossing between singlet and triplets is a possibility which cannot be discarded; however, only a few singlet excited states are found below the corresponding triplets, which suggests crossings may not form a major part of the photochemical reaction mechanism. This journal is © The Royal Society of Chemistry 2015 Conclusions In summary, we have applied a combination of solid- state and molecular quantum-chemistry calculations to model the energetics and isomerisation processes in the [Ni(Et4dien)(η2-O,ON)(η1-NO2)] system. The effect of the isomerisable ligand on the Ni 3d states and weak interactions between the ligand and the Et4dien backbone appear to both be important contributors to the energy differences between the different linkage isomers. Our results suggest that the dielectric environment of the molecular crystal likewise has a significant effect on the relative stability of the isomers, and, to a lesser extent, the isomerisation barriers. However, beyond this the interactions between the molecular units in the solid are minimal, and thus polarisable-continuum model (PCM) calculations, with a suit- able choice of the dielectric constant, may represent an efficient means of modelling this and, most likely, related systems. Considering only the states with triplet multiplicity, three isomerisation mechanisms could potentially be impor- tant following light absorption: 1) isomerisation during Transition-state modelling suggests that the nitro-to-endo- nitrito and endo-to-exo-nitrito isomerisation scan both occur via rotations of the NO2 ligand out of the plane formed by the Ni–NO2 group in the (meta)stable isomers; the latter mechanism was verified by solid-state molecular dynamics, confirming the validity of this approach. There is an apparent overestimation of the activation barrier for the GS-to-MS1 transition compared to experiment, which requires further investigation; possibly in relation to this, we found no evidence of a transition path connecting the exo-nitrito and nitro forms directly, the reasons for which are not clear at present. Fig. 7 Energy profiles for the GS →MS1 transition in the ground and photoexcited states. Triplet and singlet states are indicated by black and red lines, respectively. The excited states were computed at the TD-DFT/ M06/LANL2DZ/6-311++G(d,p) level of theory with water as a continuum. Modelling the photochemistry of the complex suggests that isomerisation could occur both in the electronically- excited state, and also in the vibrational hot-ground state after de-excitation. Distinguishing between these will require more involved non-adiabatic dynamics simulations, which will be a subject of further work. Furthermore, although Fig. 7 Energy profiles for the GS →MS1 transition in the ground and photoexcited states. Triplet and singlet states are indicated by black and red lines, respectively. The excited states were computed at the TD-DFT/ M06/LANL2DZ/6-311++G(d,p) level of theory with water as a continuum. Fig. 7 Energy profiles for the GS →MS1 transition in the ground and photoexcited states. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. None- theless, since photoexcitation is expected to populate states with significant antibonding character between the metal and the ligand, isomerisation in the excited state is also a strong possibility. Open Access Article. Published on 26 August 2014. Downloaded on 3/18/2020 3:27:48 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 11 P. Coppens, I. Novozhilova and A. Kovalevsky, Chem. Rev., 2002, 102, 861–883. 12 D. A. Johnson and V. C. Dew, Inorg. Chem., 1979, 18, 3273–3274. 13 A. Y. Kovalevsky, K. A. Bagley and P. Coppens, J. Am. Chem. Soc., 2002, 124, 9241–9248. 14 A. Y. Kovalevsky, K. A. Bagley, J. M. Cole and P. Coppens, Inorg. Chem., 2003, 42, 140–147. Acknowledgements g 15 A. E. Phillips, J. M. Cole, T. d'Almeida and K. S. Low, Phys. 15 A. E. Phillips, J. M. Cole, T. d'Almeida and K. S. Low, P Rev. B: Condens. 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The excited states were computed at the TD-DFT/ M06/LANL2DZ/6-311++G(d,p) level of theory with water as a continuum. 392 \| CrystEngComm, 2015, 17, 383–394 This journal is © The Royal Society of Chemistry 2015 View Article Online CrystEngComm Paper 9 M. R. Warren, T. L. Easun, S. K. Brayshaw, R. J. Deeth, M. W. George, A. L. Johnson, S. Schiffers, S. J. Teat, A. J. Warren, J. E. Warren, C. C. Wilson, C. H. Woodall and P. R. Raithby, Chem. – Eur. J., 2014, 20, 5468–5477. these pathways provide a plausible mechanism for the photo- chemical reaction, there are additional possibilities which merit further investigation (e.g. the possible role of other transient metastable species). 10 L. E. Hatcher, J. Christensen, M. L. Hamilton, J. 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